抑制自噬作用來加強Everolimus (RAD001) 引發膀胱癌細胞的凋亡
1新光吳火獅紀念醫院 外科部 泌尿科
Inhibition of autophagy enhances everolimus (rad001)-induced cell death in human bladder cancer cells
Thomas I-Sheng Hwang1,2,3 #, Ji-Fan Lin4#, Yi-Chia Lin1,3 , Te-Fu Tsai1,3, Hung-En Chen1, and
1Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital
2School of Medicine, Taipei Medical University
3School of Medicine, Fu-Jen Catholic University
4Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
# these authors contributed equally to this work
Mammalian target of rapamycin, mTOR, a downstream protein kinase of phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, has been recognized to play a central role in controlling cancer cell growth. The PI3K/AKT/mTOR pathway promotes tumor growth and survival while suppressing autophagy, a catabolic process in cells to sustain energy homeostasis by collecting and recycling cellular components under stress condition. Conversely, inhibitors of the mTOR pathway such as Everolimus (RAD001), induce autophagy that promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would improve the cytotoxicity of mTOR inhibitors in bladder cancer.
Materials and methods
The cytotoxicity of RT4 (grade I), 5637 (grade II), HT1376 (grade III) and T24 (grade III) human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (BafA1), chloroquine (CQ) or hydroxychloroquine (HCQ)) was accessed by WST-8 cell viability kit. The autophagy status in cells was performed by the detection of microtubule-associated light chain 3 form II (LC3-II) using immunofluorescent staining and Western blot. Acidic organelles (AVOs) formation in treated cells was determined by acridine orange (AO) vital staining. Inhibition of mTOR pathway by RAD001 was monitored by home-made QPCR gene array and the detection of phospho-mTOR by Western blot. Induced apotposis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment.
Advanced bladder cancer cells (5637, HT1376 and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001 induced autophagy. AVOs formation was detected in cells treated with RAD001 and inhibited by the addition of 3-MA or Baf A1. Co-treatment of RAD001 with autophagy inhibitors further reduced cell viability and induces apoptosis in bladder cancer cells.
Our data suggest that coordinate inhibition of the mTOR and autophagy pathway promotes apoptosis, and could be a new therapeutic paradigm for the treatment of bladder cancer.
Running title: Inhibition of autophagy enhances apoptosis in RAD001-treated bladder cancer cells
Keywords: autophagy, apoptosis, bladder cancer, chloroquine, RAD001.