Study was done on mice to confirm the carcinogenesis of BKV
They concluded that BKV promote proliferation ,invasion and migration of bladder cancer .
They focused on 2 important pathways:B catenin signaling pathway
and epithelial mesenchymal transition effect in BKV infected bladder cancer
This study is done by developing a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections The research results are : 1- BKPyV infection in bladder cancer cells is a non-lytic infection 2- BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells 3- BKPyV infection enhances bladder tumor growth and metastasis 4- BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells The authors in conclusion verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. These results may help clinical diagnoses and treatment of BKPyV- related bladder cancer.
These results revealed how TMP-SMX is currently dosed for the prevention and treatment of PCP.The low dose of TMP-SMX yields acceptable results while lowering mortality and PCP-related adverse events (AEs). Additionally, this tactic lessens the overall financial load and improves patients’ adherence to the daily routine. It may still be justified to begin a preventative treatment programme for select patient populations, such as PCP developing on TMP-SMX prophylaxis in HIV-patients. These results would enable the conduct of extensive, prospective RCTs and cohort studies to provide advice for the dosage strategies for the PCP because there is a dearth of information on the ideal dose of TMP-SMX.
Bladder tumours that expressed BKPyV-related proteins were more invasive in vitro, and BKPyV infection encourages bladder cancer proliferation, invasion, and migration. We established that the EMT impact and the -catenin signalling pathway play a part in the features of bladder cancer associated with BKPyV. The clinical diagnosis and therapy of bladder cancer linked to BKPyV may benefit from these findings.
-Level 5 evidence -This study shows that BKV infection after kidney transplantation might be related to urothelial cancer. -No current studies show evidence of viral activity to oncogenesis. – This study shows that β-catenin signaling pathway may be related to malignancy promoting invasion of malignant cells.Beta catenin blocking medications may help combating bladder malignancy in BKV infection.
This article is about the role that BKV infection plays in bladder cancer. The authors of the study researched how BKV infection promote proliferation, invasion and migration of bladder cancer cells. The role of beta catenin signaling pathway and epithelial mesenchymal transition effect in BKV infected bladder cancer was also studied.
Discussion
BKV infecting cells creates two outcomes :
hot allows virus replication leading to viral DNA amplification, progeny vision production and cell lysis
host cell does not allow viral replication, and persistent expression of LTag causes abortion of infected cell or cell transformation.
BKV integrates into the cellular genome of urothelial carcinomas in transplant recipients, thus confirming the correlation of BKV with urothelial carcinomas in the post transplant setting.
Beta catenin signaling pathway plays an important role in tumor invasion and metastasis. Beta catenin can activate genes and cause cell proliferation. Expression of beta catenin may be related to LTag, and activation can promote further tumor cell proliferation and migration invasion.
Thus blocking beta catenin signaling pathway can be an effective alternative for clinical treatment of BKV infected bladder tumors.
Conclusion
The role of BKV auto antigen expression in tumor cells needs to be studies, and the
Level of evidence
This is a narrative review, thus level of evidence is 5.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer Background:
Recent studies have confirmed the integration of the BK polyomavirus (BKPyV) gene into the cellular genome of urothelial carcinomas in transplant recipients, further confirming the correlation between BKPyV and urothelial carcinomas after transplantation. However, the role BKPyV infections play in the biological function of bladder cancer remains unclear. Introduction:
According to research, kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
In 2012, at the International Agency for Research on Cancer (IARC) meeting, BKPyV and JCPyV were classified as “possibly” carcinogenic to human (group 2B) because of the “sufficient evidence” in experimental animals and the “inadequate evidence” in humans for their carcinogenicity .
Whether BKPyV has a causal role in the development of cancer was controversial.
Gene of BKV is found in tumor cell, BKPyV reactivation in immunosuppressed environments may be considered as a transforming factor leading to urothelial carcinomas.
The study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice. Materials and methods: Cell culture:
Human bladder cancer cell lines HTB-9 and T24 were obtained and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C. BKPyV infection and inactivation:
BKPyV stocks were initially propagated in Vero cells from viruses obtained from ATC.
Both T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV. After infection cultured. Animal model:
Male BALB/c nude mice (age 5 weeks, 18–20 g; Cultures of T24 and HTB-9, including their respective BKPyV infected cells (106 cells in 100 mL PBS) were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg) (n = 5). Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
Cell counting:
Kit-8 assay The Cell Counting Kit-8 assay (CCK-8; Xiang SAM, Shanghai, China) was used to determine cell viability Colony-formation assay:
Cells were seeded in six-well plates at an initial density of 200 cells/well. Colonies were clearly visible after 10– 14 days. Selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet. Cell migration and invasion assays
After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet solution. A light microscope was used to count the number of cells in five random fields of view.
CELL WHERE EXAMONED HISTOLOGICAL AND BY if. Results:
BKPyV infection in bladder cancer cells is a non-lytic infection we used two bladder cancer cell lines, T24 and HTB-9, to infect with BKPyV. Forty-eight hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells Results from the CCK8 experiment showed that after infection by BKPyV, the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus). BKPyV infection enhances bladder tumor growth and metastasis:
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days post inoculation. Differences in tumor volume became more pronounced over time. BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKPyV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced.
These results indicate that BKPyV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells. Blocking β-catenin signal inhibits:
BKPyV infection mediated enhancement of proliferation and migration Compared to BKPyV infected cells alone, BKPyV infected cells treated with KYA1797K had significantly reduced cell proliferation migration and invasion capacities.
In vivo, expression levels of Slug were significantly reduced while expression of Claudin-1 was significantly increased. In vivo, BKPyV infection with KYA1797K applied showed significant reduction of tumor size and invasive abilities. Discussion:
With the help of the BKPyV infection tumor cell model, we were able to confirm that the proliferative capacity, migration and invasion. We also found that BKPyV infections promotes β-catenin signaling pathway activation and EMT effects. Besides, blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion. T
These results suggest that βcatenin activation in these BKPyV-infected tumor cells may be related to the overexpression of Lion ability of bladder tumors expressing BKPyV-related proteins were further increased in vitro. Xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis. Conclusions:
In summary, study described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins were more invasive in vitro. It verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
Level of this study 5. Strength:
Experimental study, show proliferation and growth of cell infected by BKV.
Verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related CANCER. Weakness: Proper study of mankind is man, difficulties inherent in extrapolating drug data from animals to man. Animal experiments are poorly designed, conducted and analyzed. Small experimental groups with inadequate statistical power Reviews and summaries of evidence from animal research are methodologically inadequate.
Introduction:
Kidney transplant recipients are3 times more likely to have urothelial cancer than the general population. BKPyV gene has been found integrated into the genome of renal and urothelial cancer cells after transplantation.
Although BKPyV large T antigens (LTag) was detected in prostate, bladder and kidney tumors in some studies, the causal role of BKPyV in the development of cancer could not be proven because BKPyV DNA could not be detected in the cancer samples.
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice. Materials and methods:
1) Human bladder cancer cell lines were obtained and cultured
2) BKPyV stocks were propagated then inactivated and thereafter cultured.
3) These were then injected into mice.
4) 30 days after implantation, the tumors were surgically dissected and
5) Processed for histopathological examination
6) Cell viability was determined and colonies were assayed
7) Cell invasion and migration were determined
8) Cells were washed, incubated and cultured
9) Histological assessment was done using Hematoxylin and Eosin (H&E) staining then immunofluorescence staining and western blot were carried out. Conclusion:
BKPyV infection promotes proliferation, invasion and migration of bladder malignancies and bladder tumors expressing BKPyV related proteins
β-catenin signaling pathway and EMT have an effect on the characteristics of BKPyV-related bladder cancer. Level of evidence is 5 Weaknesses of this article
Experimental study in animals, requires studies in humans Strengths of this article
The study explains how BKPyV infection promotes proliferation, invasion and migration of bladder malignancies.
The study also confirmed the function of the EMT and the – β-catenin signaling in BKPyV-induced bladder cancer.
In 2012, at the International Agency for Research on Cancer (IARC) meeting, BKPyV and JCPyV were classified as “possibly” carcinogenic to human (group 2B) because of the “sufficient evidence” in experimental animals and the “inadequate evidence” in humans for their carcinogenicity . So far, whether BKPyV has a causal role in the development of cancer was controversial
Author developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
Materials and methods-
Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI 1640 (Gibco Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco Life Technologies) in a humidified atmosphere with 5% CO2 at 37 °C.BKPyV stocks were initially propagated in Vero cells from viruses obtained from ATCC (VR-837, Dunlop). Viral lysates were made through three cycles of freezing the infected cells and supernatant at − 80 °C and thawing at 37 °C.
Results-
BKPyV infection in bladder cancer cells is a non-lytic infection.BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells.BKPyV infection enhances bladder tumor growth and metastasis.BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
Conclusions-In summary, author described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins were more invasive in vitro. They verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer.
What is the level of evidence provided by this article?
Evidence level is 5
What are the weaknesses and strengths of this article?
Strength-
How BK virus can promote proliferation,Invasion and migration of bladder cancer cells and how BK virus can enhance β-catenin signaling to promote proliferation
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer.
Introduction
Urothelial carcinoma is common and aggressive malignancy . it is three fold higher in post transplantation if compared to general population.
Both BKPyV and JCPyV have evidence based data to support a link to cancers especially in animals.
Some studies report the presence of BKPyV gene material urothelial malignancies, in transplant recipient. In comparison to non- transplant patient , transplant recipient has 4-11 fold higher urothelial malignancies .
Materials and methods:
Cell culture:
-Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKPyV infection and inactivation:
Viral lysates were made through three cycles of freezing the infected cells and supernatant at − 80 °C and thawing at 37 °C.
After infection for 2 hours, cells were washed three times with phosphate-buffered saline , then were cultured using a medium containing 2% serum with and without 15 μM KYA1797K , a potent and selective β-catenin inhibitor.
Animal model :
Male BALB/c nude mice were housed in sterile filter-capped cages. Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg) .
Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
Colony-formation assay :
Cells were seeded in six-well plates at an initial density of 200 cells/well. Colonies were clearly visible after 10– 14 days and selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet (Sigma-Aldrich).
Cell migration and invasion assays :
Transwell chambers (8-μm pore size; Corning, Corning, NY, USA) pre-coated with and without Matrigel (BD Biosciences) was used to determine cell invasion and migration.
Scratch wound healing assay:
Cells were inoculated in six-well plates and cultured at 37 °C in a 5% CO2 cell incubator. After the cells reached 90% confluence, wounds of approximately 1mm width were created using a sterile pipette tip. Cells were washed, incubated and continuously cultured in serum free medium. Cultures at 0, 8, and 24 h were observed under an inverted microscope .
Histological assessment :
Hematoxylin and eosin(H&E) staining was performed for histological assessments. Tumor tissues were cut into small pieces and soaked in 4% neutral formaldehyde solution. Tissue blocks were washed with distilled water and stored in 70% ethanol overnight. After being dehydrated, embedded in transparent paraffin and sectioned, slides were sealed with polylysine and stained using H&E. Tumor tissues were cut into small pieces and soaked in 4% neutral formaldehyde solution,then washed with distilled water and stored in 70% ethanol overnight. After being dehydrated, embedded in transparent paraffin and sectioned, slides were sealed with polylysine and stained using H&E.
Immunofluorescence staining:
Cell slides and frozen sections in 4% formaldehyde were diluted in PBS for 10 min and permeabilized with 0.2% Triton X100 for 10 min at room temperature . Fixed cells were blocked with a blocking buffer containing milk powder and PBS for 15 min (37 °C). Primary and secondary antibodies were diluted in the blocking buffer and incubated at RT for 50 min each. Fluorescence was detected using an inverted fluorescence microscope .
Western blot :
Membrane proteins were separated on SDS polyacrylamide gel electrophoresis and transferred to
Poly vinylidene difluoride membranes. Membranes were blocked using 5% milk and incubated overnight with anti-β-catenin, anti-cMYC, anti-Slug, anti-claudin-1 and anti-β-actin antibodies (1:1000; all from Cell SignalingTechnology).
Statistical analysis :
Statistical analyses were performed using the unpaired Student’s t-test or one-way Analysis of Variance (ANOVA) for more than two groups.
discusssion:
BKPyV infection is linked to malignancy in immunosuppressed patient.
When BKPyV infects cells, two different outcomes may occur:
(1) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virionproduction and cell lysis; or,
(2) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
High levels LTag are required for cell transformation.
By this study , it is possible to prove that BKPyV-infected cells had high proliferative, migration and invasive ability.
Previous research pointed out that LTag and STag of BKPyV can promote host cell transformation and immortalization, leading to enhanced cell proliferation capacity .
It was previously found that LTag are prooncogenic due to their ability to inactivate tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb), leading to increased cell proliferation . In
addition, STag has been shown to increase activation of the mitogenactivated protein (MAP) kinase pathway, which may also augment cell proliferation and transformation.
In this study, xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis.
Blocking the β-catenin signaling pathway can inhibit this process, which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
In BKPyV infection, the liver metastasis of bladder tumors incidence is higher, which increase the difficulty of treatment and decrease the survival rates of patients. Wnt/β-catenin signaling pathway also plays an important role in tumor invasion and metastasis. Blocking this pathway may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
Conclusions
In summary, we first described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins were more invasive in vitro. We verified the role of β-catenin signaling pathway and EMT effectin the characteristics of BKPyV-related bladder cancer. These results may help clinical diagnoses and treatmentof BKPyV-related bladder cancer.
Q 2 – what is level of evidence.
Level of evidence is 5 .
Q3 what is weakness and strength :BKV infection and malignancy. It opens the door for further future research.
Strength:
Document the link between
Weakness :
It is experimental – animal study . data is not necessarily applicable on human
Introduction Bladder cancer is the 6 th most common malignancy in males with high prevalence in immunocompromised cases. Kidney transplant recipients are three times more susceptible to have urothelial cancer compared to the general population. BKPyV reactivation can lead to BKPyV-related nephropathy in renal transplantation and haemorrhagic cystitis after hematopoietic stem cell transplantation . International Agency for Research on Cancer (IARC) meeting at 2012 stated that , BKPyV and JCPyV can be possibly carcinogenic to human but this is controversial . Some studies declared that BKPyV DNA wasnot detected in the cancer samples while others mentioned that BKPyV gene integration was noticed in the urinary tract epithelial cell tumor A study revealed that cancer bladder risk is higher in transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease compared to transplant recipients without BKPyV. The Wnt/β-catenin pathway and inhibition of β-catenin degradation in cytoplasm has a role in tumorigenesis and tumor proliferation, invasion, and metastasis . BKPyV could be oncogenic because of expression of early coding region-encoded proteins LTag and small T antigen (STag), which can stimulate neoplastic transformation. this study aimed at evaluating BKPyV infections effect on the occurrence of bladder cancer .
Materials & methods BKVPyV stockes were propagated then inactivated and cultured, thirty days after implantation the tumor were surgically dissected and processed for histopathological examination. Cells were washed, incubated and cultured at 37 degrees, then all histology was done with H&E staining, IF staining, western blot were carried out, and images were processed. All data was presented as mean+/- SD, analyzed using T test and ANOVA, P values were taken significant if <0.005.
Results They used two bladder cancer cell lines, T24 and HTB-9 to infect with BKPyV, after 48H after infection the infected cell showed larg T protien expression, the infected cell did not lyse, but continued to proliferate and grow for 9 days. The CCK8 experiment showed the ability of infected cell has increased to persist and proliferate. BKPyV infection enhances B catenin signaling pathway activation and epithelial mesenchymal transition effect in bladder cancer cells, and blocking B-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration.
Conclusion
The outcomes of the study in identifying BKPyV infection’s role in causing urinary tract cancer could aid in the identification and proper treatment of cancer resulting from the infection.
What is the level of evidence provided by this article?
Level V
What are the weaknesses and strengths of this article?
Weakness
experimental study in animals, requires studies in humans
Strength
It is a study to objectively prove role of BKV in virus induced bladder cancer.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer:
Summarise this article:
Introduction:
– urothelial carcinoma is a common and highly malignant tumor in the urinary system
– kidney transplant recipients (KTRs) are 3 times more likely to have urothelial cancer than the general population
– BKPyV undergoes reactivation in immunosuppressive states resulting in viruria, viremia, ureteral stricture , BKVAN in kidney transplant recipients and hemorrhagic cystitis in HSCT recipients
– the role of BKPyV in carcinogenesis remains controversial
– BKPyV reactivation may be considered a transforming factor leading to urothelial carcinomas in immunosuppressed states
Materials and methods:
– human bladder cancer cell lines were obtained and cultured
– BKPyV stocks were propagated then inactivated and thereafter cultured
– these were then injected into mice
– 30days after implantation, the tumors were surgically dissected and processed for histopathological examination
– cell viability was determined and colonies were assayed
– cell invasion and migration were determined
– cells were washed, incubated and cultured
– histological assessment was done using hematoxylin and eosin (H&E) staining
– immunofluorescence staining and western blot were carried out
Results:
– both bladder cancer cell lines did not lyse due to BKPyV infection but instead continued to proliferate
– BKPyV infection was shown to: –
promote proliferation, invasion and migration of bladder cancer cells
enhance bladder tumor growth and metastasis
enhance β-catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) effect in bladder cancer cells
Discussion:
– malignancies affect the long-term survival of kidney transplant recipients
– BKPyV seems to have a role in carcinogenesis based on studies which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation
– when BKPyV infects cells the host cell can either: –
allow virus replication resulting in viral DNA amplification, progeny virion production and cell lysis or
prevent virus replication causing abortion of infected cells or cell transformation
– BKPyV infection did not cause lysis of bladder cancer cells
– the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPvV-related proteins were increased in vitro Conclusion
– BKPyV infection promotes proliferation, invasion and migration of bladder malignancies and bladder tumors expressing BKPyV related proteins
– β-catenin signaling pathway and EMT have an effect on the characteristics of BKPyV-related bladder cancer
Level of evidence provided by this article:
Level V Weaknesses of this article
– experimental study in animals, requires studies in humans Strengths of this article
– the study explains how BKPyV infection promotes proliferation, invasion and migration of bladder malignancies
– the study also confirmed the function of the EMT and the – β-catenin signaling in BKPyV-induced bladder cancer
Introduction: · kidney transplant recipients are3 times more likely to have urothelial cancer than the general population. BKPyV gene has been found integrated into the genome of renal and urothelial cancer cells after transplantation. · Although BKPyV large T antigens (LTag) was detected in prostate, bladder and kidney tumors in some studies, the causal role of BKPyV in the development of cancer could not be proven because BKPyV DNA could not be detected in the cancer samples.
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Aims of the study: · Effects of BKPyV infection in proliferation and migration of bladder ca cells. · Role of wnt/B-catenin pathway and EMT in proliferation and migration of Ca cells. · Possible Causal Role of BKPyV in bladder cancer? Materials and Methods; 1. human bladder cancer cell lines (HTB-9 and T 24) were cultured – in Cell culture media 10% foetal bovine serum @ 370C 2. BKPyV stocks from Vero cells propagated – viral lysate was inactivated (@1000C x10min) and the inactive virus was maintained.
3. Bladder cancer cell lines (T24 and HTB-9) were infected with BKPyV. 2 hours of viral infection, cells were washed and cultured in 2 separate serum media – one with KYA1797K (β-catenin inhibitor) or another without inhibitor
4. Animal (Mice) model: Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice, with and without subsequent intraperitoneal injections of KYA1797K. After 30days, tumors were surgically dissected, stored in liquid nitrogen and processed for HPE. 5. Cell viability was determined (Cell counting kit-8 assay / CCK-8) and colonies counted after a special Colony Forming assay.
6. Cell migration and invasion assay performed – active and average number of cells counted using Trans-well chambers
Scratch wound healing assay – cells were washed, incubated and cultured at 370C, thereafter prepared and studied under inverted microscope.
8. Histological Examination carried out using (H&E) staining, IF 9. Western blot assay (for membrane proteins) – separated by PAG electrophoresis, fixed to PLD membrane and incubated overnight with anti-β-catenin, anti-cMYC, anti-Slug, anti-claudin-1 and anti-β-actin antibodies.
Results:
Both Bladder Cancer cell lines did not lyse due to BKPyV infection, but instead continued to proliferate with expression of LTag.
BKPyV Infected T24 and HTB9 bladder cells had more proliferation, migration and invasion compared to cells without infection.
BKPyV infection enhances bladder tumor growth and metastasis – 30 days post inoculation BKPyV infected cells had more growth rate in mice with significantly more tumor volume. Liver metastatic tumor seen in virus infected group vs no tumor in control mice.
BKPyV infection enhances Beta catenin signaling pathway activation and EMT effect in bladder cancer cells, in comparison to control – beta catenin, cMYC and slug protein in BKPyV infected cells were more while claudin 1 protein was less.
Blocking beta catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration -BKPyV Infected cells tx with KYA1797 had less proliferation, migration and infiltrating capacity compare to untreated cells.
Discussion: · BKPyV infection did not cause lysis of bladder cancer cells · BKPyV infection was shown to – o promote proliferation, invasion and migration of bladder cancer cells o enhance bladder tumor growth and metastasis to liver o Enhanced β-catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) effect in bladder cancer cells infected with BKPyV virus increases proliferation, invasion of bladder cancer cells. Conclusion § BKPyV infection promotes proliferation, invasion and migration of bladder cancer § Bladder tumors expressing BKPyV related proteins were more invasive in vitro. § The study verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. 2, Level of evidence provided by this article Level V – mechanistic studies (animal research / In Vitro studies) 3.Strengths of the study / article
· First study describing that BKPyV infection promotes proliferation, invasion and migration of bladder cancer.
· Objectively verified the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV infected bladder cancer.
· These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
4.Weaknesses of the study / article · Experimental study in in vitro (cell culture) and animal (mice) model · This result can’t be extrapolated to patients or general population · Needs further well-designed studies in human
1. Please summarise this article; According to 2018 statistics urothelial carcinoma is the most common and highly malignant tumor in the urinary system, sixth most common malignancy in male. According to literature bladder cancer is three times more common in renal transplantation. BK virus belongs to a family of polyomavirus family, small double-stranded non-enveloped DNA virus, categorized into four groups with different virulence, in 2012 IARC meeting the BKVPyV and JCPyV were classified as possible cause of carcinoma, SV40 has been weak association with malignancy. Together, these result will make more questions are will clarify the characteristic of BKV related to bladder and other related carcinomas. Materials and methods;
BKVPyV stockes were propagated then inactivated and cultured, thirty days after implantation the tumor were surgically dissected and processed for histopathological examination.
Cells were washed, incubated and cultured at 37 degrees, then all histology was done with H&E staining, IF staining, western blot were carried out, and images were processed. All data was presented as mean+/- SD, analyzed using T test and ANOVA, P values were taken significant if <0.005. Results; They used two bladder cancer cell lines, T24 and HTB-9 to infect with BKPyV, after 48H after infection the infected cell showed larg T protien expression, the infected cell did not lyse, but continued to proliferate and grow for 9 days. The CCK8 experiment showed the ability of infected cell has increased to persist and proliferate. BKPyV infection enhances B catenin signaling pathway activation and epithelial mesenchymal transition effect in bladder cancer cells, and blocking B-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration. Discussion; The correlation between BKpyV infection and tumorigeesis in immunocompromised patients has some association, by deep sequencing studies confirmed that that BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after renal transplantation. According to Querido et al, JCpyV may cause urothelial carcinoma after transplantation Conclusion; As discussed in discussion the BKPyV infection promotes proliferation, invasion, and migration of bladder cancer and expression of bladder tumor related gene. Level of evidence V
Kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Material and methods:
Both Human bladder cancer cell lines – T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV.
After infection for 2 hours, cells were washed three times with phosphate-buffered saline.
Male BALB/c nude mice were housed in sterile filter-capped cages.
Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797.
After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet solution.
A light microscope was used to count the number of cells in five random fields of view.
The mean cell number was calculated for each group.
Results:
BKPyV infection in bladder cancer cells is a non-lytic infection
Both T24 and HTB-9 cells did not lyse due to BKPyV infections, but continued to proliferate and grow for 9 days after the initial infection.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells
BKPyV infection enhances bladder tumor growth and metastasis
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
Blocking β-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration
Discussion:
When BKPyV infects cells, two different outcomes may occur: (1) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis; or, (2) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation. This result is determined by the continuous expression of LTag
Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis.
In the nucleus of tumor cells, β-catenin combined with the transcription factor family Tcf / Lefs can activate genes such as cMYC and cause cell proliferation and EMT effects.
JCPyV large T antigen can interact with β-catenin and stimulate expression of β-catenin target genes.
The study found that BKPyV infections promotes β-catenin signaling pathway activation and EMT effects.
Conclusion
The study described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins were more invasive in vitro.
The study verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer.
Strength of study:
First study to objectively prove role of BKV in virus induced bladder cancer.
