PurposeCisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC). Autophagy induction has been implied to contribute to cisplatin resistance in ovarian cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines.Materials and methodsHuman BC cells (5637 and T24) were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3)-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL) formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12) were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation.ResultsCisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose-and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1) was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis.ConclusionCollectively, the study 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.
Benzyl isothiocyanate (BITC) is a dietary chemopreventive agent that inhibits the growth of various human cancer cells by causing apoptotic cell death. In this study, we demonstrate that BITC not only induces apoptosis but also induces autophagy in human hormone-sensitive (Rv1) and -refractory (PC3) prostate cancer cells. In BITC-treated cells, the induction of autophagy was detected by monitoring the processing of an autophagy marker protein, microtubule-associated protein 1 light chain 3 (LC3), the aggregation of LC3 into granular structures and the formation of acidic organelles. Inhibition of autophagy using 3-methyladenine increased BITC-induced apoptosis, whereas the administration of caspase inhibitor suppressed BITC-induced cell death. Our data also showed that BITC inhibits mammalian target of rapamycin (mTOR) kinase activity in a dose-dependent manner. The expression of phospho-mTOR (Ser2481), an indicator of mTOR intrinsic catalytic activity, and phospho-UNC-51-like kinase 1 (Ser757), a direct substrate of mTOR, were decreased in BITC-treated cells. However, the increased expression of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and antiapoptotic Bcl-2 were detected only in PC3 cells at later stages of BITC treatment. Collectively, our results show that BITC induces a protective autophagy response in Rv1 and PC3 cells through inhibition of the mTOR signaling pathway. Activation of the AKT survival pathway was only observed in PC3 cells, representing a resistance mechanism of advanced prostate cancer upon BITC treatment. These findings could potentially contribute to the beneficial effect of BITC in prostate cancer treatments.
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