Bladder Tissue Special Oncolytic Adenovirus Influence Bladder Cancer Stem Cells’ Viability

Objective: Non- muscle invasive bladder cancer is mainly treated with TURBt or chemotherapy methods, but after a period of treatment, approximately 50% patients experience disease recurrence, and 30% patients progressed to muscle- invasive bladder cancer in 5years. Bladder tumor stem cells are a small number of cells present in bladder cancer that possess the potential of self-renewal, proliferation, and cell differentiation. Bladder tumor stem cells are closely related to chemotherapy resistance, relapse and progression of bladder cancer. We use ALDEFLUOR kit to get ALDH(+) bladder cancer cells which was called “bladder cancer stem cells” by FACS. O ur study intended to detect cancer stem sells’ CAR and CD46, and research the effect of bladder tissue special oncolytic adenovirus in Bladder Cancer Stem Cells. Methods: The bladder cancer cells T24 and EJ were cultured. Bladder cancer stem cells were assayed for aldehyde dehydrogenase(ALDH) activity using the Aldefluor kit(Stem Cell Technologies) according to the manufacturer’s instructions.,and continue to culture ALDH(+) cells a week. FACS was performed on a Beckman Moflo XDP FACS cell-sorting system. At firat, Colony formation assay was used to estimate bladder cancer stem cells’ proliferation ability, we added 40 uM cispatin, 20ng/ml EGF, 20ng/ml bFGF to bladder cancer cells, culture them in FBS-free medium. 96 h later, we detect ALDH in survival cells. C hemoresistance, cell proliferation, cell differentiation and self- renew assays were used to identify sorted bladder cancer cells’ stem cell characteristics. The second, we extracted both usual bladder cancer cells and bladder cancer stem cells’ RNA, and q RT-PCR amplification of C AR, C D46 genes was performed according to the manufacturer’s instructions. GAPDH is the housekeeping gene. Position and protein expression levels of CAR and CD46 were dectated by immunocytochemistry. MTT viability assay was used to detect bladder cancer stem cells’ survival rate after infecting with bladder tissue special oncolytic adenovirus. Result: There are 15% bladder cancer cells are cancer stem cells. Compared with normal bladder cancer cell, bladder cancer stem cells’ ability in coloney formation, self-renew, proliferation and chemo-resistant are obviousely strong. Both in gene and protein level, the expression of CD46 in ALDH(+) and ALDH(–) sorted cells hadn’t show any diversity, but the expression of CAR is lower in bladder cancer stem cell. What’s more, the expression of CD46 is higher than CAR in bladder cancer stem cells. However, a high does of MOI 80, after 48 h, Ad-PSCAE-UPⅡ-E1A-AR bladder cancer specific oncolytic adenovirus can induce cancer stem cells’ sputtering and apoptosis. Conclusion:ALDH(+)bladder cancer stem cells has such cancer stem cells’ ability as chemo-resistance, self-renew, and differentiation, it can be a candidate of biomarker. While, adding a dose of MOI 80 bladder tissue special oncolytic adenovirus to bladder cancer stem cells, 48 h later, half of cells were killed, indicates its intervention effect to some extent.