Weakness of the study:
This is animal study, and the observations need to confirm in animal studies.
● Tumors have become one of the main factors affecting the long-term survival of kidney transplant recipients.
● BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation
● Bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver
● Bladder cancer is also a prone tumor type for immunocompromised patients.
● kidney transplant recipients are three times more likely to have urothelial cancer than the general population
● BKV reactivation after renal transplant causes symptoms such as viremia, viruria, ureteral stricture and BKVN as well as hemorrhagic cystitis after HSCT
● In 2012, at the International Agency for
● BKV was classified as “possibly” carcinogenic to human
● This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Results
● BKPyV infection in bladder cancer cells is a non-lytic infection
● BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells
● BKPyV infection enhances bladder tumor growth and metastasis
● Blocking β-catenin signal inhibits BKPyV infection-mediated enhancement of proliferation and migration
● When BKPyV is activated in vivo, BKPyV triggers abnormally high expression of LTag in the host cell leading to cell transformation.
● LTag and STag of BKPyV can promote host cell transformation and immortalization, leading to enhanced cell proliferation capacity
● LTag are prooncogenic due to their ability to inactivate tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb), leading to increased cell proliferation
● β-catenin activation in BKPyV-infected tumor cells may be related to the overexpression of LTag.
● Activation of β-catenin further promotes the invasion and migration of tumor cells. it can enhance the expression of cMYC to promote cell proliferation.
● Xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis.
● Blocking the β-catenin signaling pathway can inhibit this process, which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
● Level of evidence : 5
● Strength points of study :
☆ Apromising study for more researchs in the future
☆ The study explain pathology between BKpyV infection and bladder cancer
Which may be a basic for experimental therapies later
● Weekness points of study :
☆ Experimental animal study with xenografts
III. BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Summarise this article
Introduction
– urothelial carcinoma is a common and highly malignant tumor in the urinary system
– kidney transplant recipients (KTRs) are 3 times more likely to have urothelial cancer than the general population
– BKPyV undergoes reactivation in immunosuppressive states resulting in viruria, viremia, ureteral stricture , BKVAN in kidney transplant recipients and hemorrhagic cystitis in HSCT recipients
– the role of BKPyV in carcinogenesis remains controversial
– BKPyV reactivation may be considered a transforming factor leading to urothelial carcinomas in immunosuppressed states
Materials and methods
– human bladder cancer cell lines were obtained and cultured
– BKPyV stocks were propagated then inactivated and thereafter cultured
– these were then injected into mice
– 30days after implantation, the tumors were surgically dissected and processed for histopathological examination
– cell viability was determined and colonies were assayed
– cell invasion and migration were determined
– cells were washed, incubated and cultured
– histological assessment was done using hematoxylin and eosin (H&E) staining
– immunofluorescence staining and western blot were carried out
Results
– both bladder cancer cell lines did not lyse due to BKPyV infection but instead continued to proliferate
– BKPyV infection was shown to: –
promote proliferation, invasion and migration of bladder cancer cells
enhance bladder tumor growth and metastasis
enhance β-catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) effect in bladder cancer cells
Discussion
– malignancies affect the long-term survival of kidney transplant recipients
– BKPyV seems to have a role in carcinogenesis based on studies which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation
– when BKPyV infects cells the host cell can either: –
allow virus replication resulting in viral DNA amplification, progeny virion production and cell lysis or
prevent virus replication causing abortion of infected cells or cell transformation
– BKPyV infection did not cause lysis of bladder cancer cells
– the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPvV-related proteins were increased in vitro
Conclusion
– BKPyV infection promotes proliferation, invasion and migration of bladder malignancies and bladder tumors expressing BKPyV related proteins
– β-catenin signaling pathway and EMT have an effect on the characteristics of BKPyV-related bladder cancer
Level of evidence provided by this article
Level V
Weaknesses of this article
– experimental study in animals, requires studies in humans
Strengths of this article
– the study explains how BKPyV infection promotes proliferation, invasion and migration of bladder malignancies
– the study also confirmed the function of the EMT and the – β-catenin signaling in BKPyV-induced bladder cancer
SUMMARISE ARTICLE.
9 BKV INFECTION PROMOTES GROWTH AND AGGRESSIVENESS IN BLADDER CANCER.
INTRODUCTION.
-Urothelial ca is the 6th most common Ca in males and KTR are x3 likely to have it compared to the general population.
-We have enough evidence in animals but not humans for causal role of BKV in dev of Ca.
AIMS OF THE STUDY.
To establish effects of BKPyV infection in proliferation and migration of bladder ca cells.
To establish the role of wnt/betta catenin pathway and EMT in proliferation and migration of Ca cells.
To establish xtics of BKPyV related bladder malignancies..
MATERIALS AND METHODS;
Cell culture – Human bladder ca cell lines;HTB-9 and T 24 retrieved from ATCC and cultured with 10% fetal bovine serum at 37 degrees.
BKPyV infection and inactivation -Infected cells and supernatant frozen at – 80 degrees and thawed at 37 degrees. BKPyV later inactivated at 100 degrees for 10 minutes.
Animal model -T24 and HTB-9 samples injected into mice +/- KYA1797K,Tumors were later resected at 21/7 post inoculation and stored in liquid nitrogen before processing.
Cell counting kit -8 assay -CCK-8 used to establish cell viability with all recordings reproduced in three independent experiments.
Colony forming assay- Cells were put in 6 plates, colonies identified after 10-14/7,fixed with 4% paraformaldehyde and stained with crystal violet and later the number of colonies determined.
Cell migration and invasion assay -Transwell chambers were used to determine the active and later average number of cells calculated
Scratch wound healing assay -Cells were inoculated and cultured at 37 degrees and thereafter prepared and studied via a microscope.
Histological assessment -All histology was stained by HE post preparation and thereafter studied.
IF staining -Fluorescence was determined by inverted fluorescence microscope and images processed by NIH,Bethseda,MD,USA
STATISTICAL ANALYSIS;
-All data was presented as mean +/- SD, analyzed using T test and ANOVA.P values were significant if < 0.005
RESULTS;
BKPyV Infection in bladder cancer cells in a non lytic infection – Using T24 and HTB9 ,48hr post infection, they demonstrated large T protein expression and mantained proliferation for 9 days post infection without lysis from BKPyV.
BKPyV Infection promotes proliferation, invasion and migration of bladder cancer cells.-T24 and HTB9 bladder cells had more migration and proliferation with BKPyV compared to without.
BKPyV infection enhances bladder tumor growth and metastasis -15 days post infection, BKPyV infected cells had more growth rate in mice and the difference in tumor vol was more significant with time. New lesions were also seen in liver while nil tumor seen in control mice.
BKPyV infection enhances Beta catenin signaling pathway activation and EMT effect in bladder cancer cels-In comparison to control, beta catenin,cMYC and slug protein in BKPyV infected cells were more while claudin 1 protein was less.
Blocking beta catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration -BKPyV Infected cells tx with KYA1797 had less proliferation, migration and infiltrating capacity compare to untreated cells.
DISCUSSION.
–As evidenced by BKPyV infection tumor model, BKPyV related protein in bladder tumor increases;
Proliferating capacity.
Ability to grow.
Metastatic capacity.
Risk of liver mets.
-Beta catenin signal pathway activation increases invasion and proliferation of tumor cells ang this might alter future mgt of infected BKPyV bladder malignancies.
-The information gotten from this study can be used to modify tx and better outcomes in future.
LEVEL OF EVIDENCE – 5
– STENGTHES;
-Used animal model to get us key information that will inform future studies and hopefully impact positively on pt mgt.
WEAKNESSES;
-Non human subjects means less application in human beings in managing their post transplant status.
Introduction
Urothelial carcinoma are one of the most common cancers and are highly aggressive. Kidney transplant recipients are 3 times more likely to develop urothelial cancers than general population. BKPyV and JCPyV have both being classified as possible carcinogenic supported by sufficient evidence in animal studies and insufficient in human studies.
Some studies have shown BKPyV gene integration in urothelial malignancies, this association was seen in transplant recipients and none in non-transplant population. Other studies have shown that transplant recipients are 4-11 times more prone to urothelial malignancies than general population.
Aim of the study: It explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice. Together, these results will clarify the characteristics of BKPyV- related bladder tumours, laying a foundation for further clinical trials.
Results: BKPyV infection of bladder cancer cell lines leads to an infection that is non-lytic. This BKPyV infected bladder cancer cells lines have increased proliferation ability and migration than controls. Their tumour growth rate also increased exponentially. There was an increased expression of β-catenin, cMYC and Slug proteins while the expression of Claudin-1 protein was significantly reduced.
Discussion: BKPyV infection in bladder cancer cells does not have a lytic effect. There is increased proliferative capacity, migration and invasion of the bladder tumours with BKPyV infection. BKPyV infection promotes signalling in the β-catenin pathway and blocking of this pathway may inhibit BKPyV function to promote tumour cell proliferation, migration and invasion.
Conclusion: BKPyV infected bladder cancer cell lines in vitro there was increased proliferation, invasion and migration. β-catenin signalling pathway plays a role in this infected bladder cancer cells and this may help in the diagnosis and treatment of BKPyV related bladder cancer.
Level of evidence: Animal study hence level V.
Strengths: Forms basis of future research.
Weakness: Animal study thus difficult to draw any conclusions from it.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Please summarise this article.
Introduction :
– The most common and highly malignant tumor in the urinary system is the urothelial carcinoma of which bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver .
– kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
– BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised populations, especially transplant recipients causing virus ,viremia and nephritis and ureteral stenosis in kidney transplant patients also causes hemorrhagic cystitis after hematopoietic stem cell transplantation. BKPyV and JCPyV were possibly carcinogenic to humans,when reactivated in a state of immunosuppression environments it may be considered as a transforming factor leading to urothelial carcinomas.
The risk of bladder cancer was found to be increased (4-11 times ) in kidney transplant recipients with BKPyV viremia or polyomavirus associated kidney disease when compared to transplant recipients without BKPyV. BKPyV is considered to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation. This experimental study in animals explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway ( which is implicated in cell proliferation and involved in tumorigenesis ) by establishing a model for BKPyV infections in bladder cancer cells and mice.
Materials and methods:
Cell culture: -Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKPyV infection and inactivation: The cultured BKPyV underwent three cycles of freezing and supernatant at − 80 °C and thawing at 37 °C. BKPyV was inactivated at 100 °C for 10 min. Both T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV. After infection for 2 hours, cells were washed three times with phosphate-buffered saline , then were cultured using a medium containing 2% serum with and without 15 μM KYA1797K , a potent and selective β-catenin inhibitor.
Animal model :
Male BALB/c nude mice (age 5 weeks, 18–20 g; were housed in sterile filter-capped cages. Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice . Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
Cell counting Kit-8 assay: Is the determinant of cell viability. Cells were seeded in 96-well plates . Absorbance of the medium at 450 nm was detected using a spectrophotometer by assessing cell viability.
Colony-formation assay : Cells were seeded in six-well plates at an initial density of 200 cells/well. Colonies were clearly visible after 10– 14 days and selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet . Colonies containing > 50 cells were counted using light microscopy .
Cell migration and invasion assays : Cell invasion and migration was determined by transwell chambers pre-coated with and without Matrigel . Cells (105 ) in 200 μL FBS-free medium were seeded in the upper chamber , and 600 mL medium containing 10% FBS was added to the lower chamber. After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet solution. A light microscope was used to count the number of cells in five random fields of view. Scratch wound healing assay: Cells were inoculated in six-well plates and cultured at 37 °C in a 5% CO2 cell incubator. After the cells reached 90% confluence, wounds of approximately 1 mm width were created using a sterile pipette tip. Cells were washed, incubated and continuously cultured in serum free medium and observed under an inverted microscope at 0, 8, and 24 h . Histological assessment :
Tumor tissues were cut into small pieces and soaked in 4% neutral formaldehyde solution,then washed with distilled water and stored in 70% ethanol overnight. After being dehydrated, embedded in transparent paraffin and sectioned, slides were sealed with polylysine and stained using H&E. Immunofluorescence staining:
Cell slides and frozen sections in 4% formaldehyde were diluted in PBS for 10 min and permeabilized with 0.2% Triton X100 for 10 min at room temperature . Fixed cells were blocked with a blocking buffer containing milk powder and PBS for 15 min (37 °C). Primary and secondary antibodies were diluted in the blocking buffer and incubated at RT for 50 min each. Fluorescence was detected using an inverted fluorescence microscope .
Western blot : Using SDSpolyacrylamide gel electrophoresis membrane proteins were separated and transferred to polyvinylidene difluoride membranes. Membranes were blocked using 5% milk and incubated overnight with anti-β-catenin, anti-cMYC, anti-Slug, anti-claudin-1 and anti-β-actin antibodies .
Statistical analysis : Statistical analyses were performed using the unpaired Student’s t-test or one-way Analysis of Variance (ANOVA) for more than two groups. All analyses were carried out using Graph pad prism 7 software and significant differences were considered when values had P < 0.05. Results :
Bladder cancer cell lines, T24 and HTB-9 were used to infect with BKPyV. Forty-eight hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV .BKPyV infections effect on cell fates was observed by extending the infection time . Both T24 and HTB-9 cells did not lyse due to BKPyV infections, but continued to proliferate and grow for 9 days after the initial infection .
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells: Results from the CCK8 experiment showed that after infection by BKPyV, the cells’ proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus) . Colony formation experiments further verified in the result of this study , showing that BKPyV infections promote proliferation of bladder cancer cells in vitro . T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates and higher invasive capacities than non-infected cells than the controls . BKPyV infection enhances bladder tumor growth and metastasis :
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, compared with the control group at 15 days post inoculation and became more pronounced over time . On day 30 post inoculation, new tumors were discovered in the liver of BKPyV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice . Pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream . BKPyV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells both in vivo and in vitro studies . inhibits BKPyV infection mediated enhancement of proliferation and migration Compared to BKPyV infected cells alone, BKPyV infected cells treated with KYA1797K ( Blocking β-catenin signal ) had significantly reduced cell proliferation ,reduction of tumor size , migration and invasion capacities with no metastasis occurring in vivo . Discussion : -Recently the correlation between BKPyV infection and tumorigenesis in immunocompromised individuals has drawn increased attention. -BKPyV plays an important role in cancers of the urinary system was recently confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation . – BKPyV infections did not have a lytic effect on bladder cancer cells. -Previous research pointed out that LTag and STag of BKPyV can promote host cell transformation and immortalization, leading to enhanced cell proliferation capacity . However, the role of BKPyV autoantigen expression in tumor cells has not been studied, and the biological characteristics of BKPyV-related tumors are unknown. This study was able to confirm that the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPyV-related proteins were further increased in vitro. Their ability to grow, invade and metastasize to other nearby organs in vivo was highly likely, and is of further threat to clinical patients. After BKPyV infections, liver metastases of bladder tumors are likely to occur, which will increase the difficulty of clinical treatments and seriously affect the survival rates of patients. -Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis . Blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion. as well as inhibition of bladder tumor cell distant metastasis , which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
Conclusion : BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins that were more invasive in vitro. The role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer was verified . These results is promising in clinical diagnoses and treatment of BKPyV-related bladder cancer.
What is the level of evidence provided by this article?
Level V
What are the weaknesses and strengths of this article?
weakness : is experimental study done in animal and the use of xenograft which is not applied in human .
Strength points : – -It verify the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. -It described that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors. -It gives promising results regarding treatment of BKPyV-related bladder cancer by blocking β-catenin signaling pathway .
This Article is with evidence level of 7 (animal studies-in vitro).
The causality of some Polyomaviruses to cancer is still controversial, and the relation of BK and JC though more prominent, still with low evidence. for that reason, this study was studied, but this study was performed in vitro in cell lines which may not be applicable with the same strength in vivo rather than in human. Despite that, this study showed cell proliferation and activity of T24 & HTB9 bladder cancer cells not observed in the non-infected group.
Novelty and the results comparing two groups are strengths, but applicability, being in vitro, and lack of long-term follow-up are weaknesses aspects
BK polyomavirus infection promotes growth and aggressiveness in bladder
cancer
Summary
· It was controversial to prove the role of BKV in cancer bladder, as the virus was not always isolated from cancer tissue.
· Then finding BKV DNA integrated into the DNA of urothelial cells in kidney transplant recipients with urothelial carcinoma.
· The current experimental study demonstrated the role of BKV infection in mice model.
· It concluded that it enhanced tumor growth and metastasis.
Level of evidence; experimental animal study, level V. Point of weakness and strength
· Weakness is experimental in animals needs further studies to be concluded in human.
· Strength is clarification the role of BKV in bladder cancer.
Summary of the article BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
This is an experimental study, developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
Results and Discussion
1. BKPyV infection in bladder cancer cells is a non-lytic infection. 2. BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells. 3. BKPyV infection enhances bladder tumor growth and metastasis. 4. BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells. 5. Blocking β-catenin signal inhibits BKPyV infection- mediated enhancement of proliferation and migration. 6. When BKPyV infects cells, two different outcomes may occur: a) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis…or, b) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
What are the weaknesses and strengths of this article? Weakness: a) the role of BKPyV autoantigen expression in tumor cells has not been studied. b) the biological characteristics of BKPyV-related tumors are unknown. Strength a) The study was able to confirm that the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPyV-related proteins were further increased in vitro. The level of evidence provided by this article
This is an experimental study( animal study)
Level of evidence grade 5
BKPyV and urothelial carcinoma:
It still controversial if BKPyV is causing neoplastic transformation or not. The international agency for research on cancer meeting classified BKPyV and JCPyV as possibly carcinogic to human owing to sufficient evidences in animal studies and inadequate evidences in human for their carcinogenicity. Primary evidences :
BKPyV role in malignancies stem from finding of Larg T protein [TAg] in prostate, bladder and kidney tumors.
However, No BKPyV DNA was isolated from any of the kidneys and bladders tumors. Urothelial Cancer:
Bladder cancer is the a common disease ranked 6th in its prevalence after lung, prostate, colon, stomach and liver.
Kidney transplant recipients are 3 times more prone to have urothelial cancer. Study design:
By infecting bladder cancer cells with BKPyV and studying its impact on behavior of those cells in comparison to a mice tumor cells not infected with BKPyV.
The study proved that BKPyV is promoting malignant cells to be aggressively dividing, invading and metastasizing.
Please summarise this article.
-Urothelial carcinoma is one of the most common and highly malignant tumors in the urinary system.
-Bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver and is a prone tumor type for immunocompromised patients.
-BKPyV and JCPyV were classified as “possibly” carcinogenic to human .
-Many recent studies detected BKPyV gene integration in urinary tract epithelial cell tumors, providing further evidence for the correlation between BKPyV and urinary tract tumor.
-Some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk of bladder cancer when compared to transplant recipients without BKPyV .
– BKPyV is thought to be oncogenic due to the expression
of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation.
– They used two bladder cancer cell lines, T24 and HTB-9, to infect with BKPyV. 48- hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV.
-By extending the infection time, they observed the effect of BKPyV infections on cell fates. Both T24 and HTB-9 cells did not lyse due to BKPyV infections, but continued to proliferate and grow for 9 days after the initial infection and express large T antigen after 30 days. VP1 expression in these two cell infection models increased continuously in the first few days, but maintained a stable level after.
-Results showed the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV.
-Colony formation experiments further verified their results, showing that BKPyV infections promote proliferation of bladder cancer cells in vitro.
-Transwell migration results showed that T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates than the controls.
– BKPyV infected T24 and HTB-9 cells had significantly higher invasive capacities than non-infected cells.
-The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly .
– The BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation .
-Querido et al. showed that JCpyV may cause urothelial carcinoma after transplant , which also supports the argument that human polyomavirus is closely related to bladder cancer.
-When BKPyV infects cells, two different outcomes may occur:
(1) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis.
(2) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
– A small amount of LTag however is not sufficient to cause tumors. BKPyV triggers abnormally high expression of LTag in the host cell through various mechanism, eventually leading to cell transformation.
– After BKPyV infections, liver metastases of bladder tumors are likely to occur.
– Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis . JCPyV large T antigen can interact with β-catenin and stimulate expression of β-catenin target genes.
– BKPyV infections promotes β-catenin signaling pathway activation and EMT effects.
– Besides, blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion.
-These results suggest that β- catenin activation may be related to the overexpression of LTag. Such activation of β-catenin further promotes the invasion and migration of tumor cells. What is the level of evidence provided by this article?
Level 5 What are the weaknesses and strengths of this article? Weakness: experimental study on animal. Strengths: It explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT .These results will clarify the characteristics of BKPyV related bladder tumors, laying a foundation for further clinical trials.
# Introduction:
*Carcinoma of the bladder cancer is the sixth most common cancer in males and more common in immunocompromised patients.
*The incidence of urothelial cancer is three fold higher in kidney transplant recipients compared to control group.
* BKPyV reactivation PKT lead to symptoms like viremia, viruria, ureteral stricture, BKPyV-related nephropathy and hemorrhagic cystitis post BMT.
* In 2012, the (IARC) meeting, BKPyV and JCPyV were classified as “possibly” carcinogenic to human (group 2B) due to “sufficient evidence” in experimental animals and the “inadequate evidence” in humans for their carcinogenicity.
# Materials and methods:
*The study was an experimental animal study
*Cell culture, human bladder cancer cell lines HTB-9 and T24 were obtained and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
*BKPyV infection and inactivation BKPyV were propagated in Vero cells, viral lysates were made and inactivation was performed at 100 °C for 10 min. Both T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV.
#Animal model:
*Male BALB/c nude mice were housed in sterile filter-capped cages.
*Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected s/c into nude mice.
*30 days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
*The Cell Counting Kit-8 assay was used to determine cell viability.
*Colony-formation assay cells were seeded in six-well plates at 200 cells/well and visible after 10–14 days and selected cells were fixed with 4% paraformaldehyde
for 30 min then counted using LM.
# Results:
*BKPyV infection in bladder cancer cells is a non-lytic
infection
*Further research shows the BKPyV infected T24 and HTB-9 cells xenografted
on mice still express large T antigen after 30 days.
*The cratch healing test demonstrated that BKPyV infected T24 and HTB-9 cells migrated significantly faster on plates than the control group.
* BKPyV infection promoted the migration of bladder cancer cells in vitro.
*BKPyV infected T24 and HTB-9 cells had significantly higher invasive capacities than non-infected cells.
*BKPyV infection enhances bladder tumor growth and metastasis.
*BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
*Blocking β-catenin signal inhibits BKPyV infectionmediated
enhancement of proliferation and migration.
# What is the level of evidence provided by this article?
*Level (V)
# The strength of the study:
*It was described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated
proteins were more invasive in vitro.
*The study verified the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV infected bladder cancer. These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
# The limitation of the study:
*The result obtained from the animal (mice model that differ from the human).
I agree with your analysis of strengths and limitations, and summary of this article.I agree with your analysis of the level of evidence this article provides.
Introduction
Urothelial carcinoma is a common and highly malignant tumor, for the immunocompetent and more so for the immunosuppressed population, especially kidney transplant recipients. BK polyomavirus (BKPyV) reactivation after transplantation causes viremia, viruria, ureteral stricture, hemorrhagic cystitis (in HSCT) and BKPyV-related nephropathy. It has been classified as possibly carcinogenic by the IARC. Studies have detected the presence of BKPyV large T antigens in prostate, bladder and kidney tumors, in post-transplant patients. The Wnt/β-catenin pathway is implicated in cell proliferation and transcription, and regulating pattern formation during development. This pathway is associated with tumor proliferation, invasion and metastasis. BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic formation.
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of the Wnt/β-catenin pathway in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Material and methods
Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection and cultured. Viral lysates were made through three cycles of freezing the infected cells and supernatant, and then thawing them. The inactivation of BKPyV was performed at 100°C for 10 minutes. The two cell lines were cultured and infected with BKPyV, and then re-cultured. Male BALBc nude mice were injected with the final culture. Thirty days after implantation, the tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination. The Cell Counting Kit-8 assay was used to determine cell viability. Absorbance of the medium was detected using a spectrophotometer by assessing cell viability. All observations were reproduced at least three times in independent experiments. Cells were seeded in plates and colonies were visible after 10-14 days. The average number of colonies was determined from three independent experiments. Transwell chambers were used to determine cell invasion and migration. A light microscope was used to count the number of cells in five random fields of view, and a mean cell numer was calculated for each group. The cells were then inoculated and cultured. Cultures at 0, 8 and 24 hours were observed. Hematoxylin and eosin staining was performed for histological assessments. Cell slides and frozen sections were then stained using immunofluorescence staining and processed.
Results
BKPyV infection in bladder cancer cells is a non-lytic infection
Two bladder cancer cell lines, T24 and HTB-9, were used to infect with BKPyV. 48 hours after infection, they showed large T protein expression of BKPyV.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells
After infection by BKPyV, the ability and activity of cell proliferation increased in both bladder cancer cell lines, compared to the cells not infected by BKPyV. It was also noted that BKPyV infected cells had a significantly higher invasive capacity than non-infected cells.
