(PD1-2) Inhibition of cisplatin-induced autophagy enhances apoptotic cell death in human bladder cancer cells
  • 2015-11-30,
  • 上傳者: TUA秘書處,
  •  0
抑制Cisplatin 引發的自噬作用可增強膀胱癌細胞的凋亡
新光醫療財團法人新光吳火獅紀念醫院 1外科部 2泌尿科 5中央實驗室,
3台北醫學大學 醫學院, 4輔仁大學 醫學院
Inhibition of cisplatin-induced autophagy enhances apoptotic cell death in human bladder cancer cells
Thomas I-Sheng Hwang1,2,3,4 、Ji-Fan Lin5、Yi-Chia Lin1、Te-Fu Tsai1、Hung-En Chen1、Kuang-Yu Chou1
l Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, 2Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, 3Department of Urology, Taipei Medical University, 4Division of Urology, School of Medicine, Fu-Jen Catholic University, 5Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital , Taipei, Taiwan.
Bladder cancer (BC) is a common urologic cancer with high recurrence rate. Cisplatin is the first member of a class of platinum-containing anti-cancer drugs that binding to and causing DNA cross-linking which ultimately leads to apoptosis. Cisplatin is used to treat various types of cancers including BC. However, cisplatin alone is not very effective in BC and the combinations of gemcitabine/cisplatin is now the first-line chemotherapy for muscle invasive BC. Our previous studies showed that BC cells exhibits high basal level of autophagy and treatment of chemotherapeutic agents further induces autophagy as a survival mechanism. In this study, we investigated if cisplatin induces autophagy in human BC cells and whether inhibition of cisplatin-induced autophagy enhances cancer cell death.
Materials and Methods:
The cell viability of RT4 (grade I), 5637 (grade II), and T24 (grade III) human bladder cancer cells treated with cisplatin alone or combined with autophagy inhibitor, bafilomycin A1 (BafA1), 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. Moreover, the formation of autophagolysosome was detected using transmission electron microscopy to confirm the induction of autophagy in cisplatin-treated T24 cells. To investigate the signaling pathway involved in cisplatin-induced autophagy, the activation of AKT, ERK, AMPK and MAPK and the inhibition of mTOR in cisplatin-treated cells were detected using Western blot. Induced apoptosis was determined by the detection of cleavage caspase 3, and the measurement of caspase 3/7 activity and DNA fragmentation in treated-cells.  
Advanced bladder cancer cells (5637 and T24) were more resistant to cisplatin than RT4. The processing of LC3-II was elevated in cells treated with increased concentration of cisplatin, suggesting cisplatin induces autophagy. Detection of autophagy flux by blocking autophgosome to lysosomes fusion using Baf A1 and the direct observation of autophagolysosome formation in cisplatin-treated T24 cells using TEM further confirmed that cisplatin indeed triggers autophagy in BC cells. Activation of AKT, ERK and MAPK signaling and inhibition of mTOR was detected in cisplatin treated cells. However, pretreatment of specific inhibitors of ERK, MAPK did not attenuated cisplatin-induced autophagy suggests these pathways are not involved in the induction of autophagy. Finally, reduced cell viability and induced apoptosis were observed in cisplatin-treated cells pretreated with autophagy inhibitor suggesting that inhibition of autophagy enhances cancer killing effect of cisplatin in human BC cells.
Cisplatin induces autophagy through activation of AKT and inhibition of mTOR in human BC cells. Our data suggest that autophagy inhibition promotes apoptosis in cisplatin-treated cells, and could be a new therapeutic paradigm for the treatment of bladder cancer.
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    2015-11-30 10:36:00
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