蔡德甫^1,4#、林致凡^2#、楊尚哲²、陳宏恩¹、林宜佳^1,4、仇光宇^1,4、黃一勝^1,3,4

¹新光醫院 外科部 泌尿科，²新光醫院 中央研究室， ³台北醫學大學 醫學院，⁴輔仁大學 醫學院

Te-Fu Tsai ^1,4#、Ji-Fan Lin^2#、Shan-Che Yang²、Hung-En Chen¹、Yi-Chia Lin^1,4、Kuang-Yu Chou^1,4、Thomas I-Sheng Hwang^1,3,4

¹Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital,

²Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital,

 ³ School of Medicine, Taipei Medical University, 

⁴School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.

# These authors contributed equally to this work.

Purpose: Benzyl isothiocyanate (BITC) is contained in cruciferous plants which are part of the human diet, and has been shown to induce apoptosis in varies cancer cell lines including human bladder cancer cells. In our previous work, we showed that BITC induces protective autophagy via inhibition of mTOR signaling pathway in prostate cancer cells. And the induced apoptosis and autophagy in human prostate cancer cells were mediated by BITC-induced ROS generation. Here we investigated weather inhibition of autophagy enhances apoptosis in human bladder cancer cells as a novel therapeutic strategy.

Materials and Methods: Human bladder cancer cell lines including grade II, 5637, and grad III, T24, were used in this study. We also included mouse bladder cancer cell line, MBT2, for the development of orthotopic mice bladder cancer model for further study. Bafilomycin A1 (Baf A1) and chloroquine (CQ) were used as autophagy inhibitors. Cell viability in BITC-treated cells with or without the pretreatment of autophagy inhibitors were measured by WST-1 reagent. Detection of autophagy was conducted by measuring the LC3-II processing and the accumulation of p62 by Western blotting and immunofluorescent staining of these marker protein. Detection of apoptosis was performed by the monitored the caspase 3/7 activity, Western blot of cleavage caspase3 and cell flowcytometry of DNA fragmentation in treated cells. Detection of autophagy was conducted by measuring the LC3-II processing and the accumulation of p62.

Results: Exposure of 5637, T24 and MBT2 cells to pharmacologic concentrations of BITC resulted in the decrease of cell viability and autophagy induction. Although inhibition of basal autophagy by Baf A1 or CQ alone caused cell viability loss in these bladder cancer cell lines, pre-treatment of Baf A1 or CQ to inhibit BITC-induced autophagy further enhanced the inhibition of cell growth. Enhanced apoptosis judged by the increased caspase 3/7 activity, increased amount of cleaved caspase 3 and elevated level of DNA fragmentation in BITC-treated cell with Baf A1 or CQ pretreatment suggesting inhibition of autophagy significantly potentiates the anti-cancer effect of BITC in human bladder cancer cells.

Conclusion: This is the first study showed that inhibition of autophagy induced by BITC markedly enhance apoptosis in human and mouse bladder cancer cells. We are currently using orthotopic mice bladder cancer model to translate our results from in vitro to in vivo studies. Our data may be beneficial for further development of novel therapeutic strategies against bladder cancer.