膀胱灌注azacitidine經由TNF-R1與TRAIL-R2信號路徑抑制基因毒性致癌物誘導之膀胱腫瘤生成

吳旻祐1、余家瑩1、張雅娟1、王紹全1,2、陳順郎1,2、宋文瑋1,2,*

1中山醫學大學醫學系；2中山醫學大學附設醫院泌尿科

Intravesical instillation of azacitidine suppresses tumor formation through TNF-R1 and TRAIL-R2 signaling in genotoxic carcinogen–induced bladder cancer

Min-You Wu1, Chia-Ying Yu2, Ya-Chuan Chang2, Shao-Chuan Wang1,2, Sung-Lang Chen1,2, Wen-Wei Sung1,2,*

1School of Medicine, Chung Shan Medical University, Taichung, Taiwan, 40201; 2Department of Urology, Chung Shan Medical University Hospital, Taichung, Taiwan, 40201; *Corresponding author

Purpose:

Urinary bladder urothelial carcinoma (UBUC) is the most prevalent type of bladder cancer. Azacitidine, an inhibitor of DNA methylation, has potential therapeutic effects against several malignancies. Azacitidine has an apoptosis-inducing effect and inhibits the proliferation of tumor cells. However, the anti-tumor effects of azacitidine on UBUC, especially following intravesical instillation (IVI), remain to be identified.

Materials and Methods:

The UBUC cell lines T24 and UMUC3 were used for cell assays (MTT, colony formation, and wound healing) and cell cycle analysis via flow cytometry to analyze the effects of azacitidine. Potential signaling pathways were investigated by protein arrays and western blotting. The N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat UBUC model was used for in vivo studies. The Ki67 index was used for quantitative analysis. The statistical significance of differences was determined by Student’s t test; p < 0.05 was considered statistically significant.

Results:

Azacitidine significantly inhibited DNMT expression in UBUC cell lines. MTT and colony formation assays showed significant reductions in cell viability and clonogenic activity in both cell lines treated with azacitidine. Significant cytotoxic effects, namely increased sub-G1 and Annexin V-PI populations (all p < 0.05), were seen following azacitidine treatment. The potential signaling pathways were identified with protein arrays, and the suppression of TNF-R1 and induction of TRAIL-R2 and their downstream signaling molecules were confirmed in vitro. TNF-R1 suppression reduced claspin and survivin expression, while TRAIL-R2 activation induced cytochrome C and caspase 3 expression. Rats with BBN-induced bladder cancer receiving IVI of azacitidine had significantly less tumor burden and a reduced Ki67 index (p<0.05).

Conclusion:

Our study provided evidence for a reduction in BBN-induced bladder cancer through the TRAIL-R2 and TNF-R1 signaling pathways in response to IVI of azacitidine. This therapy might provide new insights for further clinical trials.