Miconazole, an anti-fungal agent, induces autophagy via reactive oxygen species in human bladder cancer cells
咪康唑，一種抗真菌劑，通過活性氧物種誘導人類膀胱癌細胞中的自噬反應
蔡德甫1#、林致凡2#、楊尚哲2、鄧雅旻2、陳宏恩1、林宜佳1、仇光宇1、黃一勝1,2,3,4
1新光醫院 外科部 泌尿科、2新光醫院 中央研究室、3台北醫學大學 醫學院、4輔仁大學 醫學院
# these authors contributed equally to this work
Abstract
Purpose: Bladder cancer (BC) with increasing incidence is a common urological cancer worldwide. Miconazole (MIC), an antifungal agent, displays anti-tumorigenic activity in various types of cancers, and has been demonstrated to induces apoptotic cell death through the death receptor 5-dependent pathways in human bladder cancer cells. In the present study, we investigated the effect of MIC on bladder cancer cell autophagy and characterized its underlying molecular mechanism.
Materials and Methods: Human BC cell lines (5637 and T24) and immortalized urothelial cells (SV-Huc-1) were used in this study. The cell viability in cells treated with different concentrations of MIC was evaluated using WST-1 reagent. The induction of autophagy in MIC-treated cells was detected by the induction of LC3-II formation and p62 degradation using immunoblotting, the accumulation of LC3 puncta by immunofluorescent staining, and the direct observation of autophagosomes formation using transmission electron microscopy. To detect the autophagic flux and identify the role of MIC-induced autophagy, bafilomycin A1 (BafA1) was utilized to inhibit MIC-induced autophagy. The expression of apoptotic markers such as cleaved PARP, cleaved caspase-3 and cytochrome C release was detected by Western blot in MIC-treated cells with or without BafA1 pretreatment.
Results: MIC exhibits dose- and time-dependent cytotoxicity in 5637 and T24 cells, but has minimal impact on SV-Huc-1 cells. Meanwhile, MIC induces autophagy in 5637 and T24 cells, judging by the increased level of LC3-II formation, p62 degradation, LC3-positive puncta in the cytosol, and autophagosomes formation. In addition, enhanced apoptosis was detected in MIC-treated cells pretreated with BafA1, suggesting that MIC induces protective autophagy in these cells. MIC was demonstrated to induce ROS generation in 5637 and T24 cells. Inhibition of ROS using N-acetylcysteine (NAC) decreased the formation of LC3-II, indicating that MIC-induced autophagy was mediated by ROS.
Conclusions: Our results provided the first that MIC not only induces apoptosis but also protective autophagy in human bladder cancer cells. Coordinated inhibition of MIC-induced autophagy could enhance the apoptosis and should be take into consideration in further translational studies.