BKPyV infection enhances bladder tumor growth and metastasis
The growth rate of BKPyV infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days after inoculation. New tumors were detected in the liver of BKPyV infected bladder tumor mice, which were characteristic of urothelial carcinoma, showing the bladder tumor cells were transported by the blood stream.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
The levels of β-catenin and its downstream signaling molecule cMYC in BKPyV infected tumor cells were examined. It was noted that the expression of β-catenin and cMYC in BKPyV infected cells was significantly increased. The results indicate that BKPyV infection promotes β-catenin signaling activation and EMT effects in bladder cancer cells.
Blocking β-catenin signal inhibits BKPyV infection-mediated enhancement of proliferation and migration
BKPyV infected cells treated with KYA1797K had significantly reduced cell proliferation, migration and invasion capacities.
Discussion
Tumors affect the long-term survival of kidney transplant recipients. BKPyV infection has shown to increase the risk of tumors in immunocompromised patients. When BKPyV infects cells the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis, or the host prevents virus replication, and the persistent expression of the LTag causes abortion of infected cell or cell transformation. High levels LTag are required for significant cell transformation. With the help of the BKPyV infection tumor cell model, it is possible to confirm that BKPyV-infected cells had an increased proliferative capacity, migration and invasive ability. After BKPyV infection, it was also noted the liver metastasis of bladder tumors are more likely to happen, which increase the difficulty of treatment and decrease the survival rates of patients. Wnt/β-catenin signaling pathway also plays an important role in tumor invasion and metastasis. Blocking this pathway may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
Conclusion
In summary, it was noted that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors. The study also verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV–related bladder cancer. The results of this study may help clinicians better diagnose and treat BKPyV-related bladder cancer.
Level of Evidence:
This is an animal study and hence, classified as level V
Strengths:
Done under controlled conditions keeping environments for the infected and non-infected cell lines similar
The study looked at several oncogenic proteins
Limitations:
Animal study hence, difficult to draw any conclusions
I agree with your analysis of strengths and limitations, and summary of this article.I agree with your analysis of the level of evidence this article provides.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer:
Introduction:
Bladder cancer is the 6th common cause in male cancer after lung, prostate, colon and liver.
Bladder cancer is three times more in immunocompromised after kidney transplant in comparison to general population.
BK virus activated after kidney transplant due to use of immunosuppressive therapy and leading to viremia, viruria, ureteral stricture and BKPyV-related nephropathy, as well as hemorrhagic cystitis after hematopoietic stem cell transplantation.
There’s relationship between BKV virama and developing of bladder cancer. But some small studies shows no evidence or relation between DNA virama of BKV and development of bladder cancer.
B- catenin pathway is used in process of cell proliferation and transcription, and regulating pattern formation during development. This pathway is involved in tumorigenesis and is associated with many biological interactions such as tumor proliferation, invasion, and metastasis.
So inhibition of β-catenin degradation in cytoplasm can induce epithelial mesenchymal transition and promote tumorigenesis.
Materials and methods:
Cell culture were obtained from the American Type Culture Collection.
The virus of BKV collected and inactivation.
Type of animal module is mice were injected subcutaneous with infected virus and after 30 days the tumor resected and histo pathology done.
The Cell Counting Kit-8 assay was used to determine cell viability. Histology by light microscopy and electron microscopy and immunohistochemistry staining done and western blotting were done. Results and discussion:
The study shows BKV replication post kidney transplant may promote progression of development of urothelial cancer.
BKV may end with two different outcomes: 1). The host cell allows replication of the virus, resulting in viral DNA amplification, progeny production and cell lysis
2). The host prevents virus replication, and the persistent expression of the LTag cause cell transformation.
This result is enough to cause cancer. BKV dose not have effects on developing of bladder cancer. There’s no studies support effects of virus activation with proliferation and formation of tumor and still biological characteristics of BKV in relation to cancer unknown.
In the past studies BKV LTag are prooncogenic because ability to inactivate tumor suppressor proteins. Where as STag shows ability to activation of the mitogenactivated protein kinase pathway, which increase cell proliferation and transformation.
This study shows β-catenin signaling pathway plays an important role in tumor invasion and metastasis.
Activation of β-catenin may promotes the invasion and migration of tumor cells. It’s also enhance the expression of cMYC to promote cell proliferation. Blocking the β-catenin signaling pathway may possibly help to find clinical treatments of infectious BKV bladder cancer.
Conclusion:
Still there is no strong evidence between activation of BKV infection and development of bladder cancer. B catenin blocking may help in management of bladder cancer in related to BKV.
What is the level of evidence provided by this article?
Level V
What are the weaknesses and strengths of this article?
The strength of this study is address the role of BKV in relation to bladder tumor in context of cell proliferation and transformation and invasive and migration. Also address the role of B catenin in treatment of bladder tumor related to virus.
The weakness of this study is small studies and experimental on animal
The introduction ;
———————————————–
The idea that BKPyV plays an important role in cancers of the urinary system was recently confirmed by deep sequencing studies, which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation.
When BKPyV infects cells, two different outcomes may occur:
(1) The host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis .
(2) or The host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
.
LTag are prooncogenic due to their ability to inactivate tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb), leading to increased cell proliferation . In addition, STag has been shown to increase activation of the mitogen activated protein (MAP) kinase pathway, which may also augment cell proliferation and transformation .
Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis . In the nucleus of tumor cells, β-catenin combined with the transcription factor family Tcf / Lefs can activate genes such as cMYC and cause cell proliferation and EMT effects. JCPyV large T antigen can interact with β-catenin and stimulate expression of β-catenin target genes.
BKPyV infections promotes β-catenin signaling pathway activation and EMT effects. Besides, blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion.
The Aim of the Study ;
————————————————-
Is to investigate the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT .
The type of study;
——————————————–
Experimental animal study .
The Method ;
————————————–
The study group developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
The Result ;
——————————–
BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells, while the activation of β-catenin signaling pathway is one of its mediation mechanisms.
The conclusion ;
———————————————–
1-The study results suggest that β-catenin activation in BKPyV-infected tumor cells may be related to the overexpression of LTag. Such activation of β-catenin further promotes the invasion and migration of tumor cells. At the same time, it can enhance the expression of cMYC to promote cell proliferation.
2-Xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis. Blocking the β-catenin signaling pathway can inhibit this process, which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
3-This new discovery has considerable significance towards clinical treatments of infectious BKPyV bladder tumors.
What is the level of evidence provided by this article?
———————————————————————————
Level V
What are the weaknesses and strengths of this article?
—————————————————————————
The strengths;
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
I agree with your analysis of strengths and limitations, and summary of this article. I wish you could type headings and sub-headings as underline or in bold.
Introduction
·Human polyomavirus BKPyV can reactivate in immunosuppressed people, particularly transplant patients, and it can lead to symptoms including viremia, viruria, ureteral stricture, nephropathy, and hemorrhagic cystitis.
·BKPyV gene integration has been found in urinary tract epithelial cell malignancies in recent investigations, indicating a potential carcinogenic impact.
Materials and methods Cell culture
·HTB-9 and T24 were cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKPyV infection and inactivation
·BKPyV stocks were propagated in Vero cells from ATCC and viral lysates were made through three cycles of freezing and thawing, followed by inactivation at 100 °C for 10 min.
Animal model
·Male BALB/c nude mice were housed in sterile filter-capped cages and injected with T24 and HTB-9, including BKPyV-infected cells. Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen for histopathological examination.
Cell counting Kit-8 assay
·CCK-8 assay was used to assess cell viability by detecting the absorbance of the medium at 450 nm.
Colony-formation assay
·Colonies were seeded in six-well plates at 200 cells/well and stained with 4% paraformaldehyde and counted using light microscopy.
Cell migration and invasion assays
·Cells were seeded in 200 μL FBS-free medium, stained with methanol and 0.1% crystal violet, and counted using a light microscope. The mean cell number was calculated.
Scratch wound healing assay
·Cells were inoculated in six-well plates and cultured at 37 °C until 90% confluence, then washed, incubated, and continuously cultured in serum-free medium.
Histological assessment
·Hematoxylin and eosin (H&E) staining was used to assess the histological properties of tumor tissues.
Immunofluorescence staining
·Cell slides and frozen sections were diluted in PBS for 10 min and permeabilized with 0.2% Triton X-100 for 10 min at RT. Primary and secondary antibodies were diluted in blocking buffer and incubated at RT for 50 min each. Fluorescence was detected using an inverted fluorescence microscope.
Western blot
·Western blotting was used to separate membrane proteins from 5% milk and incubate them overnight with anti-β-catenin antibodies.
Statistical analysis
·Statistical analyses were performed using ANOVA for more than two groups with P < 0.05 considered significant.
Results
·BKPyV infection in bladder cancer cells is a non-lytic infection
·BKPyV infection promotes the proliferation, invasion, and migration of bladder cancer cells
·BKPyV infection enhances bladder tumor growth and metastasis
·BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
·Blocking β-catenin signal inhibits BKPyV infection- mediated enhancement of proliferation and migration
Discussion
·BKPyV infection and tumorigenesis have increased attention in immunocompromised individuals.
·BKPyV plays an important role in renal and urothelial cancers after transplantation.
·JCpyV can cause urothelial carcinoma, suggesting it is closely related to bladder cancer. When BKPyV infects cells, two different outcomes may occur:
1. Host cell allows replication of the virus, resulting in viral DNA amplification, progeny production, and cell lysis.
2. Host cell prevents virus replication, causing abortion of infection cell or cell transformation.
·BKPyV infections do not have a lytic effect on bladder cancer cells due to the continuous expression of LTag, which prevents the virus from copying normally.
·BKPyV can promote host cell transformation and immortalization, but its role in tumor cells is unknown.
·The BKPyV infection tumor cell model increased the proliferative capacity, migration, and invasion ability of bladder tumors..
·The ability to metastasize to other organs in vivo is a major threat to clinical patients.
·BKPyV infections can lead to liver metastases of bladder tumors, reducing survival rates.
·LTag inactivates tumor suppressors, leading to increased cell proliferation.
·STag increases MAP kinase activation, leading to cell proliferation and transformation.
·Blocking the β-catenin signaling pathway can be an effective treatment for BKPyV-infected bladder tumors.
Conclusions
·BKPyV infection promotes the proliferation, invasion, and migration of bladder cancer and bladder tumors.
·β-catenin signaling pathway and EMT effect in BKPyV-related bladder cancer.
·The results may help diagnose and treat BKPyV-related bladder cancer.
What is the level of evidence provided by this article?
Level V
====================================================== What are the weaknesses and strengths of this article? Weakness: the study was inducted on an animal model (mice). Experimental. Strength: It first explained how BKPyV infection promotes bladder cancer to proliferate, invade, and migrate. It confirmed the function of the epithelial-mesenchymal transition and the β-catenin signaling pathway in BKPyV-induced bladder cancer.
Introduction:
Urothelial carcinoma is the 6th common malignancy in males in the united states, prone tumor type of immunocompromised patients, kidney transplant recipients are 3 times more likely to have it than the general population.
BK polyomavirus (BKV), a virus reactivated in immunocompromised patients, causes symptoms such as viremia, viruria, ureteral stricture and BKV-related nephropathy, as well as hemorrhagic cystitis after hematopoietic stem cell transplantation, controversy among its role in urothelial cancers, by presence of T antigen, or absence of virus DNA in studies.
Some studies have shown that transplant recipients with BK viremia or polyomavirus-associated kidney disease have an increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKV.
The β-catenin pathway is involved in tumorigenesis, by cell proliferation and transcription, and regulating pattern formation during development, large T antigen binds directly to β-catenin, resulting in enhanced expression of β-catenin target genes such as c-myc and cyclin D.
Materials and methods Cell culture: Human bladder cancer cell lines HTB-9 and T24 were obtained, and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C. BKV infection and inactivation: BKV propagated in Vero cells , and inactivated by heating to 100c for 10 minutes, both T24 and HTB-9 cells were grown in culture dishes. Animal model:
Cultures of T24 and HTB-9, including their respective BKV infected cells were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg).
Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination. Cell counting Kit-8 assay:
The Cell Counting Kit-8 assay was used to determine cell viability.
All observations were reproduced at least three times in independent experiments. Colony-formation assay:
Cells were seeded in six-well plates, Colonies were clearly visible after 10– 14 days and selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet.
Colonies containing > 50 cells were counted using light microscopy. Cell migration and invasion assays:
Transwell chambers, pre-coated with and without Matrigel were used to determine cell invasion and migration.
After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and crystal violet solution.
A light microscope was used to count the number of cells in five random fields of view. The mean cell number was calculated for each group. Scratch wound healing assay:
Cells were inoculated in six-well plates and cultured at 37 °C in a 5% CO2 cell incubator.
After the cells reached 90% confluence, wounds of approximately 1 mm width were created using a sterile pipette tip.
Cells were washed, incubated and continuously cultured in serum free medium. Cultures at 0, 8, and 24 h were observed under an inverted microscope. Histological assessment: Hematoxylin and eosin(H&E) staining was performed for histological assessments. Immunofluorescence staining: Cell slides and frozen sections in 4% formaldehyde, and prepared using inverted fluorescence microscope. Western blot: was carried out and membrane proteins were separated on polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.
Results:
Both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKV, BY promoting proliferation of bladder cancer cells in vitro, and higher migration rates than the controls.The growth rate of xenografts of BKV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group, pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream.In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced.β-catenin protein expression in xenografts of BKV-infected bladder tumor cells were significantly higher than in the control, indicating that BKV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells.BKV infected cells treated with KYA1797K had significantly reduced cell proliferation, migration, and invasion capacities, levels of Slug were significantly reduced while expression of Claudin-1 was significantly increased. Conclusion:
BKV infection promotes the proliferation, invasion and migration of bladder cancer, expressing BKV related proteins were more invasive in vitro. The study verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKV-related bladder cancer, that may help clinical diagnoses and treatment of such tumors in future.
What is the level of evidence provided by this article?
Level of evidence V – animal experimental study.
What are the weaknesses and strengths of this article? Weakness:
In vitro experimental study in mice.The role of BKV autoantigen expression in tumor cells has not been studied, and the biological characteristics of BKV-related tumors.Strength:
It is the first study to clarify the activation of β-catenin promotes the invasion and migration (metastasis) of urothelial tumor cells, so may help in treating such tumors by blocking β-catenin.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
Introduction Urothelial carcinoma is one of the most common and highly malignant tumours in the urinary system. Immunocompromised individuals are at high risk of development of bladder cancer. There is three fold higher risk in post transplant patients than general population. The results from animal studies have shown association between BKV and bladder cancer. This has not been proven in humans.
Methodology The effect of BK virus was investigated by- 1- Cell culture- Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection 2-Animal Models- Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice
Results BKPyV infection in bladder cancer cells is a non-lytic infection and promotes proliferation, invasion and migration of bladder cancer cells and enhances bladder tumor growth and metastasis. BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
Conclusion This was the first study which suggested BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins It defined role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer
What is the level of evidence provided by this article? Experimental Animal study Level of evidence V
What are the weaknesses and strengths of this article?
Strengths the first study to identify the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
1. Please summarise this article. Introduction
Urothelial cancer increase 3 folds in organ transplantation as compared to normal ppls
BKPyv activation after kidney transplantation may be linked to the development of urethelial cancer
But trials results was contradictory . In addition, some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKPyV. But till now pathogenesis is not clear
The Wnt/β-catenin pathway is implicated in cell proliferation and transcription, so inhibition of degradation in cytoplasm can induce epithelial-mesenchymal transition (EMT) and promote tumor genesis
Regarding the interaction between polyom a virus and β-catenin is clear in JVPYV but this relation still not clear in case of BKPyV Materials and methods Two models: BKPyV-infected bladder cancer cell model and a mice tumor model
1. BKPyV-infected bladder cancer cell model: cultured human bladder cancer cell lines HTB-9 and T24
2. Animal model: Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice (30 days)
Tumors were surgically dissected after 30 days for histopathological examination Results BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells after infection by BKPyV, the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus)
Discussion
BKPyV promote the host cell allows replication of the virus, resulting in viral DNA amplification, and cell lysis;
Tag has been found to be carcinogenic due to its ability to inactive tumour suppressor cells
BKPyV infection also promotes beta-catenin signal pathway activation
Conclusion · BKPyV infection induce the proliferation, invasion and migration of bladder cancer · BKPyV infection enhances β-catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) effect in bladder cancer cells · When β-catenin treated with KYA1797K so become inactive signal inhibits BKPyV infection mediated enhancement of proliferation and migration 2-What is the level of evidence provided by this article? Experimental study (level V) 3- What are the weaknesses and strengths of this article? Strength: the first study proved that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells via activation of β-catenin signaling pathway Weakness: experimental (animal study)
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
1-Please summarise this article. Introduction;
-Urothelial carcinoma is one of the most common and highly malignant tumors in the urinary system.
-According to 2018 statistics from the American Cancer Society, bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver.
-Bladder cancer is also a prone tumor type for immunocompromised patients. According to research, kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
-BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised populations, especially transplant recipients.
-BKPyV reactivation after renal transplantation causes symptoms such as viremia, viruria, ureteral stricture and BKPyV-related nephropathy, as well as hemorrhagic cystitis after hematopoietic stem cell transplantation. BKPyV infection enhances bladder tumor growth and metastasis;
-The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days post inoculation.
-Differences in tumor volume became more pronounced over time.
-On day 30 post inoculation, mice were euthanized in order to observe tumor cell invasion and metastases.
-New tumors were discovered in the liver of BKPyV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice.
-Pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream. Discussion;
-Tumors have gradually become one of the main factors affecting the long-term survival of kidney transplant recipients.
-In recent years, the correlation between BKPyV infection and tumorigenesis in immunocompromised individuals has drawn increased attention.
-The idea that BKPyV plays an important role in cancers of the urinary system was recently confirmed by deep sequencing studies, which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation.
-When BKPyV infects cells, two different outcomes may occur:
(1) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis; or,
(2) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation. This result is determined by the continuous expression of LTag. Conclusions;
-BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV-related proteins were more invasive in vitro.
-The role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer is verified.
-These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer.
2-What is the level of evidence provided by this article? ( The level V evidence ) 3-What are the weaknesses and strengths of this article? Weakness; –This study is an experimental animal study with small groups, –Use of xenograft for this study which not correlate with human findings. Strength; -This study is the first study to verify the role of activation of β-catenin signaling pathway by BK virus in the pathogenesis of cancer bladder.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer. Introduction.
Kidney transplant recipients are three times more likely to have urothelial cancer than the general population, early studies detected the presence of BKPyV large T antigens in prostate, bladder- and kidney tumors but still BKPyV as a cause of malignancy is still controversial.
BKPyV reactivation in immunosuppressed environments may be considered as a transforming factor leading to urothelial carcinomas, specially some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKPyV.
The Wnt/β-catenin pathway is implicated in cell proliferation, transcription, and regulating pattern formation during development. This pathway is involved in tumorigenesis and is associated with many biological behaviors such as tumor proliferation, invasion, and metastasis, BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation, and so this study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process. Materials and methods.
Cultures of T24 and HTB-9, including their respective BKPyV infected cells (106 cells in 100 mL PBS) were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg) (n = 5). Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination, this model to discuss the role
of BKPyV infections in cancer development. Results.
1-BKPyV infection in bladder cancer cells is a non-lytic infection but continued to proliferate and grow for 9 days after the initial infection.
2-BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells and T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates than the controls.
3-BKPyV infection enhances bladder tumor growth and metastasis.
4-BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKPyV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced in vivo, β-catenin protein expression in xenografts of BKPyV-infected bladder tumor cells were significantly higher than in the control. These results indicate that BKPyV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells.
5-Blocking β-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration. Conclusions.
Try to prove that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins were more invasive in vitro in the animal models, also demonstrated the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. Strength point:
Is considered the experimental gate to discuss these issues that prove the role of BKPyV infection promotes the proliferation, invasion and migration of bladder cancer. Weakness point:
It is depends on experimental results on animal .
Not studies the association of other viral infections on the proliferation, invasion and migration of bladder cancer. Level of evidence: V (Animal and laboratory research).
Typing the whole sentence in bold amounts to shouting, however.I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Introduction
Bladder cancer is the sixth most common malignancy in males.
Kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised population, especially transplant recipients.
BKPyV and JCPyV are classified as possibly carcinogenic to human as there is sufficient evidence in experimental animals and inadequate evidence in humans for their carcinogenicity.
Many recent studies detected BKPyV gene integration in urinary tract epithelial cell tumors
The functional effects of BKPyV infections on bladder cancer and the biological characteristics of tumor cells expressing BKPyV related proteins have not yet been elucidated.
The Wnt/β-catenin pathway is involved in tumorigenesis and is associated with many biological behaviors such as tumor proliferation, invasion, and metastasis.
Previous studies have shown that JCPyV large T antigen (LTag) binds directly to β-catenin, resulting in enhanced expression of β-catenin target genes such as c-myc and cyclin D1.
Whether activation of the Wnt/β-catenin signalling pathway by BKPyV LTag induce cancer remains to be established.
· This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and epithelial-Mesenchymal transition (EMT) in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Materials and methods
They developed a BKPyV-infected bladder cancer cell model and a mice tumor model to study the role of BKPyV infections in bladder cancer and that activation of β-catenin signaling pathway is one of its mediation mechanisms.
Results
BKPyV infection in bladder cancer cells is a non-lytic infection.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells.
BKPyV infection enhances bladder tumor growth and metastasis.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
Blocking β-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration.
Discussion
The idea that BKPyV plays an important role in cancers of the urinary system was recently confirmed by deep sequencing studies, which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation.
However, the role of BKPyV autoantigen expression in tumor cells has not been studied, and the biological characteristics of BKPyV-related tumors are unknown
In this study with the help of the BKPyV infection tumor cell model, it is possible to confirm that the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPyV-related proteins are further increased in vitro.
Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis.
It is also found that BKPyV infections promotes β-catenin signaling pathway activation and EMT effects.
In addition, blocking β-catenin signaling pathways inhibits BKPyV’s function to promote tumor cell proliferation and migration invasion.
Such activation of β-catenin further promotes the invasion and migration of tumor cells and promote cell proliferation.
This new discovery has considerable significance towards clinical treatments of infectious BKPyV bladder tumors.
In this study xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis and blocking the β-catenin signaling pathway can inhibit this process, which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
Conclusions
This study described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins were more invasive in vitro.
They verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer.
The level of evidence: is 5
Weakness of this article: Being an animal (with physiology different from humans) study make the results cannot be accurately related to humans and invalid.
Strength:
This type of studies can help understand the basic principles of pathogenesis, in this study the role of wnt/β-catenin signaling pathway in tumor invasion and metastasis was seen in animal model.
Blocking the β-catenin signaling pathway can inhibit this process which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
☆ BK polyomavirus infection promotes growth and aggressiveness in bladder cancer ◇Introduction: ▪︎Kidney transplant recipients are three times more likely to have urothelial cancer than the general population. ▪︎BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised populations, especially transplant recipients. ▪︎ BKPyV reactivation after renal transplantation causes symptoms such as viremia, viruria, ureteral stricture and BKPyV-related nephropathy. ▪︎Many recent studies detected BKPyV gene integration in UT tumors, providing further evidence for the correlation between BKPyV and urinary tract tumors. ▪︎BKPyV reactivation in immunosuppressed environments may be considered as a transforming factor leading to urothelial carcinomas. ▪︎Some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk of bladder cancer when compared to transplant recipients without BKPyV. ▪︎BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen. The role BKPyV infections play in the biological function of bladder cancer is still unclear. ◇ Methods: The authors have developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
◇Results: The research proves that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells, while the activation of β-catenin signaling pathway is one of its mediation mechanisms.
◇ Conclusions: ▪︎The authors first described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer. They verified the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyVinfected bladder cancer. ▪︎These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
What is the level of evidence provided by this article? Weaknesses of the study: Strengths of the study:
◇ What is the level of evidence provided by this article?
Level of evidence: V (an experimental animal study). ◇ What are the weaknesses and strengths of this article? ¤ Weakness: this an animal study which has some limitations
Animal tests often miss the most important signs of toxicity in humans. …
Animal tests are time-consuming and expensive, limiting the number of chemicals that can be tested.
¤ The strength of the study:
• The first study of its kind to investigate how the stimulation of the beta-catenin signaling system by the BKV add to the development of the bladder cancers.
•The results of this study could provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Summary of this article:
Urothelial carcinoma is one of the most common and highly malignant tumors in the urinary system. According to 2018 statistics from the American Cancer Society,
bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver
Bladder cancer is also a prone tumor type for immunocompromised patients. According to research, kidney transplant recipients are three times more likely to have urothelial cancer than the general population
BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised populations, especially transplant recipients.
