順鉑(Cisplatin)在人類膀胱癌細胞中透過表現BECN1誘發保護型細胞自嗜
黃一勝1,2,3,4、蔡德甫1、楊尚哲2、鄧雅旻2、陳宏恩1、林宜佳1,4、仇光宇1、林致凡2
1新光醫院外科部、2新光醫院泌尿科、3台北醫學大學醫學院、4輔仁大學醫學院、
5新光醫院中央研究室
Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells
Thomas I-Sheng Hwang1,2,3,4, Te-Fu Tsai1, Shan-Che Yang2, Ya-Ming Teng2,Hung-En Chen1, Yi-Chia Lin1,4, Kuang-Yu Chou1, and Ji-Fan Lin2
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.
Background
Bladder cancer (BC) is a common urologic cancer with high recurrence rate.In muscle invasive BC (MIBC), neoadjuvant chemotherapy with methotrexate, vinblastine, doxorubicin and cisplatin (MVAC) or gemcitabine and cisplatin (GC) is the first- chemotherapeutic option, because cisplatin alone could not effectively decreased the tumor burden and certain patients may progress to cisplatin-resistant. Autophagy induction has been implied to contribute to cisplatin-resistance in ocarian cancer, and we showed previously that human BC exhibits a high basal level of autophagy. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment. In this study, we investigate if cisplatin induces autophagy and the mechanism involved.
Materials and methods
Two high grade human bladder cancer cell lines, 5637and T24, were used in this study. Cell viability was detected using WST-1 reagents. Autophagy status in cisplatin-treated cells was detected by monitoring the expression level of LC3-II and p62 by Western blot, the immunofluorescent detection of LC3-positive puncta formation, and direct observation of the autophagolysosome formation by transmission electron microscopy (TEM). Autophagy inhibition was performed by using inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy related genes (ATG7 and ATG12). Apoptosis detection was monitored by increased caspase 3/7 activity and DNA fragmentation.
Results:
Consist with previous findings, cisplatin decreased cell viability and induced apoptosis of BC cells in a dose- and time-dependent manner. We detected the induction of autophagy in cisplatin-treated cells judging by the increased LC3-II accumulation, p62 clearance, increased number of LC3-positive puncta and autophagosomes. Inhibition of cisplatin-induced autophagy by inhibitors including Baf A1, CQ or shRNAs against ATG7/12 significantly enhanced apoptosis, indicating that cisplatin induced protective autophagy. Mechanistically, cisplatin activated the expression of beclin-1 (BECN1), and knockdown of BECN1 using shRNA approaches attenuated cisplatin-induced autophagy and enhanced cisplatin-induced apoptosis, suggesting coordinate inhibition of autophagy with cisplatin effectively decreased BC cell.
Conclusion:
Our results indicated that cisplatin induced autophagy is mediated by BECN1 in BC cells. Therefore, combinative treatment using cisplatin and autophagy inhibitors could potentially overcome cisplatin-resistance related to autophagy induction.