Enhanced apoptosis by inhibition of cisplatin-induced autophagy in human bladder cancer cells

Purpose: Cisplatin has been used to treat bladder cancer (BC), however, cisplatin alone is not very effective, and the combinations of gemcitabine/cisplatin is now the first-line chemotherapy. Moreover, bladder tumor exhibits high basal level of autophagy. In this study, we investigated if cisplatin induces more autophagy in human BC cells, and whether inhibition of cisplatin-induced autophagy enhances apoptosis that leads to cancer cell death.

Materials and Methods: The autophagy status in cisplatin-treated RT4 (grade I), 5637 (grade II), and T24 (grade III) human bladder cancer cells was performed by the detection of microtubule-associated light chain 3 form II (LC3-II) and aggregation of LC3 puncta using Western blots and immunofluorescent staining, respectively. Moreover, the formation of autophagolysosome was detected using transmission electron microscopy to confirm the increased number of autophagosomes in cisplatin-treated T24 cells. The cell viability in cells treated with cisplatin with or without the autophagy inhibitor, bafilomycin A1 (BafA1), was accessed by WST-1 cell viability kit. 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 by Western blot. Induced apoptosis was determined by the detection of cleaved caspase 3, cleaved PARP, the caspase 3/7 activity and the level DNA fragmentation in treated-cells.

Results: 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) in 5637 and T24 cells, and the direct observation of autophagolysosome formation in cisplatin-treated T24 cells using TEM further confirmed that cisplatin indeed triggers autophagy. Advanced bladder cancer cells (5637 and T24) were more resistant to cisplatin than RT4, suggesting autophagy acts as a survival mechanism in high grade BC cells. While no response was found in AMPK, the 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 detected in cisplatin-treated cells pretreated with autophagy inhibitor suggesting that inhibition of autophagy enhances cancer killing effect of cisplatin in human BC cells.

Conclusion: Cisplatin induces autophagy in human BC cells, and autophagy inhibition enhances apoptosis in cisplatin-treated cells. This study suggests a new therapeutic paradigm for the treatment of bladder cancer.