Bladder cancer is 3 times more common in transplant recipients than general population
The rule of BK virus in the development of genitourinary malignancies remains debatable and unclear, some reported association and others reported causation
Those who suggested causation, rely on detection of BK large T antigens in kidney, bladder and prostate tumors and on the fact that BK gene was found to be integrated into the cellular genome of urothelial carcinomas in renal transplant recipients
The Objective of the current study:
It is an animal study evaluating the rule of BK virus infection in bladder cancer through injecting a mouse (subcutaneously) with human bladder cancer cell lines (HTB-9 and T24) obtained through culture and BK virus infected cells => to explored the effects of BKV infections on the proliferation and migration of bladder cancer cells and the role of β-catenin pathway and endothelial mesenchyme transition (EMT).
Materials and methods:
The two human bladder cancer cell lines HTB-9 and T24 were obtained and cultured in culture dishes in-vitro.
Both cell lines were infected with BKV with and without β-catenin selective inhibitor.
Cultured bladder cancer cells infected with BKV or not (control) were inject SC in mice with and without intra-peritoneal injection of β-catenin antagonist.
Animals were killed after 30 days, and tumor was resected for examination.
Results:
BKV infection of bladder cancer cells:
Both cell lines infected with BKV showed large T protein expression of BKV after 48 hrs.
Infected cells did not lyse but continue to grow and proliferate for 9 days after infection.
BKV infection promotes proliferation, invasion and migration of bladder cancer cells
The proliferation ability and activity of infected cell lines was significantly higher in infected cells. Also, they had higher migration activity compared to control.
BKPyV infected T24 and HTB-9 cells migrated significantly faster on plates than the control group.
BKV infection enhances bladder tumor growth and metastasis:
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly.
On day 30, tumor metastasis was discovered in the liver of BKV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells:
In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced.
In vivo, β-catenin protein expression in xenografts of BKV-infected bladder tumor cells were significantly higher than in the control.
Blocking β-catenin signal inhibits BKV infection mediated enhancement of proliferation and migration.
Conclusions:
BKV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKV related proteins were more invasive in vitro.
The study results verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKV-related bladder cancer.
These results may impact diagnoses & treatment of BKPyV-related bladder cancer.
2. level of evidence => V (experimental animal study) 3. What are the weaknesses and strengths of this article?
Strength:
This is the first study to describe that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and that bladder tumors expressing BKPyV related proteins were more invasive in vitro.
Weakness:
It is an animal study which is poorly correlated with human.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
Introduction Urothelial carcinoma is one of the most common malignant tumors in the urinary system. BKPyV and JCPyV were classified as “possibly” carcinogenic. The underlying role of BKPyV in the development of cancer was controversial. Aim of the study: -Explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and explore the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice. Materials and methods Theye developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections. Cultures of BKPyV infected human bladder cell were injected subcutaneously into nude mice Cell migration and invasion assessed by incubating the cells in Transwell chambers pre-coated with and without Matrigel Results: – BKPyV infected bladder cells did not lyse due to BKPyV infections, they express T antigen and continued to proliferate and grow, the cells proliferation ability and activity in infected bladder cancer cells were significantly increased compared to cells not infected. – BKPyV infections promote invasion and migration of bladder cancer cells in vitro.Also, it promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells. Conclusions: -BKPyV infection promotes the proliferation, invasion and migration of bladder cancer. while the activation of β-catenin signaling pathway and Epithelial-Mesenchymal Transition is one of its mechanisms -These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer Limitation: Animal study model and the results may not be applied in humans studies. Strength: The first study to explore the role of B catenin signaling pathway in development of bladder cancer, inhibition of this pathway may provide promise for future treatment. Level of evidence: level V animal study
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
Urothelial carcinoma is the sixth most common malignancy in males, and BKPyV reactivation after renal transplantation can cause symptoms such as viremia, viruria, ureteral stricture, nephropathy, and hemorrhagic cystitis.
BKPyV and JCPyV were classified as “possibly” carcinogenic to human due to evidence in experimental animals and inadequate evidence in humans.
BKPyV reactivation in immunosuppressed environments may be an atransforming factor leading to urothelial carcinomas, and transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk of bladder cancer.
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of the Wnt/β-catenin pathway and EMT in this process.
Results will help clarify the characteristics of bladder tumors and lay a foundation for further clinical trials.
Cell culture Cell culture of HTB-9 and T24 was obtained from ATCC and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with CO 2 at 37 °C.
BKPyV infection and inactivation BKPyV infection and inactivation was propagated in Vero cells from ATCC viruses, with viral lysates made through three cycles of freezing, thawing, and inactivation.
Animal model Animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.
Cell counting Kit-8 assay CCK-8 assay was used to measure cell viability and absorbance of medium at 450 nm.
Colony-formation assay Colonies were clearly visible after 10-14 days and stained with 4 mg/mL crystal violet, and counted using light microscopy.
Cell migration and invasion assays were used to determine cell invasion and migration, seeded in 200 μL FBS-free medium and stained with methanol and 0.1% crystal violet solution.
Scratch wound healing assay Cells were inoculated and cultured at 37 °C to create wounds of 1 mm width. Cultures were observed under an inverted microscope.
Histological assessment Hematoxylin and eosin (H&E) staining was used for histological assessment of tumor tissues.
Immunofluorescence staining of cells and frozen sections in formaldehyde and permeabilized with 0.2% Triton X-100. Primary and secondary antibodies were diluted in blocking buffer and incubated at RT for 50 min.
Western blotting was used to separate membrane proteins and incubate them overnight with anti-β-catenin antibodies.
Statistical analyses were performed using ANOVA for more than two groups, with P < 0.05 for vales.
BKPyV infection in bladder cancer cells is a non-lytic infection, with T24 and HTB-9 cells showing large T protein expression after 9 days and VP1 expression remaining stable after 30 days.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells in vitro, with higher migration rates and higher invasive capacities than non-infected cells.
BKPyV infection increases the growth rate of xenografts of BKPyV-infected blad-der tumor cells in mice, with differences in tumor volume becoming more pronounced over time.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
BKPyV infection promotes β-catenin signaling activation and EMT effects in bladder cancer cells.
Blocking β-catenin signal inhibits BKPyV infection-mediated enhancement of proliferation and migration, reducing tumor size and invasive abilities.
BKPyV plays an important role in cancers of the urinary system, as confirmed by deep sequencing studies and JCpyV.
When BKPyV is activated in vivo, it triggers abnormally high expression of LTag in the host cell, leading to cell transformation.
However, it does not have a lytic effect on bladder cancer cells, suggesting that these two tumor cell lines are already in an infinite proliferation state.
Previous research has shown that BKPyV can promote host cell transformation and immortalization, leading to enhanced cell proliferation capacity.
However, the role of autoanti-gen expression in tumor cells has not been studied. With the help of a tumor cell model, we were able to confirm that the proliferative capacity, migration and invasion ability of bladder tumors expressing bKPyV-related proteins were further increased in vitro.
LTag and STag are prooncogenic due to their ability to inactivate tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb).
STag has been shown to increase activation of the mitogenactivated protein (MAP) kinase pathway, which may also augment cell proliferation and trans formation.
Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis, and JCPyV large T antigen can interact with β-Catenin and stimulate expression of β-catenins target genes.
These results suggest that BKPV infections can promote tumor cell proliferation and migration invasion.
Blocking β-catenin signaling pathway can inhibit distant metastasis of BKPyV-infected bladder tumors.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer; Introduction: Urothelialcarcinoma is the most common and highly malignant tumor in the urinary system. American Cancer Society statistics in 2018; (Bladder cancer is the 6th most common malignant tumor in males after lung, prostate, colorectal, stomach, and liver. Immunocompromised patients is the risk factors of bladder cancer, and the urothelial cancer is 3 times more prevalent than in general population. In 2012 at the international Agency for Research on Cancer (IARC) meeting; BKPyV and JCPyV were classified as possibly carcinogenic to human, as there is a sufficient evidence in experimental animals and inadequate evidence in humans for their carcinogenicity. Early studies detected the presence of BKPyV large T antigen in prostate, bladder, and kidney tumors, but several researchers thought BKPyV was unlikely to be involved in the etiology of most renal and bladder tumors because they had failed to detect BKPyV DNA in the cancer samples. Many recent studies detected BKPyV gene integration in urinary tract epithelial cell tumors, providing further evidence for the correlation between BKPyV and urinary tract tumors. Interestingly, these changes occur in post transplant tumor, but not in non-transplant tumors, therefore BKPyV considered as a transforming factor leading to urothelial carcinoma. Somestudies, show that recipient with BKPyV viremia or polyomavirus-associated kidney disease has an increased risk (4-11 times) of bladder cancer, when compared to to transplant recipients without BKPyV, without full clear evidence of te underline role. BKPyV is thought to be oncogenic due to the expression of the early coding region encoded proteins LTag and small T antigen (STag) which can initiate or derive neoplastic transformation. Results: BKPyV infection in bladder cancer cells is a non-lytic infection;
Tow bladder cell lines had been used, T24 and HTB-9, to infect with BKPyV.
48 hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV.
By extended the infection time, the effect of BKPyV infection on cell fates.
Both T24 and HTB-9 cells did not lyse due to BKPyV infections, but continued to proliferate and grow for 9 days after the initial infection, and this is extended till 30 days.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells:
After infection with BKPyV, the cell proliferate and show activity in both T24 and HTB-9 bladder cancer cells and increased significantly compared to non-infected cells.
T24 and HTB-9 showed higher migration rate than the non-infected cells.
T24 and HTB-9 cell had promotes migration of bladder cell cancer.
T24 and HTB-9 had higher invasion capacity.
BKPyV infection enhances bladder tumor growth and metastesis;
The growth rate of the BKPyV infected tumor cells was increased significantly, with the tumor volume larger in both cell line compared to control.
Liver mass discovered in BKPyV infected tumor cell, found that the types of migerating cells was of urothelial origin.
BKPyV infection enhances B-catenin signaling pathway activation and epithelial -Mesenchymal transition(EMT) effect in bladder cancer cells.
Conclusion:
BKPyV infected cells associated with promotion, proliferation, migration and invasion of bladder cancer tumor.
Level of evidence: Level ((V)). Strtength of the Article: Expermental studies. Weakness of the article: Small group and case study All result are mostly an association rather than clear direct effects
In 2012, the International Agency for Research on Cancer (IARC) classified BKPyV & JCPyV as “possibly” carcinogenic to human (group 2B) because of the “sufficient evidence” in experimental animals & the “inadequate evidence” in humans for their carcinogenicity.
Deep sequencing studies had confirmed that the BKPyV gene was integrated into the genome of renal cancer & urothelial carcinoma cells after TX.
JCpyV may cause urothelial carcinoma after transplant (Querido et al.).
The study
This is an experimental animal study.
The authors developed a BKPyV-infected bladder cancer cell model & a mice tumor model to assess the role of BKPyV infections.
Materials and methods
Two bladder cancer cell lines, T24 & HTB-9, were used to infect with BKPyV. 48 hours after infection, T24 & HTB-9 cells showed large T protein expression of BKPyV.
The effect of BKPyV infections on cell fates was observed by extending the infection time.
Both cells lines did not lyse due to BKPyV infections, but continued to proliferate & grow for 9 days after initial infection. Further research shows the
BKPyV infected T24 & HTB-9 cells xenografted on mice still expressed large T antigen after 30 days.
Results:
BKPyV infection in bladder cancer cells is a non-lytic infection.
BKPyV infection promotes proliferation, invasion & migration of bladder cancer cells.
BKPyV infection enhances β-catenin signaling pathway activation & epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
Blocking β-catenin signal inhibits BKPyV infection mediated enhancement of proliferation & migration.
Conclusions:
BKPyV infection promotes the proliferation, invasion & migration of bladder cancer & bladder tumors expressing BKPyV related proteins were more invasive in vitro.
The study verified the role of β-catenin signaling pathway & EMT effect in the characteristics of BKPyV-related bladder cancer.
These results may impact diagnoses & treatment of BKPyV-related bladder cancer.
============================ 2. What is the level of evidence provided by this article?
Level V: Animal and laboratory research
============================ 3. What are the weaknesses and strengths of this article? Weaknesses:
It is difficult to extrapolate the results of this in vitro study to human patients.
Strengths:
This is the first study to describe that BKPyV infection promotes the proliferation, invasion & migration of bladder cancer & that bladder tumors expressing BKPyV related proteins were more invasive in vitro.
I- Summary: Objectives: to explored the effects of BKV infectionson the proliferation and migration of bladdercancer cells and the role of β-catenin pathway and endothelial mesenchyme transition (EMT) in this process by establishing a model for BKV infections in bladder cancer cells and mice. Methods:
Human bladder cancer cell lines HTB-9 and T24 were obtained and cultured in culture dishes in-vitro.
Both cell lines were infected with BKV with and without β-catenin selective inhibitor.
Cultured bladder cancer cells infected with BKV or not (control) were inject SC in mice with and without intra-peritoneal injection of β-catenin antagonist.
Animals were killed after 30 days and tumor was resected for examination.
Results: BKV infection of bladder cancer cells:
Both cell lines infected with BKV showed large T protein expression of BKV after 48 hrs.
Infected cells did not lyse but continue to grow and proliferate for 9 days after infection.
BKV infection promotes proliferation, invasion and migration of bladder cancer cells
The proliferation ability and activity of infected cell lines was significantly higher in infected cells. Also, they had higher migration activity compared to control.
BKPyV infected T24 and HTB-9 cells migrated significantly faster on plates than the control group.
BKV infection enhances bladder tumor growth and metastasis:
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly.
On day 30, tumor metastasis was discovered in the liver of BKV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells:
In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced.
In vivo, β-catenin protein expression in xenografts of BKV-infected bladder tumor cells were significantly higher than in the control.
Blocking β-catenin signal inhibits BKV infection mediated enhancement of proliferation and migration.
Conclusions:
BKV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKV related proteins were more invasive in vitro.
The study results verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKV-related bladder cancer.
II- level of evidence: V III- strength of the study:
The first study to explore the underlying mechanism of the BKV induced bladder
cancer cell pathway. IV- weakness:
It is an animal study which is poorly correlated with human.
Urothelial cancer is a common malignancy, and in USA the urinary bladder cancer is the 6th common cancer in male.
Renal transplant recipients have 3 fold increase of urothelial cancer compared to general population.
BKV infection considered as possible carcinogenic (inadequate evidence in human & sufficient evidence in experimental animal studies).
BKPyV reactivation in immunosuppressive environment may be a transformer factor lead to urothelial cancer.
Aim of the study:
Establishing the effect of BKPyV infection on proliferation & migration of bladder cancer.
Role of Int/B catinin pathway & EMT in bladder cancer cells & mice.
Result & discussion:
Presence of malignant tumor in SOT recipients can affect patient outcome, so establishing the correlation between BKPyV infection & tumorigenesis had great attention.
Recent data confirm the important role ion BKPyV infection in urinary system cancer through integrated of BKPyV gene into genome of renal cancer & urothelial cancer cells after transplantation.
BKPyV infection outcome:
replication of virus inside the host cells result in viral DNA amplification, progeny vision production & cell lysis.
prevention of viral replication (by host cells) & persistent expression of LTag leading to abortion of infection & cell transformation.
Low level of LTag is not sufficient to cause tumor.
The study result show that BKPyV infection didn’t have lytic effect on bladder cancer cells.
Also the study confirm that the proliferation capacity, migration & invasion of bladder cancer expressing BKpYV-related protein increased in vitro, so growth, invasion & metastasis to other organs in vivo was highly likely.
LTag are prooncogenic & increase activation of MAP kinase pathway (increase cell proliferation & transformation).
Wnt/B-catenin signaling pathway have a role in tumor invasion & metastasis.
BKPyV infection enhance B-catinin signaling pathway activation & EMT effects, so blocking of B-catenin signal can inhibit BKPyV function & inhibit distant metastasis.
Level of evidence is 5.
Strength :
First study describe BKPyV infection can increase risk of invasion & migration of bladder cancer.
Limitation:
Animal studies correlate very poorly to real human patients, & it fail to predict real human outcome in 50-99% of cases.
Bladder cancer is the sixth most common malignancy in males according to the 2018 American Cancer Society. Furthermore, bladder cancer has been observed to be common among those that are immunocompromised like those with kidney transplants that are three times more common to have uroepithelia tumours than the general population.
The reactivation of BKPyv after kidney transplantation is noticed with accompanying symptoms like viremia, viruria, ureteral stricture, and hemorrhagic cystitis among those with hematopoietic stem cell transplants. However, there is still a debate if BKPyV has a causal role in the development of urinary tract malignancy, although early studies detected the presence of BKPyV large T antigens in prostate-, bladder, and kidney tumours. Also, several researchers thought BKPyV was unlikely to be involved in the etiology of most renal and bladder tumors because they had failed to detect BKPyV DNA in the cancer samples.
Materials and Methods
Human bladder cancer cell lines HTB-9 and T24 were obtained and cultured in RPMI 1640
Male BALB/c nude mice (age 5 weeks, 18–20 g; were housed in sterile filter-capped cages.
Cultures of T24 and HTB-9, including their respective BKPyV infected cells (106 cells in 100 mL PBS) were injected subcutaneously into nude mice.
Hematoxylin and eosin(H&E) staining was performed for histological assessments
Immunofluorescence and western blot were also carried out on the cells
Results
Forty-eight hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV
.Both T24 and HTB-9 cells did not lyse due to BKPyV infections, but continued to proliferate and grow for 9 days after the initial infection
The cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV
The two experiments consistently showed that BKPyV infection promoted the migration of bladder cancer cells in vitro.
BKPyV-infected T24 and HTB-9 cells had significantly higher invasive capacities than non-infected cells
Discussion
The deep sequencing study has confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation.
The invasion of BKPyV into a cell can cause; the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production, and cell lysis; or, the host prevents virus replication and the persistent expression of the LTag
LTag has been found now to be prooncogenenic due to its ability to inactive tumour suppressor cells
BKPyV infection also promotes beta-catenin signal pathway activation
Conclusion
The outcomes of the study in identifying BKPyV infection’s role in causing urinary tract cancer could aid in the identification and proper treatment of cancer resulting from the infection.
The level of evidence is 5
Strength of the study
The molecular explanation of the aetiopathogenesis of the role of BKPyV in causing urinary bladder cancer
Limitations of the study
Use of xenograft for the study
Animals like monkey will be closer to human than the use of rat
Introduction
Kidney transplant recipients are three times more likely to have urothelial cancer than the general population
In 2012, BK polyomavirus (BKPyV) and JCPyV were classified as “possibly” carcinogenic to human
The causal role of BKPyV infections in the biological function of bladder cancer remains unclear
Aim of the study: discuss the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and epithelial-mesenchymal transition (EMT)
Materials and methods Two models: BKPyV-infected bladder cancer cell model and a mice tumor model
1. BKPyV-infected bladder cancer cell model: cultured human bladder cancer cell lines HTB-9 and T24
2. Animal model: Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice (30 days)
Tumors were surgically dissected after 30 days for histopathological examination
Results
BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells when compared to cells not infected with BKPyV (inactivated BKPyV virus). The activation of β-catenin signaling pathway is one of its mediation mechanisms
Conclusions
· BKPyV infection promotes the proliferation, invasion and migration of bladder cancer
· BKPyV infection enhances bladder tumor growth and metastasis
· BKPyV infection enhances β-catenin signaling pathway activation and EMT effect in bladder cancer cells
· Blocking β-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration (treated with KYA1797K)
What is the level of evidence provided by this article?
Experimental study (level V)
What are the weaknesses and strengths of this article? Weakness: experimental (animal study) Strength: the first study proved that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells via activation of β-catenin signaling pathway
Introduction Urothelial carcinoma is a frequent and fatal urinary malignancy. Bladder cancer ranks sixth among male malignancies after lung, prostate, colorectal, stomach, and liver, according to 2018 American Cancer Society data. BKPyV and JCPyV were classified as “probably” carcinogenic to humans at the 2012 IARC meeting due to “sufficient evidence” in experimental animals and “inadequate evidence” in humans. This study established a model for BKPyV infections in bladder cancer cells and mice to investigate the impact of BKPyV on bladder cancer cell proliferation, migration, and Wnt/β-catenin pathway and EMT. These findings will illuminate BKPyV-related bladder cancers, paving the way for clinical studies.
Methods A BKPyV-infected bladder cancer cell model and a mouse tumor model were developed to discuss the role of BKPyV infections.
Results In bladder cancer cells, BKPyV infection is a non-lytic infection. Infection with BKPyV enhances the growth, invasion, and migration of bladder cancer cells. Infection with BKPyV promotes bladder tumor growth and metastasis. In bladder cancer cells, BKPyV infection increases -catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) impact. Blocking β-catenin signal inhibits BKPyV infectionmediated enhancement of proliferation and migration.
Discussion The BKPyV gene was found to be integrated into the genomes of renal cancer and urothelial carcinoma cells after transplantation, in agreement with a previous study that demonstrated a link between human polyomavirus and bladder cancer. When BKPyV infection occurs, the host cell permits virus reproduction and cell lysis; alternatively, the host resists virus replication; nonetheless, prolonged expression of the LTag can result in cell transformation or abortive infection. Unstudied was the function of BKPyV autoantigen expression in tumor cells. Bladder tumors frequently spread to the liver following a BKPyV infection. LTag are prooncogenic as a result of the inactivation of tumor suppressor proteins such as p53 and retinoblastoma protein (pRb) and the enhancement of the mitogen-activated protein kinase (MAP kinase) pathway, which results in enhanced cell proliferation. The Wnt/-catenin signaling pathway facilitates tumor invasion and dissemination. Blocking -catenin signaling pathways can inhibit the action of BKPyV. This discovery can be utilized to treat infectious BKPyV bladder cancers.
In conclusion, bladder tumors expressing BKPyV-related proteins were more invasive in vitro. BKPyV infection enhances the proliferation, invasion, and migration of bladder cancer. The role of β-catenin signaling pathway and EMT effect was verified in the features of BKPyV-related bladder cancer. These findings may aid in the clinical diagnosis and treatment of bladder cancer caused by BKPyV.
Introduction
Many recent studies detected BKPyV gene integration in urinary tract epithelial cell tumors
BKPyV reactivation in immunosuppressed considered as a transforming factor leading to urothelial carcinomas. increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKPyV
BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation. Whether constitutive activation of the Wnt/β-catenin signalling pathway by BKPyV LTag induce cancer remains to be established
AIM OF STUDY (YZ and TZ designed the experiments)
BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins were more invasive in vitro. We verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer
RESULTS
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells after infection by BKPyV, the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus)
Colony formation experiments further showing that BKPyV infections promote proliferation of bladder cancer cells in vitro
Transwell migration results showed that T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates than the controls
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days post inoculation.
Differences in tumor volume became more pronounced over time On day 30 post inoculation, mice were euthanized in order to observe tumor cell invasion and metastases. New tumors were discovered in the liver of BKPyV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice . Pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream
2-What is the level of evidence provided by this article? Should they be regarded as being similar to “bench studies” and thus be viewed as a Level 5 (CEBM) study, or given a lower number (for example 1b or 2b), or none at all?
3-What are the weaknesses and strengths of this article?
main weakness of animal studies is that animals have a different physiology to humans. This means that any studies on animals cannot be accurately related to humans, making them invalid.
STRENGHT The choosing of good control groups are useful. the formulation of new questions to be further responded is expected.
Introduction:
Urothelial carcinoma is a frequent and deadly urinary malignancy. According to 2018 American Cancer Society data, bladder cancer is the sixth most prevalent male malignancy after lung, prostate, colorectal, stomach, and liver. Immunocompromised people can develop bladder cancer. Research shows that kidney transplant patients are three times more likely to develop urothelial carcinoma.
Methods:
In order to investigate the possible effects of BKPyV infections, we created a bladder cancer cell model that was infected with BKPyV as well as a mouse tumor model.
Sterile filter-capped cages contained male BALB/c nude mice (5 weeks, 18–20 g; Shanghai LC Laboratory Animal Co. Ltd., Shanghai, China). T24 and HTB-9 cultures, comprising BKPyV-infected cells (106 cells in 100 mL PBS), were administered subcutaneously into nude mice with and without intraperitoneal KYA1797K (25 mg/kg) (n = 5).
Thirty days after implantation, tumors were surgically excised and frozen in liquid nitrogen before histological evaluation.
Results:
The findings of our study demonstrate that BKPyV infections encourage the proliferation, invasion, and migration of bladder cancer cells, with the stimulation of the β-catenin signaling pathway serving as one of the mediation mechanisms responsible for this effect.
conclusion :
that BKPyV infection is responsible for promoting the proliferation, invasion, and migration of bladder cancer. In BKPyV-infected bladder cancer, we were able to confirm the function that the -catenin signaling pathway and the Epithelial-Mesenchymal Transition affect play. These findings contribute significantly to our understanding of how best to diagnose and treat patients with bladder cancer in clinical settings.
