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

¹新光醫院 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

Hung-En Chen^1#, Ji-Fan Lin^2#, Te-Fu Tsai ^1,4, Yi-Chia Lin^1,4, Kuang-Yu Chou^1,4,
and Thomas I-Sheng Hwang^1,3,4

¹Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, ²Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, ³School of Medicine, Taipei Medical University

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

# These authors contributed equally to this work.

Purpose: Allyl isothiocyanate (AITC) is one of the most widely studies members of the ITC family which are enriched in cruciferous vegetables. It has recently be demonstrate to inhibit survival of human prostate cancer cells while has minimal effects on a normal prostate epithelial cell line. In our previous studies, we showed that AITC induces reactive oxygen species (ROS)-mediated apoptosis and protective autophagy. Here, we investigate the underlying mechanism of AITC induced autophagy in human prostate cancer cells.

Materials and Methods: Total protein from androgen-sensitive (Rv1) and -refractory (PC3) human prostate cancer cells treated with AITC for 24 hours were extracted and subjected to the detection of the activation status of mTOR, AMPK, ERK, JNK and p-38 (MAPK) that are reported to be related to autophagy induction. Protein level of beclin-1 and Bcl-2 was also detected to determine the involvement of autophagy/apoptosis switch. The small chemical drugs, PD184352, Dorsomorphin, and SP600125 were used to specific inhibit AITC-activated ERK, AMPK and JNK, respectively. Inhibition of AITC-induced ROS generation was performed by pretreatment of N-acetylcysteine (NAC).

Results: ERK activation was observed only in Rv1 cells, while AMPK, and JNK activation was observed in both Rv1 and PC3 cells upon AITC treatment. However, pre-treatment of chemical inhibitors specific for either ERK, AMPK, or JNK did not attenuated AITC-induced autophagy that judged from the increased LC3-II processing. Therefore, AITC induced autophagy was independent to these cell signaling pathways. We next detected the expression of beclin-1/Bcl-2 that had been prove to be an important switch in cell fate determination regarding to autophagy and apoptosis. We discovered a significantly increased level of beclin-1/Bcl-2 in AITC-treated prostate cancer cells. Pre-treatment of NAC decreased the expression level of beclin-1 in AITC-treated cells, this indicates that beclin-1 which is a downstream target of ROS was responsible for the autophagy induction in AITC-treated prostate cancer cells.

Conclusion: We for the first time provide evidences that AITC induces ROS-mediated protective autophagy was through the up-regulation of beclin-1 expression regardless of androgen sensitivity in human prostate cancer cells. We also found that ERK, AMPK and JNK activation that is independent for autophagy induction in cells upon AITC treatment. The signaling pathways which lead to up-regulation of beclin-1 in AITC-treated cells remains further investigation.