Suppression of Quercetin-Induced Autophagy Enhances Cytotoxicity through Elevating Apoptotic Cell Death in Human Bladder Cancer Cells

Objective: Quercetin, a natural dietary compound, has been demonstrated with antitumor activities against several types of cancers by disrupting cell cycle and inducing apoptotic cell death. However, human bladder cancer cells such as 5637 and T24 cells expressing mutant p53 are resistant to a 24 hrs quercetin treatment. In this study, the anticancer effect of quercetin was evaluated in these bladder cancer cells. Materials and Methods: The bladder cancer cells treated with quercetin were subjected to evaluated cell apoptosis by caspase activity, TUNEL assay and cell viability assay. The cell autophagy was assessed by detecting procession of LC3-II autophagic marker protein. Results: After 48 and 72 hrs of incubation, quercetin was found to be significantly effective in inhibiting proliferation of 5637 and T24 cells in a dose-dependent manner. Quercetin treatment increased the caspase 3/7 activities, percentage of subG0/G1 cells, and DNA fragmentation, indicating an induced apoptotic cell death. Pretreatment of a pan-caspase inhibitor, Z-VAD-FMK, attenuated the quercetin-decreased cell viability, suggesting that the cytotoxicity caused by quercetin mainly via apoptotic cell death. We also found that quercetin induced autophagy, as evidenced by the increased processing of LC3-II, a specific marker of autophagy. The disruption of autophagic flux by using bafilomycin A1, an autophagy inhibitor, caused significant accumulation of cellular p62 and LC3-II. In addition, the pretreatment of autophagy inhibitors, Baf A1 and chloroquine, strongly augmented apoptosis in 5637 and T24 cells, indicating the suppression of quercetin-induced autophagy enhanced apoptosis. Furthermore, the decreased cell viability and increased LC3-II processing were attenuated in quercetin-treated cells which pretreated with a reactive oxygen species (ROS) scavenger, N-acetyl cystine (NAC) suggested that quercetin-induced cytotoxicity and autophagy were initiated by the generation of ROS. Conclusion: This study proposes that combined treatment of autophagy inhibitor which sensitizes cells to quercetin treatment may be a better therapeutic approach to reduce bladder cancer cells proliferation.