What is the level of evidence provided by this article?
It is an experimental animal study.
Level of evidence 5
What are the weaknesses and strengths of this article?
Weakness: Much animal research into potential treatments for humans is wasted because it is poorly conducted and not evaluated through systematic reviews
The strength: is that this is the first research of its kind to investigate how the stimulation of the beta-catenin signaling system by the BK virus contributes to the development of cancer in the bladder.
1- Summary Introduction
Bladder cancer is the 6 th most common malignancy in males with high prevalence in immunocompromised cases.
Kidney transplant recipients are three times more susceptible to have urothelial cancer compared to the general population.
BKPyV reactivation can lead to BKPyV-related nephropathy in renal transplantation and haemorrhagic cystitis after hematopoietic stem cell transplantation .
International Agency for Research on Cancer (IARC) meeting at 2012 stated that , BKPyV and JCPyV
can be possibly carcinogenic to human but this is controversial .
Some studies declared that BKPyV DNA wasnot detected in the cancer samples while others mentioned that BKPyV gene integration was noticed in the urinary tract epithelial cell tumor
A study revealed that cancer bladder risk is higher in transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease compared to transplant recipients without BKPyV.
The Wnt/β-catenin pathway and inhibition of β-catenin degradation in cytoplasm has a role in tumorigenesis and tumor proliferation, invasion, and metastasis .
BKPyV could be oncogenic because of expression of early coding region-encoded proteins
LTag and small T antigen (STag), which can stimulate neoplastic transformation.
this study aimed at evaluating BKPyV infections effect on the occurrence of bladder cancer . Methods
BKPyV-infected bladder cancer cell model and a mice tumor model was created to evaluate BKPyV infections role. Results
BKPyV infection in bladder cancer cells is a non-lytic infection with large T protein expression
of BKPyV and proliferation of cancer cells with time .
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells in vitro.
BKPyV infected T24 and HTB-9 cells had significantly higher invasive potentials than non-infected cells.
BKPyV infection promoted bladder tumor growth and metastasis.
BKPyV infection activated β-catenin signaling pathway and increased epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
Blocking β-catenin signal inhibits BKPyV infection mediated augmentation of proliferation and migration. Discussion
Studies declared that BKPyV gene was integrated into the genome of renal cancer and
urothelial carcinoma cells after transplantation in accordance with another study that showed that human polyomavirus can be related to cancer bladder.
When BKPyV infection occur the host cell allows replication of the virus and cell lysis; or the host prevents virus replication , but persistent expression of the LTag can lead to abortion of infection cell or cell transformation.
The role of BKPyV autoantigen expression in tumor cells was not studied.
After BKPyV infections, bladder tumors tend to metastasis to the liver .
LTag are prooncogenic through inactivation of tumor suppressor proteins as p53 and retinoblastoma protein (pRb), and enhances mitogen activated protein (MAP) kinase pathway leading to increased cell proliferation.
Wnt/β-catenin signaling pathway enhances tumor invasion and metastasis.
Blocking β-catenin signaling pathways can suppress BKPyV’s function.
Such finding can be used for treatment of infectious BKPyV bladder tumors. Conclusion
BKPyV infection enhances the proliferation, invasion of cancer bladder .
β-catenin signaling pathway and EMT effect have a role in BKPyV-related bladder cancer affecting diagnosis and therapy of cancer bladder.
2-level of evidence is V ;as it is conducted on animals without human enrollment with high possibility of bias
3- Strength is that it is a pioneer study conducted to access BKV role in cancer bladder involving the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV infected bladder cancer.
Limitation is using animal tumor model which varies from human models and tumor cells therefore some results may not be applicable for humans
Bladder cancer is 3 times more common in transplant recipients than general population
The rule of BK virus in the development of genitourinary malignancies remains debatable and unclear, some reported association and others reported causation
Those who suggested causation, rely on detection of BK large T antigens in kidney, bladder and prostate tumors and on the fact that BK gene was found to be integrated into the cellular genome of urothelial carcinomas in renal transplant recipients
The current study is an animal study evaluating the rule of BK virus infection in bladder cancer through injecting a mice (subcutaneously) with human bladder cancer cell lines (HTB-9 and T24) obtained through culture and BK virus infected cells.
Results
BK infection may promotes growth and aggressiveness in bladder cancer through multiple mechanisms, one of them is the activation of β-catenin signaling pathway
Blocking β-catenin signaling pathway can prevent distant metastasis
Conclusion
BK virus infection may have a rule in the growth and behavior of bladder cancer
Blocking β-catenin signaling pathway may be used in the future in the treatment of bladder cancer
What is the level of evidence provided by this article?
It is an experimental animal study, it is sorted under foundational evidence not included in the 5 levels of evidance
What are the weaknesses and strengths of this article?
Weakness of the study arise from being an animal study
Strength of the study is that it is the first study to explore the effect of activation of β-catenin signaling pathway by BK virus in the pathogenesis of cancer bladder
Data from animal studies were suggestive of carcinoghenic potential of the BKV specially urothelial carcinoma but this was not proved in human studies and remained uncertain.
Methodology:
Tow things were considered to study the effects of BKV infections; 1)BKV-infected human bladder cell lines ( taken from the American type culture collection) 2) Animal model (mice tumour).
Results:
BKV infection promote cellular proliferation and activities in bladder cancer cells but this was not seen in cells not infected by BKV.
BKV infection activates both beta-catenin signalling pathway & epithelial-mesenchymal transition (EMT) in bladder cancer cells.
Conclusion:
This was the first study to show that BKV infection promotes cellular proliferation, and metastasis of bladder cancer through beta-catenin pathway. This may shade lights regarding the diagnosis & treatment of bladder cancer.
2.What is the level of evidence provided by this article?
This was well design control trial without randomization = quasi-experimental study, level III.
3.What are the weaknesses and strengths of this article?.
Strength; this was the first study to demonstrate that BKV can promote cell growth & carcinogenesis. It opened an avenue for possible future therapies e.g. blocking the beta-catenin pathway.
Weakness: the results we got from mice model may not necessarily applied to human. The level of evidence may not be strong enough since we are not talking about RCT in this case.
Urothelial carcinoma is three times as common in kidney transplant patients than in the general population.
The classification of BKPyV and JCPyV is as “probably” human carcinogenic.
There is a clear association between BKPyV and urinary tract malignancies since several recent research have found BKPyV gene integration in these tumours.
Direct binding of JCPyV large T antigen (LTag) to beta catenin results in increased expression of beta catenin target genes such c-myc and cyclin D1.
Epithelial-mesenchymal transition (EMT) and cancer can both be induced by inhibiting beta catenin breakdown in the cytoplasm.
The early coding region-encoded proteins LTag and small T antigen (STag), which can start or fuel neoplastic transformation, are expressed by BKPyV and are presumed to be carcinogenic.
By deep sequencing research, which demonstrated that following transplantation, renal malignancy and urothelial carcinoma cells have the BKPyV gene incorporated into their genomes.
After transplant, JCpyV may result in urothelial cancer.
There are two possible consequences when BKPyV infects cells: either the host cell permits virus reproduction, leading to viral DNA amplification, progeny virion generation, and cell lysis; or the host suppresses virus replication.
BKPyV causes unusually high levels of LTag expression in the host cell when it is triggered in vivo, which results in cell transformation.
The BKPyV LTag and STag can encourage host cell immortalization and transformation, which increases the ability of cells to proliferate.
BKPyV infection tumour cell model, we were able to confirm that bladder tumours expressing BKPyV-related proteins had further improved proliferative, migratory, and invasion abilities in vitro.
Due to their capacity to deactivate tumour suppressor proteins including p53 and pRb, which promote enhanced cell proliferation, LTag are prooncogenic.
STag has been demonstrated to boost MAP kinase pathway activation, which may potentially promote cell proliferation and transformation.
A combination of beta catenin and the transcription factor family Tcf / Lefs in the nucleus of tumour cells can activate genes like cMYC, leading to cell proliferation and EMT effects.
By interacting with beta catenin, JCPyV big T antigen can promote the expression of beta catenin target genes.
BKPyV infections encourage the activation of the beta catenin signalling pathway and the consequences of EMT.
The overexpression of LTag in these BKPyV-infected tumour cells may be attributed to catenin activation.
Such -catenin activation facilitates the invasion of malignant cell even further and also their migration and can increase cMYC expression to encourage cell growth.
BKPyV-infected bladder tumour cell xenografts are susceptible to distant metastases.
level 5 evidence
strength – by showing the role played. by beta catenin in future agents which block its function can be developed as a part of therapy.
limitation – findings in animal studies do not corelate with findings in the human studies.
Study was done on mice to confirm the carcinogenesis of BKV
They concluded that BKV promote proliferation ,invasion and migration of bladder cancer .
They focused on 2 important pathways:B catenin signaling pathway
and epithelial mesenchymal transition effect in BKV infected bladder cancer
This study is done by developing a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections
The research results are :
1- BKPyV infection in bladder cancer cells is a non-lytic infection
2- BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells
3- BKPyV infection enhances bladder tumor growth and metastasis
4- BKPyV infection enhances β-catenin signaling pathway
activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
The authors in conclusion verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
These results may help clinical diagnoses and treatment of BKPyV- related bladder cancer.
Level 5 of evidence
These results revealed how TMP-SMX is currently dosed for the prevention and treatment of PCP. The low dose of TMP-SMX yields acceptable results while lowering mortality and PCP-related adverse events (AEs). Additionally, this tactic lessens the overall financial load and improves patients’ adherence to the daily routine. It may still be justified to begin a preventative treatment programme for select patient populations, such as PCP developing on TMP-SMX prophylaxis in HIV-patients. These results would enable the conduct of extensive, prospective RCTs and cohort studies to provide advice for the dosage strategies for the PCP because there is a dearth of information on the ideal dose of TMP-SMX.
Bladder tumours that expressed BKPyV-related proteins were more invasive in vitro, and BKPyV infection encourages bladder cancer proliferation, invasion, and migration. We established that the EMT impact and the -catenin signalling pathway play a part in the features of bladder cancer associated with BKPyV. The clinical diagnosis and therapy of bladder cancer linked to BKPyV may benefit from these findings.
Level 5 evidence
-Level 5 evidence
-This study shows that BKV infection after kidney transplantation might be related to urothelial cancer.
-No current studies show evidence of viral activity to oncogenesis.
– This study shows that β-catenin signaling pathway may be related to malignancy promoting invasion of malignant cells.Beta catenin blocking medications may help combating bladder malignancy in BKV infection.
Summary
Introduction
This article is about the role that BKV infection plays in bladder cancer. The authors of the study researched how BKV infection promote proliferation, invasion and migration of bladder cancer cells. The role of beta catenin signaling pathway and epithelial mesenchymal transition effect in BKV infected bladder cancer was also studied.
Discussion
BKV infecting cells creates two outcomes :
BKV integrates into the cellular genome of urothelial carcinomas in transplant recipients, thus confirming the correlation of BKV with urothelial carcinomas in the post transplant setting.
Beta catenin signaling pathway plays an important role in tumor invasion and metastasis. Beta catenin can activate genes and cause cell proliferation. Expression of beta catenin may be related to LTag, and activation can promote further tumor cell proliferation and migration invasion.
Thus blocking beta catenin signaling pathway can be an effective alternative for clinical treatment of BKV infected bladder tumors.
Conclusion
The role of BKV auto antigen expression in tumor cells needs to be studies, and the
Level of evidence
This is a narrative review, thus level of evidence is 5.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Background:
Recent studies have confirmed the integration of the BK polyomavirus (BKPyV) gene into the cellular genome of urothelial carcinomas in transplant recipients, further confirming the correlation between BKPyV and urothelial carcinomas after transplantation. However, the role BKPyV infections play in the biological function of bladder cancer remains unclear.
Introduction:
According to research, kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
In 2012, at the International Agency for Research on Cancer (IARC) meeting, BKPyV and JCPyV were classified as “possibly” carcinogenic to human (group 2B) because of the “sufficient evidence” in experimental animals and the “inadequate evidence” in humans for their carcinogenicity .
Whether BKPyV has a causal role in the development of cancer was controversial.
Gene of BKV is found in tumor cell, BKPyV reactivation in immunosuppressed environments may be considered as a transforming factor leading to urothelial carcinomas.
The study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Materials and methods:
Cell culture:
Human bladder cancer cell lines HTB-9 and T24 were obtained and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKPyV infection and inactivation:
BKPyV stocks were initially propagated in Vero cells from viruses obtained from ATC.
Both T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV. After infection cultured.
Animal model:
Male BALB/c nude mice (age 5 weeks, 18–20 g; Cultures of T24 and HTB-9, including their respective BKPyV infected cells (106 cells in 100 mL PBS) were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg) (n = 5). Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
Cell counting:
Kit-8 assay The Cell Counting Kit-8 assay (CCK-8; Xiang SAM, Shanghai, China) was used to determine cell viability
Colony-formation assay:
Cells were seeded in six-well plates at an initial density of 200 cells/well. Colonies were clearly visible after 10– 14 days. Selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet.
Cell migration and invasion assays
After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet solution. A light microscope was used to count the number of cells in five random fields of view.
CELL WHERE EXAMONED HISTOLOGICAL AND BY if.
Results:
BKPyV infection in bladder cancer cells is a non-lytic infection we used two bladder cancer cell lines, T24 and HTB-9, to infect with BKPyV. Forty-eight hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells Results from the CCK8 experiment showed that after infection by BKPyV, the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus).
BKPyV infection enhances bladder tumor growth and metastasis:
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days post inoculation. Differences in tumor volume became more pronounced over time.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKPyV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced.
These results indicate that BKPyV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells.
Blocking β-catenin signal inhibits:
BKPyV infection mediated enhancement of proliferation and migration Compared to BKPyV infected cells alone, BKPyV infected cells treated with KYA1797K had significantly reduced cell proliferation migration and invasion capacities.
In vivo, expression levels of Slug were significantly reduced while expression of Claudin-1 was significantly increased. In vivo, BKPyV infection with KYA1797K applied showed significant reduction of tumor size and invasive abilities.
Discussion:
With the help of the BKPyV infection tumor cell model, we were able to confirm that the proliferative capacity, migration and invasion. We also found that BKPyV infections promotes β-catenin signaling pathway activation and EMT effects. Besides, blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion. T
These results suggest that βcatenin activation in these BKPyV-infected tumor cells may be related to the overexpression of Lion ability of bladder tumors expressing BKPyV-related proteins were further increased in vitro. Xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis.
Conclusions:
In summary, study described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins were more invasive in vitro. It verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
Level of this study 5.
Strength:
Experimental study, show proliferation and growth of cell infected by BKV.
Verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related CANCER.
Weakness:
Proper study of mankind is man, difficulties inherent in extrapolating drug data from animals to man.
Animal experiments are poorly designed, conducted and analyzed.
Small experimental groups with inadequate statistical power
Reviews and summaries of evidence from animal research are methodologically inadequate.
Introduction:
Kidney transplant recipients are3 times more likely to have urothelial cancer than the general population. BKPyV gene has been found integrated into the genome of renal and urothelial cancer cells after transplantation.
Although BKPyV large T antigens (LTag) was detected in prostate, bladder and kidney tumors in some studies, the causal role of BKPyV in the development of cancer could not be proven because BKPyV DNA could not be detected in the cancer samples.
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Materials and methods:
1) Human bladder cancer cell lines were obtained and cultured
2) BKPyV stocks were propagated then inactivated and thereafter cultured.
3) These were then injected into mice.
4) 30 days after implantation, the tumors were surgically dissected and
5) Processed for histopathological examination
6) Cell viability was determined and colonies were assayed
7) Cell invasion and migration were determined
8) Cells were washed, incubated and cultured
9) Histological assessment was done using Hematoxylin and Eosin (H&E) staining then immunofluorescence staining and western blot were carried out.
Conclusion:
BKPyV infection promotes proliferation, invasion and migration of bladder malignancies and bladder tumors expressing BKPyV related proteins
β-catenin signaling pathway and EMT have an effect on the characteristics of BKPyV-related bladder cancer.
Level of evidence is 5
Weaknesses of this article
Experimental study in animals, requires studies in humans
Strengths of this article
The study explains how BKPyV infection promotes proliferation, invasion and migration of bladder malignancies.
The study also confirmed the function of the EMT and the – β-catenin signaling in BKPyV-induced bladder cancer.
In 2012, at the International Agency for Research on Cancer (IARC) meeting, BKPyV and JCPyV were classified as “possibly” carcinogenic to human (group 2B) because of the “sufficient evidence” in experimental animals and the “inadequate evidence” in humans for their carcinogenicity . So far, whether BKPyV has a causal role in the development of cancer was controversial
Author developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
Materials and methods-
Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI 1640 (Gibco Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco Life Technologies) in a humidified atmosphere with 5% CO2 at 37 °C.BKPyV stocks were initially propagated in Vero cells from viruses obtained from ATCC (VR-837, Dunlop). Viral lysates were made through three cycles of freezing the infected cells and supernatant at − 80 °C and thawing at 37 °C.
Results-
BKPyV infection in bladder cancer cells is a non-lytic infection.BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells.BKPyV infection enhances bladder tumor growth and metastasis.BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
Conclusions-In summary, author described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins were more invasive in vitro. They verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer.
What is the level of evidence provided by this article?
Evidence level is 5
What are the weaknesses and strengths of this article?
Strength-
How BK virus can promote proliferation,Invasion and migration of bladder cancer cells and how BK virus can enhance β-catenin signaling to promote proliferation
Weakness–
Animal study(mice tumor model)
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer.
Introduction
Urothelial carcinoma is common and aggressive malignancy . it is three fold higher in post transplantation if compared to general population.
Both BKPyV and JCPyV have evidence based data to support a link to cancers especially in animals.
Some studies report the presence of BKPyV gene material urothelial malignancies, in transplant recipient. In comparison to non- transplant patient , transplant recipient has 4-11 fold higher urothelial malignancies .
Materials and methods:
Cell culture:
-Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKPyV infection and inactivation:
Viral lysates were made through three cycles of freezing the infected cells and supernatant at − 80 °C and thawing at 37 °C.
After infection for 2 hours, cells were washed three times with phosphate-buffered saline , then were cultured using a medium containing 2% serum with and without 15 μM KYA1797K , a potent and selective β-catenin inhibitor.
Animal model :
Male BALB/c nude mice were housed in sterile filter-capped cages. Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg) .
Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
Colony-formation assay :
Cells were seeded in six-well plates at an initial density of 200 cells/well. Colonies were clearly visible after 10– 14 days and selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet (Sigma-Aldrich).
Cell migration and invasion assays :
Transwell chambers (8-μm pore size; Corning, Corning, NY, USA) pre-coated with and without Matrigel (BD Biosciences) was used to determine cell invasion and migration.
Scratch wound healing assay:
Cells were inoculated in six-well plates and cultured at 37 °C in a 5% CO2 cell incubator. After the cells reached 90% confluence, wounds of approximately 1mm width were created using a sterile pipette tip. Cells were washed, incubated and continuously cultured in serum free medium. Cultures at 0, 8, and 24 h were observed under an inverted microscope .
Histological assessment :
Hematoxylin and eosin(H&E) staining was performed for histological assessments. Tumor tissues were cut into small pieces and soaked in 4% neutral formaldehyde solution. Tissue blocks were washed with distilled water and stored in 70% ethanol overnight. After being dehydrated, embedded in transparent paraffin and sectioned, slides were sealed with polylysine and stained using H&E. Tumor tissues were cut into small pieces and soaked in 4% neutral formaldehyde solution,then washed with distilled water and stored in 70% ethanol overnight. After being dehydrated, embedded in transparent paraffin and sectioned, slides were sealed with polylysine and stained using H&E.
Immunofluorescence staining:
Cell slides and frozen sections in 4% formaldehyde were diluted in PBS for 10 min and permeabilized with 0.2% Triton X100 for 10 min at room temperature . Fixed cells were blocked with a blocking buffer containing milk powder and PBS for 15 min (37 °C). Primary and secondary antibodies were diluted in the blocking buffer and incubated at RT for 50 min each. Fluorescence was detected using an inverted fluorescence microscope .
Western blot :
Membrane proteins were separated on SDS polyacrylamide gel electrophoresis and transferred to
Poly vinylidene difluoride membranes. Membranes were blocked using 5% milk and incubated overnight with anti-β-catenin, anti-cMYC, anti-Slug, anti-claudin-1 and anti-β-actin antibodies (1:1000; all from Cell SignalingTechnology).
Statistical analysis :
Statistical analyses were performed using the unpaired Student’s t-test or one-way Analysis of Variance (ANOVA) for more than two groups.
discusssion:
BKPyV infection is linked to malignancy in immunosuppressed patient.
When BKPyV infects cells, two different outcomes may occur:
(1) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virionproduction and cell lysis; or,
(2) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
High levels LTag are required for cell transformation.
By this study , it is possible to prove that BKPyV-infected cells had high proliferative, migration and invasive ability.
Previous research pointed out that LTag and STag of BKPyV can promote host cell transformation and immortalization, leading to enhanced cell proliferation capacity .
It was previously found that LTag are prooncogenic due to their ability to inactivate tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb), leading to increased cell proliferation . In
addition, STag has been shown to increase activation of the mitogenactivated protein (MAP) kinase pathway, which may also augment cell proliferation and transformation.
In this study, xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis.
Blocking the β-catenin signaling pathway can inhibit this process, which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
In BKPyV infection, the liver metastasis of bladder tumors incidence is higher, which increase the difficulty of treatment and decrease the survival rates of patients. Wnt/β-catenin signaling pathway also plays an important role in tumor invasion and metastasis. Blocking this pathway may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
Conclusions
In summary, we first described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins were more invasive in vitro. We verified the role of β-catenin signaling pathway and EMT effectin the characteristics of BKPyV-related bladder cancer. These results may help clinical diagnoses and treatmentof BKPyV-related bladder cancer.
Q 2 – what is level of evidence.
Level of evidence is 5 .
Q3 what is weakness and strength :BKV infection and malignancy. It opens the door for further future research.
Strength:
Document the link between
Weakness :
It is experimental – animal study . data is not necessarily applicable on human
Introduction
Bladder cancer is the 6 th most common malignancy in males with high prevalence in immunocompromised cases.
Kidney transplant recipients are three times more susceptible to have urothelial cancer compared to the general population.
BKPyV reactivation can lead to BKPyV-related nephropathy in renal transplantation and haemorrhagic cystitis after hematopoietic stem cell transplantation .
International Agency for Research on Cancer (IARC) meeting at 2012 stated that , BKPyV and JCPyV
can be possibly carcinogenic to human but this is controversial .
Some studies declared that BKPyV DNA wasnot detected in the cancer samples while others mentioned that BKPyV gene integration was noticed in the urinary tract epithelial cell tumor
A study revealed that cancer bladder risk is higher in transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease compared to transplant recipients without BKPyV.
The Wnt/β-catenin pathway and inhibition of β-catenin degradation in cytoplasm has a role in tumorigenesis and tumor proliferation, invasion, and metastasis .
BKPyV could be oncogenic because of expression of early coding region-encoded proteins
LTag and small T antigen (STag), which can stimulate neoplastic transformation.
this study aimed at evaluating BKPyV infections effect on the occurrence of bladder cancer .
Materials & methods
BKVPyV stockes were propagated then inactivated and cultured, thirty days after implantation the tumor were surgically dissected and processed for histopathological examination.
Cells were washed, incubated and cultured at 37 degrees, then all histology was done with H&E staining, IF staining, western blot were carried out, and images were processed.
All data was presented as mean+/- SD, analyzed using T test and ANOVA, P values were taken significant if <0.005.
Results
They used two bladder cancer cell lines, T24 and HTB-9 to infect with BKPyV, after 48H after infection the infected cell showed larg T protien expression, the infected cell did not lyse, but continued to proliferate and grow for 9 days.
The CCK8 experiment showed the ability of infected cell has increased to persist and proliferate.
BKPyV infection enhances B catenin signaling pathway activation and epithelial mesenchymal transition effect in bladder cancer cells, and blocking B-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration.
Conclusion
The outcomes of the study in identifying BKPyV infection’s role in causing urinary tract cancer could aid in the identification and proper treatment of cancer resulting from the infection.
