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

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 Tsai¹,Shan-Che Yang², Ya-Ming Teng², Yi-Chia Lin^1,4, Kuang-Yu Chou¹, and Thomas I-Sheng Hwang^1,2,3,4

Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital¹, Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital², Department of Urology, Taipei Medical University³, Division of Urology, School of Medicine, Fu-Jen Catholic University⁴, Taipei, Taiwan.

# These authors contributed equally to this work.

 

Purpose: Allyl isothiocyanate (AITC), one of the most widely studies members of the ITC family, 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 demonstrated that benzyl isothiocyanate (BITC) induces protective autophagy in prostate cancer cell. We previously demonstrated that AITC induces autophagy in prostate cancer cells. Here, we investigate the signaling pathways that involved in AITC-induced autophagy.

Materials and Methods: The cell viability of PC-3 and CRW22Rv1 (androgen-independent and sensitive human prostate cancer cell lines, respectively) was detected by WST-1 upon AITC exposure. The induced autophagy in AITC-treated cells was monitored by immunefluorescent or Western blot detection of LC3-II. Signalling pathways which involved in autophagy induction such as MEK/ERK, mTOR, AMPK, JNK, MAPK and beclin-1 (BECN1) were detected by Western blot. Detection of caspase 3/7 activities, caspase 3 cleavages and double DNA breaks (TUNEL) were conducted to monitor increased apoptosis in treated-cells.

Results: Increased LC3-II processing was detected in cells treated with AITC in a dose- and time-dependent manner. In 20-80 μM AITC-treated cells, we detected the phosphorylation forms of ERK, mTOR, AMPK and JNK; only phospho-ERK showed a significant and increased level in cells upon AITC treatment. However, inhibition of MEK/ERK signaling with 10 μM of PD98058 did not decreased AITC induced LC3-II processing, indicating the induction of autophagy in AITC-treated cells was independent on ERK signalling. Treatment of AITC induces ROS generation, and inhibition of ROS by antioxidant NAC attenuated autophagy and apoptosis induced by AITC. Decreased cell viability, increased caspase 3/7 activities, increased caspase 3 cleavage and increased TUNEL-positive cells in AITC-treated cells pretreated with Bafilomycin A1, suggesting inhibition of autophagy increased apoptosis in prostate cancer cells. Finally, increased beclin1 expression in both mRNA and protein levels was detected in AITC-treated cells, indicating AITC induced autophagy was through activation of beclin-1.

Conclusion: We provide evidences that AITC induces ROS-mediated protective autophagy in PC-3 and CRW22Rv1 cells. Inhibition of BECN1 significantly increases the anti-cancer effects of AITC in prostate cancer cells. Our results could potentially contribute to the beneficial effect of AITC in prostate cancer patients.