Level V
Weakness
experimental study in animals, requires studies in humans
Strength
It is a study to objectively prove role of BKV in virus induced bladder cancer.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer:
Summarise this article:
Introduction:
– urothelial carcinoma is a common and highly malignant tumor in the urinary system
– kidney transplant recipients (KTRs) are 3 times more likely to have urothelial cancer than the general population
– BKPyV undergoes reactivation in immunosuppressive states resulting in viruria, viremia, ureteral stricture , BKVAN in kidney transplant recipients and hemorrhagic cystitis in HSCT recipients
– the role of BKPyV in carcinogenesis remains controversial
– BKPyV reactivation may be considered a transforming factor leading to urothelial carcinomas in immunosuppressed states
Materials and methods:
– human bladder cancer cell lines were obtained and cultured
– BKPyV stocks were propagated then inactivated and thereafter cultured
– these were then injected into mice
– 30days after implantation, the tumors were surgically dissected and processed for histopathological examination
– cell viability was determined and colonies were assayed
– cell invasion and migration were determined
– cells were washed, incubated and cultured
– histological assessment was done using hematoxylin and eosin (H&E) staining
– immunofluorescence staining and western blot were carried out
Results:
– both bladder cancer cell lines did not lyse due to BKPyV infection but instead continued to proliferate
– BKPyV infection was shown to: –
Discussion:
– malignancies affect the long-term survival of kidney transplant recipients
– BKPyV seems to have a role in carcinogenesis based on studies which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation
– when BKPyV infects cells the host cell can either: –
– BKPyV infection did not cause lysis of bladder cancer cells
– the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPvV-related proteins were increased in vitro
Conclusion
– BKPyV infection promotes proliferation, invasion and migration of bladder malignancies and bladder tumors expressing BKPyV related proteins
– β-catenin signaling pathway and EMT have an effect on the characteristics of BKPyV-related bladder cancer
Level of evidence provided by this article:
Level V
Weaknesses of this article
– experimental study in animals, requires studies in humans
Strengths of this article
– the study explains how BKPyV infection promotes proliferation, invasion and migration of bladder malignancies
– the study also confirmed the function of the EMT and the – β-catenin signaling in BKPyV-induced bladder cancer
Introduction:
· kidney transplant recipients are3 times more likely to have urothelial cancer than the general population. BKPyV gene has been found integrated into the genome of renal and urothelial cancer cells after transplantation.
· Although BKPyV large T antigens (LTag) was detected in prostate, bladder and kidney tumors in some studies, the causal role of BKPyV in the development of cancer could not be proven because BKPyV DNA could not be detected in the cancer samples.
Aims of the study:
· Effects of BKPyV infection in proliferation and migration of bladder ca cells.
· Role of wnt/B-catenin pathway and EMT in proliferation and migration of Ca cells.
· Possible Causal Role of BKPyV in bladder cancer?
Materials and Methods;
1. human bladder cancer cell lines (HTB-9 and T 24) were cultured – in Cell culture media 10% foetal bovine serum @ 370C
2. BKPyV stocks from Vero cells propagated – viral lysate was inactivated (@1000C x10min) and the inactive virus was maintained.
3. Bladder cancer cell lines (T24 and HTB-9) were infected with BKPyV. 2 hours of viral infection, cells were washed and cultured in 2 separate serum media – one with KYA1797K (β-catenin inhibitor) or another without inhibitor
4. Animal (Mice) model: Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice, with and without subsequent intraperitoneal injections of KYA1797K. After 30days, tumors were surgically dissected, stored in liquid nitrogen and processed for HPE.
5. Cell viability was determined (Cell counting kit-8 assay / CCK-8) and colonies counted after a special Colony Forming assay.
6. Cell migration and invasion assay performed – active and average number of cells counted using Trans-well chambers
8. Histological Examination carried out using (H&E) staining, IF
9. Western blot assay (for membrane proteins) – separated by PAG electrophoresis, fixed to PLD membrane and incubated overnight with anti-β-catenin, anti-cMYC, anti-Slug, anti-claudin-1 and anti-β-actin antibodies.
Results:
Discussion:
· BKPyV infection did not cause lysis of bladder cancer cells
· BKPyV infection was shown to –
o promote proliferation, invasion and migration of bladder cancer cells
o enhance bladder tumor growth and metastasis to liver
o Enhanced β-catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) effect in bladder cancer cells infected with BKPyV virus increases proliferation, invasion of bladder cancer cells.
Conclusion
§ BKPyV infection promotes proliferation, invasion and migration of bladder cancer
§ Bladder tumors expressing BKPyV related proteins were more invasive in vitro.
§ The study verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
2, Level of evidence provided by this article
Level V – mechanistic studies (animal research / In Vitro studies)
3.Strengths of the study / article
· First study describing that BKPyV infection promotes proliferation, invasion and migration of bladder cancer.
· Objectively verified the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV infected bladder cancer.
· These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
4.Weaknesses of the study / article
· Experimental study in in vitro (cell culture) and animal (mice) model
· This result can’t be extrapolated to patients or general population
· Needs further well-designed studies in human
1. Please summarise this article;
According to 2018 statistics urothelial carcinoma is the most common and highly malignant tumor in the urinary system, sixth most common malignancy in male.
According to literature bladder cancer is three times more common in renal transplantation.
BK virus belongs to a family of polyomavirus family, small double-stranded non-enveloped DNA virus, categorized into four groups with different virulence, in 2012 IARC meeting the BKVPyV and JCPyV were classified as possible cause of carcinoma, SV40 has been weak association with malignancy.
Together, these result will make more questions are will clarify the characteristic of BKV related to bladder and other related carcinomas.
Materials and methods;
BKVPyV stockes were propagated then inactivated and cultured, thirty days after implantation the tumor were surgically dissected and processed for histopathological examination.
Cells were washed, incubated and cultured at 37 degrees, then all histology was done with H&E staining, IF staining, western blot were carried out, and images were processed.
All data was presented as mean+/- SD, analyzed using T test and ANOVA, P values were taken significant if <0.005.
Results;
They used two bladder cancer cell lines, T24 and HTB-9 to infect with BKPyV, after 48H after infection the infected cell showed larg T protien expression, the infected cell did not lyse, but continued to proliferate and grow for 9 days.
The CCK8 experiment showed the ability of infected cell has increased to persist and proliferate.
BKPyV infection enhances B catenin signaling pathway activation and epithelial mesenchymal transition effect in bladder cancer cells, and blocking B-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration.
Discussion;
The correlation between BKpyV infection and tumorigeesis in immunocompromised patients has some association, by deep sequencing studies confirmed that that BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after renal transplantation.
According to Querido et al, JCpyV may cause urothelial carcinoma after transplantation
Conclusion;
As discussed in discussion the BKPyV infection promotes proliferation, invasion, and migration of bladder cancer and expression of bladder tumor related gene.
Level of evidence V
Introduction:
Material and methods:
Results:
Discussion:
Conclusion
Strength of study:
First study to objectively prove role of BKV in virus induced bladder cancer.
Weakness of the study:
This is animal study, and the observations need to confirm in animal studies.
Level of evidence: NA (Animal Study)
● Tumors have become one of the main factors affecting the long-term survival of kidney transplant recipients.
● BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation
● Bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver
● Bladder cancer is also a prone tumor type for immunocompromised patients.
● kidney transplant recipients are three times more likely to have urothelial cancer than the general population
● BKV reactivation after renal transplant causes symptoms such as viremia, viruria, ureteral stricture and BKVN as well as hemorrhagic cystitis after HSCT
● In 2012, at the International Agency for
● BKV was classified as “possibly” carcinogenic to human
● This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Results
● BKPyV infection in bladder cancer cells is a non-lytic infection
● BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells
● BKPyV infection enhances bladder tumor growth and metastasis
● Blocking β-catenin signal inhibits BKPyV infection-mediated enhancement of proliferation and migration
● When BKPyV is activated in vivo, BKPyV triggers abnormally high expression of LTag in the host cell leading to cell transformation.
● LTag and STag of BKPyV can promote host cell transformation and immortalization, leading to enhanced cell proliferation capacity
● LTag are prooncogenic due to their ability to inactivate tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb), leading to increased cell proliferation
● β-catenin activation in BKPyV-infected tumor cells may be related to the overexpression of LTag.
● Activation of β-catenin further promotes the invasion and migration of tumor cells. it can enhance the expression of cMYC to promote cell proliferation.
● Xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis.
● Blocking the β-catenin signaling pathway can inhibit this process, which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
● Level of evidence : 5
● Strength points of study :
☆ Apromising study for more researchs in the future
☆ The study explain pathology between BKpyV infection and bladder cancer
Which may be a basic for experimental therapies later
● Weekness points of study :
☆ Experimental animal study with xenografts
III. BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Summarise this article
Introduction
– urothelial carcinoma is a common and highly malignant tumor in the urinary system
– kidney transplant recipients (KTRs) are 3 times more likely to have urothelial cancer than the general population
– BKPyV undergoes reactivation in immunosuppressive states resulting in viruria, viremia, ureteral stricture , BKVAN in kidney transplant recipients and hemorrhagic cystitis in HSCT recipients
– the role of BKPyV in carcinogenesis remains controversial
– BKPyV reactivation may be considered a transforming factor leading to urothelial carcinomas in immunosuppressed states
Materials and methods
– human bladder cancer cell lines were obtained and cultured
– BKPyV stocks were propagated then inactivated and thereafter cultured
– these were then injected into mice
– 30days after implantation, the tumors were surgically dissected and processed for histopathological examination
– cell viability was determined and colonies were assayed
– cell invasion and migration were determined
– cells were washed, incubated and cultured
– histological assessment was done using hematoxylin and eosin (H&E) staining
– immunofluorescence staining and western blot were carried out
Results
– both bladder cancer cell lines did not lyse due to BKPyV infection but instead continued to proliferate
– BKPyV infection was shown to: –
Discussion
– malignancies affect the long-term survival of kidney transplant recipients
– BKPyV seems to have a role in carcinogenesis based on studies which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation
– when BKPyV infects cells the host cell can either: –
– BKPyV infection did not cause lysis of bladder cancer cells
– the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPvV-related proteins were increased in vitro
Conclusion
– BKPyV infection promotes proliferation, invasion and migration of bladder malignancies and bladder tumors expressing BKPyV related proteins
– β-catenin signaling pathway and EMT have an effect on the characteristics of BKPyV-related bladder cancer
Level of evidence provided by this article
Level V
Weaknesses of this article
– experimental study in animals, requires studies in humans
Strengths of this article
– the study explains how BKPyV infection promotes proliferation, invasion and migration of bladder malignancies
– the study also confirmed the function of the EMT and the – β-catenin signaling in BKPyV-induced bladder cancer
SUMMARISE ARTICLE.
9
BKV INFECTION PROMOTES GROWTH AND AGGRESSIVENESS IN BLADDER CANCER.
INTRODUCTION.
-Urothelial ca is the 6th most common Ca in males and KTR are x3 likely to have it compared to the general population.
-We have enough evidence in animals but not humans for causal role of BKV in dev of Ca.
AIMS OF THE STUDY.
MATERIALS AND METHODS;
STATISTICAL ANALYSIS;
-All data was presented as mean +/- SD, analyzed using T test and ANOVA.P values were significant if < 0.005
RESULTS;
DISCUSSION.
–As evidenced by BKPyV infection tumor model, BKPyV related protein in bladder tumor increases;
-Beta catenin signal pathway activation increases invasion and proliferation of tumor cells ang this might alter future mgt of infected BKPyV bladder malignancies.
-The information gotten from this study can be used to modify tx and better outcomes in future.
LEVEL OF EVIDENCE – 5
–
STENGTHES;
-Used animal model to get us key information that will inform future studies and hopefully impact positively on pt mgt.
WEAKNESSES;
-Non human subjects means less application in human beings in managing their post transplant status.
Summary
Introduction
Urothelial carcinoma are one of the most common cancers and are highly aggressive. Kidney transplant recipients are 3 times more likely to develop urothelial cancers than general population. BKPyV and JCPyV have both being classified as possible carcinogenic supported by sufficient evidence in animal studies and insufficient in human studies.
Some studies have shown BKPyV gene integration in urothelial malignancies, this association was seen in transplant recipients and none in non-transplant population. Other studies have shown that transplant recipients are 4-11 times more prone to urothelial malignancies than general population.
Aim of the study: It explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice. Together, these results will clarify the characteristics of BKPyV- related bladder tumours, laying a foundation for further clinical trials.
Results: BKPyV infection of bladder cancer cell lines leads to an infection that is non-lytic. This BKPyV infected bladder cancer cells lines have increased proliferation ability and migration than controls. Their tumour growth rate also increased exponentially. There was an increased expression of β-catenin, cMYC and Slug proteins while the expression of Claudin-1 protein was significantly reduced.
Discussion: BKPyV infection in bladder cancer cells does not have a lytic effect. There is increased proliferative capacity, migration and invasion of the bladder tumours with BKPyV infection. BKPyV infection promotes signalling in the β-catenin pathway and blocking of this pathway may inhibit BKPyV function to promote tumour cell proliferation, migration and invasion.
Conclusion: BKPyV infected bladder cancer cell lines in vitro there was increased proliferation, invasion and migration. β-catenin signalling pathway plays a role in this infected bladder cancer cells and this may help in the diagnosis and treatment of BKPyV related bladder cancer.
Level of evidence: Animal study hence level V.
Strengths: Forms basis of future research.
Weakness: Animal study thus difficult to draw any conclusions from it.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Introduction :
– The most common and highly malignant tumor in the urinary system is the urothelial carcinoma of which bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver .
– kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
– BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised populations, especially transplant recipients causing virus ,viremia and nephritis and ureteral stenosis in kidney transplant patients also causes hemorrhagic cystitis after hematopoietic stem cell transplantation.
BKPyV and JCPyV were possibly carcinogenic to humans,when reactivated in a state of immunosuppression environments it may be considered as a transforming factor leading to urothelial carcinomas.
The risk of bladder cancer was found to be increased (4-11 times ) in kidney transplant recipients with BKPyV viremia or polyomavirus associated kidney disease when compared to transplant recipients without BKPyV.
BKPyV is considered to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation.
This experimental study in animals explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway ( which is implicated in cell proliferation and involved in tumorigenesis ) by establishing a model for BKPyV infections in bladder cancer cells and mice.
Materials and methods:
Cell culture:
-Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKPyV infection and inactivation:
The cultured BKPyV underwent three cycles of freezing and supernatant at − 80 °C and thawing at 37 °C. BKPyV was inactivated at 100 °C for 10 min. Both T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV.
After infection for 2 hours, cells were washed three times with phosphate-buffered saline , then were cultured using a medium containing 2% serum with and without 15 μM KYA1797K , a potent and selective β-catenin inhibitor.
Animal model :
Male BALB/c nude mice (age 5 weeks, 18–20 g; were housed in sterile filter-capped cages.
Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice .
Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
Cell counting Kit-8 assay: Is the determinant of cell viability. Cells were seeded in 96-well plates . Absorbance of the medium at 450 nm was detected using a spectrophotometer by assessing cell viability.
Colony-formation assay :
Cells were seeded in six-well plates at an initial density of 200 cells/well. Colonies were clearly visible after 10– 14 days and selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet . Colonies containing > 50 cells were counted using light microscopy .
Cell migration and invasion assays :
Cell invasion and migration was determined by transwell chambers pre-coated with and without Matrigel . Cells (105 ) in 200 μL FBS-free medium were seeded in the upper chamber , and 600 mL medium containing 10% FBS was added to the lower chamber.
After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet solution.
A light microscope was used to count the number of cells in five random fields of view.
Scratch wound healing assay:
Cells were inoculated in six-well plates and cultured at 37 °C in a 5% CO2 cell incubator. After the cells reached 90% confluence, wounds of approximately 1 mm width were created using a sterile pipette tip. Cells were washed, incubated and continuously cultured in serum free medium and observed under an inverted microscope at 0, 8, and 24 h .
Histological assessment :
Tumor tissues were cut into small pieces and soaked in 4% neutral formaldehyde solution,then washed with distilled water and stored in 70% ethanol overnight. After being dehydrated, embedded in transparent paraffin and sectioned, slides were sealed with polylysine and stained using H&E.
Immunofluorescence staining:
Cell slides and frozen sections in 4% formaldehyde were diluted in PBS for 10 min and permeabilized with 0.2% Triton X100 for 10 min at room temperature .
Fixed cells were blocked with a blocking buffer containing milk powder and PBS for 15 min (37 °C). Primary and secondary antibodies were diluted in the blocking buffer and incubated at RT for 50 min each. Fluorescence was detected using an inverted fluorescence microscope .
Western blot :
Using SDSpolyacrylamide gel electrophoresis membrane proteins were separated and transferred to polyvinylidene difluoride membranes. Membranes were blocked using 5% milk and incubated overnight with anti-β-catenin, anti-cMYC, anti-Slug, anti-claudin-1 and anti-β-actin antibodies .
Statistical analysis :
Statistical analyses were performed using the unpaired Student’s t-test or one-way Analysis of Variance (ANOVA) for more than two groups. All analyses were carried out using Graph pad prism 7 software and significant differences were considered when values had P < 0.05.
Results :
Bladder cancer cell lines, T24 and HTB-9 were used to infect with BKPyV. Forty-eight hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV .BKPyV infections effect on cell fates was observed by extending the infection time . Both T24 and HTB-9 cells did not lyse due to BKPyV infections, but continued to proliferate and grow for 9 days after the initial infection .
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells: Results from the CCK8 experiment showed that after infection by BKPyV, the cells’ proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus) .
Colony formation experiments further verified in the result of this study , showing that BKPyV infections promote proliferation of bladder cancer cells in vitro . T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates and higher invasive capacities than non-infected cells than the controls
.
BKPyV infection enhances bladder tumor growth and metastasis :
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, compared with the control group at 15 days post inoculation and became more pronounced over time .
On day 30 post inoculation, new tumors were discovered in the liver of BKPyV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice .
Pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream .
BKPyV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells both in vivo and in vitro studies .
inhibits BKPyV infection mediated enhancement of proliferation and migration
Compared to BKPyV infected cells alone, BKPyV infected cells treated with KYA1797K ( Blocking β-catenin signal ) had significantly reduced cell proliferation ,reduction of tumor size , migration and invasion capacities with no metastasis occurring in vivo .
Discussion :
-Recently the correlation between BKPyV infection and tumorigenesis in immunocompromised individuals has drawn increased attention.
-BKPyV plays an important role in cancers of the urinary system was recently confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation .
– BKPyV infections did not have a lytic effect on bladder cancer cells.
-Previous research pointed out that LTag and STag of BKPyV can promote host cell transformation and immortalization, leading to enhanced cell proliferation capacity . However, the role of BKPyV autoantigen expression in tumor cells has not been studied, and the biological characteristics of BKPyV-related tumors are unknown.
This study was able to confirm that the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPyV-related proteins were further increased in vitro. Their ability to grow, invade and metastasize to other nearby organs in vivo was highly likely, and is of further threat to clinical patients.
After BKPyV infections, liver metastases of bladder tumors are likely to occur, which will increase the difficulty of clinical treatments and seriously affect the survival rates of patients.
-Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis .
Blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion. as well as inhibition of bladder tumor cell distant metastasis , which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
Conclusion :
BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins that were more invasive in vitro. The role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer was verified . These results is promising in clinical diagnoses and treatment of BKPyV-related bladder cancer.
Level V
weakness : is experimental study done in animal and the use of xenograft which is not applied in human .
Strength points : –
-It verify the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
-It described that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors.
-It gives promising results regarding treatment of BKPyV-related bladder cancer by blocking β-catenin signaling pathway .
This Article is with evidence level of 7 (animal studies-in vitro).
The causality of some Polyomaviruses to cancer is still controversial, and the relation of BK and JC though more prominent, still with low evidence. for that reason, this study was studied, but this study was performed in vitro in cell lines which may not be applicable with the same strength in vivo rather than in human. Despite that, this study showed cell proliferation and activity of T24 & HTB9 bladder cancer cells not observed in the non-infected group.
Novelty and the results comparing two groups are strengths, but applicability, being in vitro, and lack of long-term follow-up are weaknesses aspects
BK polyomavirus infection promotes growth and aggressiveness in bladder
cancer
Summary
· It was controversial to prove the role of BKV in cancer bladder, as the virus was not always isolated from cancer tissue.
· Then finding BKV DNA integrated into the DNA of urothelial cells in kidney transplant recipients with urothelial carcinoma.
· The current experimental study demonstrated the role of BKV infection in mice model.
· It concluded that it enhanced tumor growth and metastasis.
Level of evidence; experimental animal study, level V.
Point of weakness and strength
· Weakness is experimental in animals needs further studies to be concluded in human.
· Strength is clarification the role of BKV in bladder cancer.
Summary of the article
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
This is an experimental study, developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
Results and Discussion
1. BKPyV infection in bladder cancer cells is a non-lytic infection.
2. BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells.
3. BKPyV infection enhances bladder tumor growth and metastasis.
4. BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
5. Blocking β-catenin signal inhibits BKPyV infection- mediated enhancement of proliferation and migration.
6. When BKPyV infects cells, two different outcomes may occur:
a) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis…or,
b) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
What are the weaknesses and strengths of this article?
Weakness:
a) the role of BKPyV autoantigen expression in tumor cells has not been studied.
b) the biological characteristics of BKPyV-related tumors are unknown.
Strength
a) The study was able to confirm that the proliferative capacity, migration and invasion ability of bladder tumors expressing BKPyV-related proteins were further increased in vitro.
The level of evidence provided by this article
This is an experimental study( animal study)
Level of evidence grade 5
BKPyV and urothelial carcinoma:
It still controversial if BKPyV is causing neoplastic transformation or not. The international agency for research on cancer meeting classified BKPyV and JCPyV as possibly carcinogic to human owing to sufficient evidences in animal studies and inadequate evidences in human for their carcinogenicity.
Primary evidences :
BKPyV role in malignancies stem from finding of Larg T protein [TAg] in prostate, bladder and kidney tumors.
However, No BKPyV DNA was isolated from any of the kidneys and bladders tumors.
Urothelial Cancer:
Bladder cancer is the a common disease ranked 6th in its prevalence after lung, prostate, colon, stomach and liver.
Kidney transplant recipients are 3 times more prone to have urothelial cancer.
Study design:
By infecting bladder cancer cells with BKPyV and studying its impact on behavior of those cells in comparison to a mice tumor cells not infected with BKPyV.
The study proved that BKPyV is promoting malignant cells to be aggressively dividing, invading and metastasizing.
Please summarise this article.
-Urothelial carcinoma is one of the most common and highly malignant tumors in the urinary system.
-Bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver and is a prone tumor type for immunocompromised patients.
-BKPyV and JCPyV were classified as “possibly” carcinogenic to human .
-Many recent studies detected BKPyV gene integration in urinary tract epithelial cell tumors, providing further evidence for the correlation between BKPyV and urinary tract tumor.
-Some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk of bladder cancer when compared to transplant recipients without BKPyV .
– BKPyV is thought to be oncogenic due to the expression
of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation.
– They used two bladder cancer cell lines, T24 and HTB-9, to infect with BKPyV. 48- hours after infection, T24 and HTB-9 cells showed large T protein expression of BKPyV.
-By extending the infection time, they observed the effect of BKPyV infections on cell fates. Both T24 and HTB-9 cells did not lyse due to BKPyV infections, but continued to proliferate and grow for 9 days after the initial infection and express large T antigen after 30 days. VP1 expression in these two cell infection models increased continuously in the first few days, but maintained a stable level after.
-Results showed the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV.
-Colony formation experiments further verified their results, showing that BKPyV infections promote proliferation of bladder cancer cells in vitro.
-Transwell migration results showed that T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates than the controls.
– BKPyV infected T24 and HTB-9 cells had significantly higher invasive capacities than non-infected cells.
-The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly .
– The BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation .
-Querido et al. showed that JCpyV may cause urothelial carcinoma after transplant , which also supports the argument that human polyomavirus is closely related to bladder cancer.
-When BKPyV infects cells, two different outcomes may occur:
(1) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis.
(2) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
– A small amount of LTag however is not sufficient to cause tumors. BKPyV triggers abnormally high expression of LTag in the host cell through various mechanism, eventually leading to cell transformation.
– After BKPyV infections, liver metastases of bladder tumors are likely to occur.
– Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis . JCPyV large T antigen can interact with β-catenin and stimulate expression of β-catenin target genes.
– BKPyV infections promotes β-catenin signaling pathway activation and EMT effects.
– Besides, blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion.
-These results suggest that β- catenin activation may be related to the overexpression of LTag. Such activation of β-catenin further promotes the invasion and migration of tumor cells.
What is the level of evidence provided by this article?
Level 5
What are the weaknesses and strengths of this article?
Weakness: experimental study on animal.
Strengths: It explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT .These results will clarify the characteristics of BKPyV related bladder tumors, laying a foundation for further clinical trials.
# Please summarise this article.
# Introduction:
*Carcinoma of the bladder cancer is the sixth most common cancer in males and more common in immunocompromised patients.
*The incidence of urothelial cancer is three fold higher in kidney transplant recipients compared to control group.
* BKPyV reactivation PKT lead to symptoms like viremia, viruria, ureteral stricture, BKPyV-related nephropathy and hemorrhagic cystitis post BMT.
* In 2012, the (IARC) meeting, BKPyV and JCPyV were classified as “possibly” carcinogenic to human (group 2B) due to “sufficient evidence” in experimental animals and the “inadequate evidence” in humans for their carcinogenicity.
# Materials and methods:
*The study was an experimental animal study
*Cell culture, human bladder cancer cell lines HTB-9 and T24 were obtained and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
*BKPyV infection and inactivation BKPyV were propagated in Vero cells, viral lysates were made and inactivation was performed at 100 °C for 10 min. Both T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV.
#Animal model:
*Male BALB/c nude mice were housed in sterile filter-capped cages.
*Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected s/c into nude mice.
*30 days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
*The Cell Counting Kit-8 assay was used to determine cell viability.
*Colony-formation assay cells were seeded in six-well plates at 200 cells/well and visible after 10–14 days and selected cells were fixed with 4% paraformaldehyde
for 30 min then counted using LM.
# Results:
*BKPyV infection in bladder cancer cells is a non-lytic
infection
*Further research shows the BKPyV infected T24 and HTB-9 cells xenografted
on mice still express large T antigen after 30 days.
*The cratch healing test demonstrated that BKPyV infected T24 and HTB-9 cells migrated significantly faster on plates than the control group.
* BKPyV infection promoted the migration of bladder cancer cells in vitro.
*BKPyV infected T24 and HTB-9 cells had significantly higher invasive capacities than non-infected cells.
*BKPyV infection enhances bladder tumor growth and metastasis.
*BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
*Blocking β-catenin signal inhibits BKPyV infectionmediated
enhancement of proliferation and migration.
# What is the level of evidence provided by this article?
*Level (V)
# The strength of the study:
*It was described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated
proteins were more invasive in vitro.
*The study verified the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV infected bladder cancer. These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
# The limitation of the study:
*The result obtained from the animal (mice model that differ from the human).
I agree with your analysis of strengths and limitations, and summary of this article.I agree with your analysis of the level of evidence this article provides.
Introduction
Urothelial carcinoma is a common and highly malignant tumor, for the immunocompetent and more so for the immunosuppressed population, especially kidney transplant recipients. BK polyomavirus (BKPyV) reactivation after transplantation causes viremia, viruria, ureteral stricture, hemorrhagic cystitis (in HSCT) and BKPyV-related nephropathy. It has been classified as possibly carcinogenic by the IARC. Studies have detected the presence of BKPyV large T antigens in prostate, bladder and kidney tumors, in post-transplant patients. The Wnt/β-catenin pathway is implicated in cell proliferation and transcription, and regulating pattern formation during development. This pathway is associated with tumor proliferation, invasion and metastasis. BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic formation.
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of the Wnt/β-catenin pathway in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Material and methods
Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection and cultured. Viral lysates were made through three cycles of freezing the infected cells and supernatant, and then thawing them. The inactivation of BKPyV was performed at 100°C for 10 minutes. The two cell lines were cultured and infected with BKPyV, and then re-cultured. Male BALBc nude mice were injected with the final culture. Thirty days after implantation, the tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination. The Cell Counting Kit-8 assay was used to determine cell viability. Absorbance of the medium was detected using a spectrophotometer by assessing cell viability. All observations were reproduced at least three times in independent experiments. Cells were seeded in plates and colonies were visible after 10-14 days. The average number of colonies was determined from three independent experiments. Transwell chambers were used to determine cell invasion and migration. A light microscope was used to count the number of cells in five random fields of view, and a mean cell numer was calculated for each group. The cells were then inoculated and cultured. Cultures at 0, 8 and 24 hours were observed. Hematoxylin and eosin staining was performed for histological assessments. Cell slides and frozen sections were then stained using immunofluorescence staining and processed.
Results
BKPyV infection in bladder cancer cells is a non-lytic infection
Two bladder cancer cell lines, T24 and HTB-9, were used to infect with BKPyV. 48 hours after infection, they showed large T protein expression of BKPyV.
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells
After infection by BKPyV, the ability and activity of cell proliferation increased in both bladder cancer cell lines, compared to the cells not infected by BKPyV. It was also noted that BKPyV infected cells had a significantly higher invasive capacity than non-infected cells.
BKPyV infection enhances bladder tumor growth and metastasis
The growth rate of BKPyV infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days after inoculation. New tumors were detected in the liver of BKPyV infected bladder tumor mice, which were characteristic of urothelial carcinoma, showing the bladder tumor cells were transported by the blood stream.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
The levels of β-catenin and its downstream signaling molecule cMYC in BKPyV infected tumor cells were examined. It was noted that the expression of β-catenin and cMYC in BKPyV infected cells was significantly increased. The results indicate that BKPyV infection promotes β-catenin signaling activation and EMT effects in bladder cancer cells.
Blocking β-catenin signal inhibits BKPyV infection-mediated enhancement of proliferation and migration
BKPyV infected cells treated with KYA1797K had significantly reduced cell proliferation, migration and invasion capacities.
Discussion
Tumors affect the long-term survival of kidney transplant recipients. BKPyV infection has shown to increase the risk of tumors in immunocompromised patients. When BKPyV infects cells the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis, or the host prevents virus replication, and the persistent expression of the LTag causes abortion of infected cell or cell transformation. High levels LTag are required for significant cell transformation. With the help of the BKPyV infection tumor cell model, it is possible to confirm that BKPyV-infected cells had an increased proliferative capacity, migration and invasive ability. After BKPyV infection, it was also noted the liver metastasis of bladder tumors are more likely to happen, which increase the difficulty of treatment and decrease the survival rates of patients. Wnt/β-catenin signaling pathway also plays an important role in tumor invasion and metastasis. Blocking this pathway may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
Conclusion
In summary, it was noted that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors. The study also verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV–related bladder cancer. The results of this study may help clinicians better diagnose and treat BKPyV-related bladder cancer.
Level of Evidence:
This is an animal study and hence, classified as level V
Strengths:
Done under controlled conditions keeping environments for the infected and non-infected cell lines similar
The study looked at several oncogenic proteins
Limitations:
Animal study hence, difficult to draw any conclusions
I agree with your analysis of strengths and limitations, and summary of this article.I agree with your analysis of the level of evidence this article provides.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer:
Introduction:
Bladder cancer is the 6th common cause in male cancer after lung, prostate, colon and liver.
Bladder cancer is three times more in immunocompromised after kidney transplant in comparison to general population.
BK virus activated after kidney transplant due to use of immunosuppressive therapy and leading to viremia, viruria, ureteral stricture and BKPyV-related nephropathy, as well as hemorrhagic cystitis after hematopoietic stem cell transplantation.
There’s relationship between BKV virama and developing of bladder cancer. But some small studies shows no evidence or relation between DNA virama of BKV and development of bladder cancer.
B- catenin pathway is used in process of cell proliferation and transcription, and regulating pattern formation during development. This pathway is involved in tumorigenesis and is associated with many biological interactions such as tumor proliferation, invasion, and metastasis.
So inhibition of β-catenin degradation in cytoplasm can induce epithelial mesenchymal transition and promote tumorigenesis.
Materials and methods:
Cell culture were obtained from the American Type Culture Collection.
The virus of BKV collected and inactivation.
Type of animal module is mice were injected subcutaneous with infected virus and after 30 days the tumor resected and histo pathology done.
The Cell Counting Kit-8 assay was used to determine cell viability. Histology by light microscopy and electron microscopy and immunohistochemistry staining done and western blotting were done.
Results and discussion:
The study shows BKV replication post kidney transplant may promote progression of development of urothelial cancer.
BKV may end with two different outcomes: 1). The host cell allows replication of the virus, resulting in viral DNA amplification, progeny production and cell lysis
2). The host prevents virus replication, and the persistent expression of the LTag cause cell transformation.
This result is enough to cause cancer. BKV dose not have effects on developing of bladder cancer. There’s no studies support effects of virus activation with proliferation and formation of tumor and still biological characteristics of BKV in relation to cancer unknown.
In the past studies BKV LTag are prooncogenic because ability to inactivate tumor suppressor proteins. Where as STag shows ability to activation of the mitogenactivated protein kinase pathway, which increase cell proliferation and transformation.
This study shows β-catenin signaling pathway plays an important role in tumor invasion and metastasis.
Activation of β-catenin may promotes the invasion and migration of tumor cells. It’s also enhance the expression of cMYC to promote cell proliferation. Blocking the β-catenin signaling pathway may possibly help to find clinical treatments of infectious BKV bladder cancer.
Conclusion:
Still there is no strong evidence between activation of BKV infection and development of bladder cancer. B catenin blocking may help in management of bladder cancer in related to BKV.
Level V
The strength of this study is address the role of BKV in relation to bladder tumor in context of cell proliferation and transformation and invasive and migration. Also address the role of B catenin in treatment of bladder tumor related to virus.
The weakness of this study is small studies and experimental on animal
I agree with your analysis of strengths and limitations, and summary of this article.
The introduction ;
———————————————–
The idea that BKPyV plays an important role in cancers of the urinary system was recently confirmed by deep sequencing studies, which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation.
When BKPyV infects cells, two different outcomes may occur:
(1) The host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis .
(2) or The host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation.
.
LTag are prooncogenic due to their ability to inactivate tumor suppressor proteins, such as p53 and retinoblastoma protein (pRb), leading to increased cell proliferation . In addition, STag has been shown to increase activation of the mitogen activated protein (MAP) kinase pathway, which may also augment cell proliferation and transformation .
Wnt/β-catenin signaling pathway plays an important role in tumor invasion and metastasis . In the nucleus of tumor cells, β-catenin combined with the transcription factor family Tcf / Lefs can activate genes such as cMYC and cause cell proliferation and EMT effects. JCPyV large T antigen can interact with β-catenin and stimulate expression of β-catenin target genes.
BKPyV infections promotes β-catenin signaling pathway activation and EMT effects. Besides, blocking β-catenin signaling pathways can inhibit BKPyV’s function to promote tumor cell proliferation and migration invasion.
The Aim of the Study ;
————————————————-
Is to investigate the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT .
The type of study;
——————————————–
Experimental animal study .
The Method ;
————————————–
The study group developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
The Result ;
——————————–
BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells, while the activation of β-catenin signaling pathway is one of its mediation mechanisms.
The conclusion ;
———————————————–
1-The study results suggest that β-catenin activation in BKPyV-infected tumor cells may be related to the overexpression of LTag. Such activation of β-catenin further promotes the invasion and migration of tumor cells. At the same time, it can enhance the expression of cMYC to promote cell proliferation.
2-Xenografts of BKPyV-infected bladder tumor cells are prone to distant metastasis. Blocking the β-catenin signaling pathway can inhibit this process, which may be an effective alternative for clinical treatment of BKPyV-infected bladder tumors.
3-This new discovery has considerable significance towards clinical treatments of infectious BKPyV bladder tumors.
What is the level of evidence provided by this article?
———————————————————————————
Level V
What are the weaknesses and strengths of this article?
—————————————————————————
The strengths;
This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
The weakness ;
It is an experimental study .
I agree with your analysis of strengths and limitations, and summary of this article. I wish you could type headings and sub-headings as underline or in bold.
Please summarise this article.
Introduction
·Human polyomavirus BKPyV can reactivate in immunosuppressed people, particularly transplant patients, and it can lead to symptoms including viremia, viruria, ureteral stricture, nephropathy, and hemorrhagic cystitis.
·BKPyV gene integration has been found in urinary tract epithelial cell malignancies in recent investigations, indicating a potential carcinogenic impact.
Materials and methods
Cell culture
·HTB-9 and T24 were cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKPyV infection and inactivation
·BKPyV stocks were propagated in Vero cells from ATCC and viral lysates were made through three cycles of freezing and thawing, followed by inactivation at 100 °C for 10 min.
Animal model
·Male BALB/c nude mice were housed in sterile filter-capped cages and injected with T24 and HTB-9, including BKPyV-infected cells. Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen for histopathological examination.
Cell counting Kit-8 assay
·CCK-8 assay was used to assess cell viability by detecting the absorbance of the medium at 450 nm.
Colony-formation assay
·Colonies were seeded in six-well plates at 200 cells/well and stained with 4% paraformaldehyde and counted using light microscopy.
Cell migration and invasion assays
·Cells were seeded in 200 μL FBS-free medium, stained with methanol and 0.1% crystal violet, and counted using a light microscope. The mean cell number was calculated.
Scratch wound healing assay
·Cells were inoculated in six-well plates and cultured at 37 °C until 90% confluence, then washed, incubated, and continuously cultured in serum-free medium.
Histological assessment
·Hematoxylin and eosin (H&E) staining was used to assess the histological properties of tumor tissues.
Immunofluorescence staining
·Cell slides and frozen sections were diluted in PBS for 10 min and permeabilized with 0.2% Triton X-100 for 10 min at RT. Primary and secondary antibodies were diluted in blocking buffer and incubated at RT for 50 min each. Fluorescence was detected using an inverted fluorescence microscope.
Western blot
·Western blotting was used to separate membrane proteins from 5% milk and incubate them overnight with anti-β-catenin antibodies.
Statistical analysis
·Statistical analyses were performed using ANOVA for more than two groups with P < 0.05 considered significant.
Results
·BKPyV infection in bladder cancer cells is a non-lytic infection
·BKPyV infection promotes the proliferation, invasion, and migration of bladder cancer cells
·BKPyV infection enhances bladder tumor growth and metastasis
·BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
·Blocking β-catenin signal inhibits BKPyV infection- mediated enhancement of proliferation and migration
Discussion
·BKPyV infection and tumorigenesis have increased attention in immunocompromised individuals.
·BKPyV plays an important role in renal and urothelial cancers after transplantation.
·JCpyV can cause urothelial carcinoma, suggesting it is closely related to bladder cancer. When BKPyV infects cells, two different outcomes may occur:
1. Host cell allows replication of the virus, resulting in viral DNA amplification, progeny production, and cell lysis.
2. Host cell prevents virus replication, causing abortion of infection cell or cell transformation.
·BKPyV infections do not have a lytic effect on bladder cancer cells due to the continuous expression of LTag, which prevents the virus from copying normally.
·BKPyV can promote host cell transformation and immortalization, but its role in tumor cells is unknown.
·The BKPyV infection tumor cell model increased the proliferative capacity, migration, and invasion ability of bladder tumors..
·The ability to metastasize to other organs in vivo is a major threat to clinical patients.
·BKPyV infections can lead to liver metastases of bladder tumors, reducing survival rates.
·LTag inactivates tumor suppressors, leading to increased cell proliferation.
·STag increases MAP kinase activation, leading to cell proliferation and transformation.
·Blocking the β-catenin signaling pathway can be an effective treatment for BKPyV-infected bladder tumors.
Conclusions
·BKPyV infection promotes the proliferation, invasion, and migration of bladder cancer and bladder tumors.
·β-catenin signaling pathway and EMT effect in BKPyV-related bladder cancer.
·The results may help diagnose and treat BKPyV-related bladder cancer.
======================================================
What is the level of evidence provided by this article?
Level V
======================================================
What are the weaknesses and strengths of this article?
Weakness: the study was inducted on an animal model (mice). Experimental.
Strength: It first explained how BKPyV infection promotes bladder cancer to proliferate, invade, and migrate. It confirmed the function of the epithelial-mesenchymal transition and the β-catenin signaling pathway in BKPyV-induced bladder cancer.
Typing the whole sentence in bold amounts to shouting, however.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article.
Please summarise this article.
Introduction:
Urothelial carcinoma is the 6th common malignancy in males in the united states, prone tumor type of immunocompromised patients, kidney transplant recipients are 3 times more likely to have it than the general population.
BK polyomavirus (BKV), a virus reactivated in immunocompromised patients, causes symptoms such as viremia, viruria, ureteral stricture and BKV-related nephropathy, as well as hemorrhagic cystitis after hematopoietic stem cell transplantation, controversy among its role in urothelial cancers, by presence of T antigen, or absence of virus DNA in studies.
Some studies have shown that transplant recipients with BK viremia or polyomavirus-associated kidney disease have an increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKV.
The β-catenin pathway is involved in tumorigenesis, by cell proliferation and transcription, and regulating pattern formation during development, large T antigen binds directly to β-catenin, resulting in enhanced expression of β-catenin target genes such as c-myc and cyclin D.
Materials and methods
Cell culture: Human bladder cancer cell lines HTB-9 and T24 were obtained, and cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C.
BKV infection and inactivation: BKV propagated in Vero cells , and inactivated by heating to 100c for 10 minutes, both T24 and HTB-9 cells were grown in culture dishes.
Animal model:
Cultures of T24 and HTB-9, including their respective BKV infected cells were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg).
Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination.
Cell counting Kit-8 assay:
The Cell Counting Kit-8 assay was used to determine cell viability.
All observations were reproduced at least three times in independent experiments.
Colony-formation assay:
Cells were seeded in six-well plates, Colonies were clearly visible after 10– 14 days and selected cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 4 mg/mL crystal violet.
Colonies containing > 50 cells were counted using light microscopy.
Cell migration and invasion assays:
Transwell chambers, pre-coated with and without Matrigel were used to determine cell invasion and migration.
After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and crystal violet solution.
A light microscope was used to count the number of cells in five random fields of view. The mean cell number was calculated for each group.
Scratch wound healing assay:
Cells were inoculated in six-well plates and cultured at 37 °C in a 5% CO2 cell incubator.
After the cells reached 90% confluence, wounds of approximately 1 mm width were created using a sterile pipette tip.
Cells were washed, incubated and continuously cultured in serum free medium. Cultures at 0, 8, and 24 h were observed under an inverted microscope.
Histological assessment: Hematoxylin and eosin(H&E) staining was performed for histological assessments.
Immunofluorescence staining: Cell slides and frozen sections in 4% formaldehyde, and prepared using inverted fluorescence microscope.
Western blot: was carried out and membrane proteins were separated on polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.
Results:
Both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKV, BY promoting proliferation of bladder cancer cells in vitro, and higher migration rates than the controls.The growth rate of xenografts of BKV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group, pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream.In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced.β-catenin protein expression in xenografts of BKV-infected bladder tumor cells were significantly higher than in the control, indicating that BKV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells.BKV infected cells treated with KYA1797K had significantly reduced cell proliferation, migration, and invasion capacities, levels of Slug were significantly reduced while expression of Claudin-1 was significantly increased.
Conclusion:
BKV infection promotes the proliferation, invasion and migration of bladder cancer, expressing BKV related proteins were more invasive in vitro. The study verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKV-related bladder cancer, that may help clinical diagnoses and treatment of such tumors in future.
What is the level of evidence provided by this article?
Level of evidence V – animal experimental study.
What are the weaknesses and strengths of this article?
Weakness:
In vitro experimental study in mice.The role of BKV autoantigen expression in tumor cells has not been studied, and the biological characteristics of BKV-related tumors.Strength:
It is the first study to clarify the activation of β-catenin promotes the invasion and migration (metastasis) of urothelial tumor cells, so may help in treating such tumors by blocking β-catenin.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
Please summarise this article.
Introduction
Urothelial carcinoma is one of the most common and highly malignant tumours in the urinary system. Immunocompromised individuals are at high risk of development of bladder cancer. There is three fold higher risk in post transplant patients than general population. The results from animal studies have shown association between BKV and bladder cancer. This has not been proven in humans.
Methodology
The effect of BK virus was investigated by-
1- Cell culture- Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection
2-Animal Models- Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice
Results
BKPyV infection in bladder cancer cells is a non-lytic infection and promotes proliferation, invasion and migration of bladder cancer cells and enhances bladder tumor growth and metastasis.
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
Conclusion
This was the first study which suggested BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins
It defined role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer
What is the level of evidence provided by this article?
Experimental Animal study
Level of evidence V
What are the weaknesses and strengths of this article?
Strengths
the first study to identify the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer
Weaknesses
Study on animals
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
1. Please summarise this article.
Introduction
Urothelial cancer increase 3 folds in organ transplantation as compared to normal ppls
BKPyv activation after kidney transplantation may be linked to the development of urethelial cancer
But trials results was contradictory . In addition, some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKPyV. But till now pathogenesis is not clear
The Wnt/β-catenin pathway is implicated in cell proliferation and transcription, so inhibition of degradation in cytoplasm can induce epithelial-mesenchymal transition (EMT) and promote tumor genesis
Regarding the interaction between polyom a virus and β-catenin is clear in JVPYV but this relation still not clear in case of BKPyV
Materials and methods
Two models: BKPyV-infected bladder cancer cell model and a mice tumor model
1. BKPyV-infected bladder cancer cell model: cultured human bladder cancer cell lines HTB-9 and T24
2. Animal model: Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice (30 days)
Tumors were surgically dissected after 30 days for histopathological examination
Results
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells after infection by BKPyV, the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus)
Discussion
Conclusion
· BKPyV infection induce the proliferation, invasion and migration of bladder cancer
· BKPyV infection enhances β-catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) effect in bladder cancer cells
· When β-catenin treated with KYA1797K so become inactive signal inhibits BKPyV infection mediated enhancement of proliferation and migration
2-What is the level of evidence provided by this article?
Experimental study (level V)
3- What are the weaknesses and strengths of this article?
Strength: the first study proved that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells via activation of β-catenin signaling pathway
Weakness: experimental (animal study)
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
I agree with your brief analysis.
I wish you could type headings and sub-headings as underline or in bold.
1-Please summarise this article.
Introduction;
-Urothelial carcinoma is one of the most common and highly malignant tumors in the urinary system.
-According to 2018 statistics from the American Cancer Society, bladder cancer is the sixth most common malignancy in males after lung, prostate, colorectal, stomach and liver.
-Bladder cancer is also a prone tumor type for immunocompromised patients. According to research, kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
-BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised populations, especially transplant recipients.
-BKPyV reactivation after renal transplantation causes symptoms such as viremia, viruria, ureteral stricture and BKPyV-related nephropathy, as well as hemorrhagic cystitis after hematopoietic stem cell transplantation.
BKPyV infection enhances bladder tumor growth and metastasis;
-The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days post inoculation.
-Differences in tumor volume became more pronounced over time.
-On day 30 post inoculation, mice were euthanized in order to observe tumor cell invasion and metastases.
-New tumors were discovered in the liver of BKPyV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice.
-Pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream.
Discussion;
-Tumors have gradually become one of the main factors affecting the long-term survival of kidney transplant recipients.
-In recent years, the correlation between BKPyV infection and tumorigenesis in immunocompromised individuals has drawn increased attention.
-The idea that BKPyV plays an important role in cancers of the urinary system was recently confirmed by deep sequencing studies, which confirmed that the BKPyV gene was integrated into the genome of renal cancer and urothelial carcinoma cells after transplantation.
-When BKPyV infects cells, two different outcomes may occur:
(1) the host cell allows replication of the virus, resulting in viral DNA amplification, progeny virion production and cell lysis; or,
(2) the host prevents virus replication, and the persistent expression of the LTag causes abortion of infection cell or cell transformation. This result is determined by the continuous expression of LTag.
Conclusions;
-BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV-related proteins were more invasive in vitro.
-The role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer is verified.
-These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer.
2-What is the level of evidence provided by this article?
( The level V evidence )
3-What are the weaknesses and strengths of this article?
Weakness;
–This study is an experimental animal study with small groups,
–Use of xenograft for this study which not correlate with human findings.
Strength;
-This study is the first study to verify the role of activation of β-catenin signaling pathway by BK virus in the pathogenesis of cancer bladder.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer.
Introduction.
Kidney transplant recipients are three times more likely to have urothelial cancer than the general population, early studies detected the presence of BKPyV large T antigens in prostate, bladder- and kidney tumors but still BKPyV as a cause of malignancy is still controversial.
BKPyV reactivation in immunosuppressed environments may be considered as a transforming factor leading to urothelial carcinomas, specially some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKPyV.
The Wnt/β-catenin pathway is implicated in cell proliferation, transcription, and regulating pattern formation during development. This pathway is involved in tumorigenesis and is associated with many biological behaviors such as tumor proliferation, invasion, and metastasis, BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation, and so this study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and EMT in this process.
Materials and methods.
Cultures of T24 and HTB-9, including their respective BKPyV infected cells (106 cells in 100 mL PBS) were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg) (n = 5). Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination, this model to discuss the role
of BKPyV infections in cancer development.
Results.
1-BKPyV infection in bladder cancer cells is a non-lytic infection but continued to proliferate and grow for 9 days after the initial infection.
2-BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells and T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates than the controls.
3-BKPyV infection enhances bladder tumor growth and metastasis.
4-BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells.
In vitro, when compared to the control, the expression of β-catenin, cMYC and Slug proteins in BKPyV infected cells were significantly increased, while the expression of Claudin-1 protein was significantly reduced in vivo, β-catenin protein expression in xenografts of BKPyV-infected bladder tumor cells were significantly higher than in the control. These results indicate that BKPyV infection promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells.
5-Blocking β-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration.
Conclusions.
Try to prove that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyV related proteins were more invasive in vitro in the animal models, also demonstrated the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer.
Strength point:
Is considered the experimental gate to discuss these issues that prove the role of BKPyV infection promotes the proliferation, invasion and migration of bladder cancer.
Weakness point:
It is depends on experimental results on animal .
Not studies the association of other viral infections on the proliferation, invasion and migration of bladder cancer.
Level of evidence: V (Animal and laboratory research).
Typing the whole sentence in bold amounts to shouting, however.I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Introduction
· This study explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and epithelial-Mesenchymal transition (EMT) in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Materials and methods
They developed a BKPyV-infected bladder cancer cell model and a mice tumor model to study the role of BKPyV infections in bladder cancer and that activation of β-catenin signaling pathway is one of its mediation mechanisms.
Results
Discussion
Conclusions
The level of evidence: is 5
Weakness of this article: Being an animal (with physiology different from humans) study make the results cannot be accurately related to humans and invalid.
Strength:
☆ BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
◇ Introduction:
▪︎Kidney transplant recipients are three times more likely to have urothelial cancer than the general population.
▪︎BK polyomavirus (BKPyV) is a human polyomavirus prone to reactivation in immunocompromised populations, especially transplant recipients.
▪︎ BKPyV reactivation after renal transplantation causes symptoms such as viremia, viruria, ureteral stricture and BKPyV-related nephropathy.
▪︎Many recent studies detected BKPyV gene integration in UT tumors, providing further evidence for the correlation between BKPyV and urinary tract tumors.
▪︎BKPyV reactivation in immunosuppressed environments may be considered as a transforming factor leading to urothelial carcinomas.
▪︎Some studies have shown that transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease have an increased risk of bladder cancer
when compared to transplant recipients without BKPyV.
▪︎BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen.
The role BKPyV infections play in the biological function of bladder cancer is still unclear.
◇ Methods:
The authors have developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections.
◇ Results:
The research proves that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells, while the activation of β-catenin signaling pathway is one of its mediation mechanisms.
◇ Conclusions:
▪︎The authors first described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer. They verified the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyVinfected bladder cancer.
▪︎These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
What is the level of evidence provided by this article?
Weaknesses of the study:
Strengths of the study:
◇ What is the level of evidence provided by this article?
Level of evidence: V (an experimental animal study).
◇ What are the weaknesses and strengths of this article?
¤ Weakness: this an animal study which has some limitations
¤ The strength of the study:
• The first study of its kind to investigate how the stimulation of the beta-catenin signaling system by the BKV add to the development of the bladder cancers.
•The results of this study could provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer
Summary of this article:
The Objective of the current study:
Materials and methods:
Results:
BKV infection of bladder cancer cells:
BKV infection promotes proliferation, invasion and migration of bladder cancer cells
BKV infection enhances bladder tumor growth and metastasis:
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells:
Blocking β-catenin signal inhibits BKV infection mediated enhancement of proliferation and migration.
Conclusions:
2. level of evidence => V (experimental animal study)
3. What are the weaknesses and strengths of this article?
Strength:
This is the first study to describe that BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and that bladder tumors expressing BKPyV related proteins were more invasive in vitro.
Weakness:
It is an animal study which is poorly correlated with human.
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
Introduction
Urothelial carcinoma is one of the most common malignant tumors in the urinary system.
BKPyV and JCPyV were classified as “possibly” carcinogenic. The underlying role of BKPyV in the development of cancer was controversial.
Aim of the study:
-Explored the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and explore the role of Wnt/β-catenin pathway and EMT in this process by establishing a model for BKPyV infections in bladder cancer cells and mice.
Materials and methods
Theye developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role
of BKPyV infections.
Cultures of BKPyV infected human bladder cell were injected subcutaneously into nude mice
Cell migration and invasion assessed by incubating the cells in Transwell chambers pre-coated with and without Matrigel
Results:
– BKPyV infected bladder cells did not lyse due to BKPyV infections, they express T antigen and continued to proliferate and grow, the cells proliferation ability and activity in infected bladder cancer cells were significantly increased compared to cells not infected.
– BKPyV infections promote invasion and migration of bladder cancer cells in vitro.Also, it promotes β-catenin signaling activation and Epithelial-Mesenchymal Transition (EMT) effects in bladder cancer cells.
Conclusions:
-BKPyV infection promotes the proliferation, invasion and migration of bladder cancer. while the activation of β-catenin signaling pathway and Epithelial-Mesenchymal Transition is one of its mechanisms
-These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer
Limitation:
Animal study model and the results may not be applied in humans studies.
Strength:
The first study to explore the role of B catenin signaling pathway in development of bladder cancer, inhibition of this pathway may provide promise for future treatment.
Level of evidence: level V animal study
I appreciate your views on carcinogenic potential of BKV. I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
III.BK polyomavirus infection promotes growth and aggressiveness in bladder cancer.
====================================================================
Please summarise this article.
Introduction
====================================================================
Materials and methods
====================================================================
Results
====================================================================
Discussion
====================================================================
Conclusions
====================================================================
What is the level of evidence provided by this article?
The level of evidence is V
====================================================================
What are the weaknesses and strengths of this article?
Strength:
According to the first study, BKPyV infection increases the chance of bladder cancer invasion and migration.
weakness
The study has weaknesses because it was conducted on animals.
I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
Many thank Prof.Sharma
BK polyomavirus infection promotes growth and aggressiveness in bladder cancer;
Introduction:
Urothelial carcinoma is the most common and highly malignant tumor in the urinary system.
American Cancer Society statistics in 2018; (Bladder cancer is the 6th most common malignant tumor in males after lung, prostate, colorectal, stomach, and liver.
Immunocompromised patients is the risk factors of bladder cancer, and the urothelial cancer is 3 times more prevalent than in general population.
In 2012 at the international Agency for Research on Cancer (IARC) meeting; BKPyV and JCPyV were classified as possibly carcinogenic to human, as there is a sufficient evidence in experimental animals and inadequate evidence in humans for their carcinogenicity.
Early studies detected the presence of BKPyV large T antigen in prostate, bladder, and kidney tumors, but several researchers thought BKPyV was unlikely to be involved in the etiology of most renal and bladder tumors because they had failed to detect BKPyV DNA in the cancer samples.
Many recent studies detected BKPyV gene integration in urinary tract epithelial cell tumors, providing further evidence for the correlation between BKPyV and urinary tract tumors.
Interestingly, these changes occur in post transplant tumor, but not in non-transplant tumors, therefore BKPyV considered as a transforming factor leading to urothelial carcinoma.
Some studies, show that recipient with BKPyV viremia or polyomavirus-associated kidney disease has an increased risk (4-11 times) of bladder cancer, when compared to to transplant recipients without BKPyV, without full clear evidence of te underline role.
BKPyV is thought to be oncogenic due to the expression of the early coding region encoded proteins LTag and small T antigen (STag) which can initiate or derive neoplastic transformation.
Results:
BKPyV infection in bladder cancer cells is a non-lytic infection;
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells:
BKPyV infection enhances bladder tumor growth and metastesis;
BKPyV infection enhances B-catenin signaling pathway activation and epithelial -Mesenchymal transition(EMT) effect in bladder cancer cells.
Conclusion:
Level of evidence:
Level ((V)).
Strtength of the Article:
Expermental studies.
Weakness of the article:
Small group and case study
All result are mostly an association rather than clear direct effects
I agree with your analysis of strengths and limitations, and summary of this article. Typing the whole sentence in bold amounts to shouting, however.
1. Please summarise this article.
Introduction
The study
Materials and methods
Results:
Conclusions:
============================
2. What is the level of evidence provided by this article?
============================
3. What are the weaknesses and strengths of this article?
Weaknesses:
Strengths:
I agree with your analysis of strengths and limitations, and summary of this article
I- Summary:
Objectives: to explored the effects of BKV infections on the proliferation and migration of bladder cancer cells and the role of β-catenin pathway and endothelial mesenchyme transition (EMT) in this process by establishing a model for BKV infections in bladder cancer cells and mice.
Methods:
Results:
BKV infection of bladder cancer cells:
BKV infection promotes proliferation, invasion and migration of bladder cancer cells
BKV infection enhances bladder tumor growth and metastasis:
BKPyV infection enhances β-catenin signaling pathway activation and epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells:
Blocking β-catenin signal inhibits BKV infection mediated enhancement of proliferation and migration.
Conclusions:
II- level of evidence: V
III- strength of the study:
The first study to explore the underlying mechanism of the BKV induced bladder
cancer cell pathway.
IV- weakness:
It is an animal study which is poorly correlated with human.
I agree with your analysis of strengths and limitations, and summary of this article
Introduction:
Aim of the study:
Result & discussion:
Level of evidence is 5.
Strength :
Limitation:
I agree with your analysis of strengths and limitations, and summary of this article
SUMMARY
Introduction
Bladder cancer is the sixth most common malignancy in males according to the 2018 American Cancer Society. Furthermore, bladder cancer has been observed to be common among those that are immunocompromised like those with kidney transplants that are three times more common to have uroepithelia tumours than the general population.
The reactivation of BKPyv after kidney transplantation is noticed with accompanying symptoms like viremia, viruria, ureteral stricture, and hemorrhagic cystitis among those with hematopoietic stem cell transplants. However, there is still a debate if BKPyV has a causal role in the development of urinary tract malignancy, although early studies detected the presence of BKPyV large T antigens in prostate-, bladder, and kidney tumours. Also, several researchers thought BKPyV was unlikely to be involved in the etiology of most renal and bladder tumors because they had failed to detect BKPyV DNA in the cancer samples.
Materials and Methods
Results
Discussion
Conclusion
The outcomes of the study in identifying BKPyV infection’s role in causing urinary tract cancer could aid in the identification and proper treatment of cancer resulting from the infection.
The level of evidence is 5
Strength of the study
The molecular explanation of the aetiopathogenesis of the role of BKPyV in causing urinary bladder cancer
Limitations of the study
I agree with your analysis of strengths and limitations, and summary of this article
Please summarise this article.
Introduction
Kidney transplant recipients are three times more likely to have urothelial cancer than the general population
In 2012, BK polyomavirus (BKPyV) and JCPyV were classified as “possibly” carcinogenic to human
The causal role of BKPyV infections in the biological function of bladder cancer remains unclear
Aim of the study: discuss the effects of BKPyV infections on the proliferation and migration of bladder cancer cells and the role of Wnt/β-catenin pathway and epithelial-mesenchymal transition (EMT)
Materials and methods
Two models: BKPyV-infected bladder cancer cell model and a mice tumor model
1. BKPyV-infected bladder cancer cell model: cultured human bladder cancer cell lines HTB-9 and T24
2. Animal model: Cultures of T24 and HTB-9, including their respective BKPyV infected cells were injected subcutaneously into nude mice (30 days)
Tumors were surgically dissected after 30 days for histopathological examination
Results
BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells when compared to cells not infected with BKPyV (inactivated BKPyV virus). The activation of β-catenin signaling pathway is one of its mediation mechanisms
Conclusions
· BKPyV infection promotes the proliferation, invasion and migration of bladder cancer
· BKPyV infection enhances bladder tumor growth and metastasis
· BKPyV infection enhances β-catenin signaling pathway activation and EMT effect in bladder cancer cells
· Blocking β-catenin signal inhibits BKPyV infection mediated enhancement of proliferation and migration (treated with KYA1797K)
What is the level of evidence provided by this article?
Experimental study (level V)
What are the weaknesses and strengths of this article?
Weakness: experimental (animal study)
Strength: the first study proved that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells via activation of β-catenin signaling pathway
I agree with your analysis of strengths and weaknesses, and summary of this article
Introduction
Urothelial carcinoma is a frequent and fatal urinary malignancy. Bladder cancer ranks sixth among male malignancies after lung, prostate, colorectal, stomach, and liver, according to 2018 American Cancer Society data.
BKPyV and JCPyV were classified as “probably” carcinogenic to humans at the 2012 IARC meeting due to “sufficient evidence” in experimental animals and “inadequate evidence” in humans.
This study established a model for BKPyV infections in bladder cancer cells and mice to investigate the impact of BKPyV on bladder cancer cell proliferation, migration, and Wnt/β-catenin pathway and EMT. These findings will illuminate BKPyV-related bladder cancers, paving the way for clinical studies.
Methods
A BKPyV-infected bladder cancer cell model and a mouse tumor model were developed to discuss the role of BKPyV infections.
Results
In bladder cancer cells, BKPyV infection is a non-lytic infection.
Infection with BKPyV enhances the growth, invasion, and migration of bladder cancer cells.
Infection with BKPyV promotes bladder tumor growth and metastasis.
In bladder cancer cells, BKPyV infection increases -catenin signaling pathway activation and epithelial-mesenchymal transition (EMT) impact.
Blocking β-catenin signal inhibits BKPyV infectionmediated enhancement of proliferation and migration.
Discussion
The BKPyV gene was found to be integrated into the genomes of renal cancer and urothelial carcinoma cells after transplantation, in agreement with a previous study that demonstrated a link between human polyomavirus and bladder cancer.
When BKPyV infection occurs, the host cell permits virus reproduction and cell lysis; alternatively, the host resists virus replication; nonetheless, prolonged expression of the LTag can result in cell transformation or abortive infection.
Unstudied was the function of BKPyV autoantigen expression in tumor cells.
Bladder tumors frequently spread to the liver following a BKPyV infection.
LTag are prooncogenic as a result of the inactivation of tumor suppressor proteins such as p53 and retinoblastoma protein (pRb) and the enhancement of the mitogen-activated protein kinase (MAP kinase) pathway, which results in enhanced cell proliferation.
The Wnt/-catenin signaling pathway facilitates tumor invasion and dissemination.
Blocking -catenin signaling pathways can inhibit the action of BKPyV.
This discovery can be utilized to treat infectious BKPyV bladder cancers.
In conclusion, bladder tumors expressing BKPyV-related proteins were more invasive in vitro. BKPyV infection enhances the proliferation, invasion, and migration of bladder cancer. The role of β-catenin signaling pathway and EMT effect was verified in the features of BKPyV-related bladder cancer.
These findings may aid in the clinical diagnosis and treatment of bladder cancer caused by BKPyV.
I like summary of this article
1-Please summarise this article.
Introduction
Many recent studies detected BKPyV gene integration in urinary tract epithelial cell tumors
BKPyV reactivation in immunosuppressed considered as a transforming factor leading to urothelial carcinomas. increased risk (4–11 times) of bladder cancer when compared to transplant recipients without BKPyV
BKPyV is thought to be oncogenic due to the expression of the early coding region-encoded proteins LTag and small T antigen (STag), which can initiate or drive neoplastic transformation. Whether constitutive activation of the Wnt/β-catenin signalling pathway by BKPyV LTag induce cancer remains to be established
AIM OF STUDY (YZ and TZ designed the experiments)
BKPyV infection promotes the proliferation, invasion and migration of bladder cancer and bladder tumors expressing BKPyVrelated proteins were more invasive in vitro. We verified the role of β-catenin signaling pathway and EMT effect in the characteristics of BKPyV-related bladder cancer. These results may help clinical diagnoses and treatment of BKPyV-related bladder cancer
RESULTS
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells after infection by BKPyV, the cells proliferation ability and activity in both T24 and HTB-9 bladder cancer cells were significantly increased compared to cells not infected with BKPyV (inactivated BKPyV virus)
Colony formation experiments further showing that BKPyV infections promote proliferation of bladder cancer cells in vitro
Transwell migration results showed that T24 and HTB-9 cells infected with BKPyV had significantly higher migration rates than the controls
The growth rate of xenografts of BKPyV-infected bladder tumor cells in mice increased significantly, with the tumor volume of the two cell lines being significantly larger than that of the control group at 15 days post inoculation.
Differences in tumor volume became more pronounced over time On day 30 post inoculation, mice were euthanized in order to observe tumor cell invasion and metastases. New tumors were discovered in the liver of BKPyV infected bladder tumor mice, while no tumor tissue was observed in other organs of the control mice . Pathological morphology revealed that tumor tissue present in the livers were characteristic of urothelial carcinoma, identified as bladder tumor cells transported by the blood stream
2-What is the level of evidence provided by this article?
Should they be regarded as being similar to “bench studies” and thus be viewed as a Level 5 (CEBM) study, or given a lower number (for example 1b or 2b), or none at all?
3-What are the weaknesses and strengths of this article?
main weakness of animal studies is that animals have a different physiology to humans. This means that any studies on animals cannot be accurately related to humans, making them invalid.
STRENGHT
The choosing of good control groups are useful.
the formulation of new questions to be further responded is expected.
I agree with your analysis of strengths and weaknesses, and summary of this article
THANKS FOR YOUR SUPPORT
Prof. Ajay Sharma
summarise this article
Introduction:
Urothelial carcinoma is a frequent and deadly urinary malignancy. According to 2018 American Cancer Society data, bladder cancer is the sixth most prevalent male malignancy after lung, prostate, colorectal, stomach, and liver. Immunocompromised people can develop bladder cancer. Research shows that kidney transplant patients are three times more likely to develop urothelial carcinoma.
Methods:
In order to investigate the possible effects of BKPyV infections, we created a bladder cancer cell model that was infected with BKPyV as well as a mouse tumor model.
Sterile filter-capped cages contained male BALB/c nude mice (5 weeks, 18–20 g; Shanghai LC Laboratory Animal Co. Ltd., Shanghai, China). T24 and HTB-9 cultures, comprising BKPyV-infected cells (106 cells in 100 mL PBS), were administered subcutaneously into nude mice with and without intraperitoneal KYA1797K (25 mg/kg) (n = 5).
Thirty days after implantation, tumors were surgically excised and frozen in liquid nitrogen before histological evaluation.
Results:
The findings of our study demonstrate that BKPyV infections encourage the proliferation, invasion, and migration of bladder cancer cells, with the stimulation of the β-catenin signaling pathway serving as one of the mediation mechanisms responsible for this effect.
conclusion :
that BKPyV infection is responsible for promoting the proliferation, invasion, and migration of bladder cancer. In BKPyV-infected bladder cancer, we were able to confirm the function that the -catenin signaling pathway and the Epithelial-Mesenchymal Transition affect play. These findings contribute significantly to our understanding of how best to diagnose and treat patients with bladder cancer in clinical settings.
What is the level of evidence provided by this article?
It is an experimental animal study.
Level of evidence 5
What are the weaknesses and strengths of this article?
The strength: is that this is the first research of its kind to investigate how the stimulation of the beta-catenin signaling system by the BK virus contributes to the development of cancer in the bladder.
I agree with your analysis of strengths and weaknesses, and summary of this article
1- Summary
Introduction
Bladder cancer is the 6 th most common malignancy in males with high prevalence in immunocompromised cases.
Kidney transplant recipients are three times more susceptible to have urothelial cancer compared to the general population.
BKPyV reactivation can lead to BKPyV-related nephropathy in renal transplantation and haemorrhagic cystitis after hematopoietic stem cell transplantation .
International Agency for Research on Cancer (IARC) meeting at 2012 stated that , BKPyV and JCPyV
can be possibly carcinogenic to human but this is controversial .
Some studies declared that BKPyV DNA wasnot detected in the cancer samples while others mentioned that BKPyV gene integration was noticed in the urinary tract epithelial cell tumor
A study revealed that cancer bladder risk is higher in transplant recipients with BKPyV viremia or polyomavirus-associated kidney disease compared to transplant recipients without BKPyV.
The Wnt/β-catenin pathway and inhibition of β-catenin degradation in cytoplasm has a role in tumorigenesis and tumor proliferation, invasion, and metastasis .
BKPyV could be oncogenic because of expression of early coding region-encoded proteins
LTag and small T antigen (STag), which can stimulate neoplastic transformation.
this study aimed at evaluating BKPyV infections effect on the occurrence of bladder cancer .
Methods
BKPyV-infected bladder cancer cell model and a mice tumor model was created to evaluate BKPyV infections role.
Results
BKPyV infection in bladder cancer cells is a non-lytic infection with large T protein expression
of BKPyV and proliferation of cancer cells with time .
BKPyV infection promotes proliferation, invasion and migration of bladder cancer cells in vitro.
BKPyV infected T24 and HTB-9 cells had significantly higher invasive potentials than non-infected cells.
BKPyV infection promoted bladder tumor growth and metastasis.
BKPyV infection activated β-catenin signaling pathway and increased epithelial-Mesenchymal transition (EMT) effect in bladder cancer cells
Blocking β-catenin signal inhibits BKPyV infection mediated augmentation of proliferation and migration.
Discussion
Studies declared that BKPyV gene was integrated into the genome of renal cancer and
urothelial carcinoma cells after transplantation in accordance with another study that showed that human polyomavirus can be related to cancer bladder.
When BKPyV infection occur the host cell allows replication of the virus and cell lysis; or the host prevents virus replication , but persistent expression of the LTag can lead to abortion of infection cell or cell transformation.
The role of BKPyV autoantigen expression in tumor cells was not studied.
After BKPyV infections, bladder tumors tend to metastasis to the liver .
LTag are prooncogenic through inactivation of tumor suppressor proteins as p53 and retinoblastoma protein (pRb), and enhances mitogen activated protein (MAP) kinase pathway leading to increased cell proliferation.
Wnt/β-catenin signaling pathway enhances tumor invasion and metastasis.
Blocking β-catenin signaling pathways can suppress BKPyV’s function.
Such finding can be used for treatment of infectious BKPyV bladder tumors.
Conclusion
BKPyV infection enhances the proliferation, invasion of cancer bladder .
β-catenin signaling pathway and EMT effect have a role in BKPyV-related bladder cancer affecting diagnosis and therapy of cancer bladder.
2-level of evidence is V ;as it is conducted on animals without human enrollment with high possibility of bias
3- Strength is that it is a pioneer study conducted to access BKV role in cancer bladder involving the role of β-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV infected bladder cancer.
Limitation is using animal tumor model which varies from human models and tumor cells therefore some results may not be applicable for humans
I agree with your analysis of strengths and limitations, and summary of this article
Please summarise this article.
Results
Conclusion
What is the level of evidence provided by this article?
What are the weaknesses and strengths of this article?
I agree with your analysis of strengths and weaknesses, and summary of this article
1.Please summarize this article.
Introduction:
Methodology:
Results:
Conclusion:
2.What is the level of evidence provided by this article?
3.What are the weaknesses and strengths of this article?.
I agree with your analysis of strengths and weaknesses, and summary of this article
Urothelial carcinoma is three times as common in kidney transplant patients than in the general population.
The classification of BKPyV and JCPyV is as “probably” human carcinogenic.
There is a clear association between BKPyV and urinary tract malignancies since several recent research have found BKPyV gene integration in these tumours.
Direct binding of JCPyV large T antigen (LTag) to beta catenin results in increased expression of beta catenin target genes such c-myc and cyclin D1.
Epithelial-mesenchymal transition (EMT) and cancer can both be induced by inhibiting beta catenin breakdown in the cytoplasm.
The early coding region-encoded proteins LTag and small T antigen (STag), which can start or fuel neoplastic transformation, are expressed by BKPyV and are presumed to be carcinogenic.
By deep sequencing research, which demonstrated that following transplantation, renal malignancy and urothelial carcinoma cells have the BKPyV gene incorporated into their genomes.
After transplant, JCpyV may result in urothelial cancer.
There are two possible consequences when BKPyV infects cells: either the host cell permits virus reproduction, leading to viral DNA amplification, progeny virion generation, and cell lysis; or the host suppresses virus replication.
BKPyV causes unusually high levels of LTag expression in the host cell when it is triggered in vivo, which results in cell transformation.
The BKPyV LTag and STag can encourage host cell immortalization and transformation, which increases the ability of cells to proliferate.
BKPyV infection tumour cell model, we were able to confirm that bladder tumours expressing BKPyV-related proteins had further improved proliferative, migratory, and invasion abilities in vitro.
Due to their capacity to deactivate tumour suppressor proteins including p53 and pRb, which promote enhanced cell proliferation, LTag are prooncogenic.
STag has been demonstrated to boost MAP kinase pathway activation, which may potentially promote cell proliferation and transformation.
A combination of beta catenin and the transcription factor family Tcf / Lefs in the nucleus of tumour cells can activate genes like cMYC, leading to cell proliferation and EMT effects.
By interacting with beta catenin, JCPyV big T antigen can promote the expression of beta catenin target genes.
BKPyV infections encourage the activation of the beta catenin signalling pathway and the consequences of EMT.
The overexpression of LTag in these BKPyV-infected tumour cells may be attributed to catenin activation.
Such -catenin activation facilitates the invasion of malignant cell even further and also their migration and can increase cMYC expression to encourage cell growth.
BKPyV-infected bladder tumour cell xenografts are susceptible to distant metastases.
level 5 evidence
strength –
by showing the role played. by beta catenin in future agents which block its function can be developed as a part of therapy.
limitation –
findings in animal studies do not corelate with findings in the human studies.
I agree with you analysis, summary and level of evidence this article provides.
You should type headings and subheadings in underline or bold to make it easier to read.
I agree with you analysis of strengths and weaknesses of this article.