抗黴菌藥物Miconazole在膀胱癌之抑癌效果及凋亡機制
袁小雲、王賢祥、蔡淦仁1、蕭明裕2、歐宴泉
台中榮總 外科部 泌尿外科; 1中山醫學大學 醫學檢驗暨生物技術學系;2弘光科技大學 護理系
Inhibition of cell growth and induction of apoptosis on human bladder cancer cells with the antifungal drug miconazole
Sheau-yun yuan, shian-shiang wang, kan-jen tsai1, ming-yuh shiau2, yen-chuan ou
Department of Surgery, Taichung Veterans General Hospital;1Department of Medical Technology, Chung Shan Medical University;2Department of Nursing, HungKung University, Taichung, Taiwan
Purpose: Miconazole (MIC) is an imidazole antifungal agent that is commonly applied topically to the skin or mucous membranes to improve fungal infections. Recent studies have demonstrated that MIC exhibits anti-tumor effects on human colon cancers and leukemia. The aim of this study was to determine the effects of MIC on the growth inhibition and apoptosis of human bladder cancer cells.
Materials and Methods: Human bladder cancer cell lines (T24 and TSGH-8301) were used in this study. The T24 and TSGH-8301 cells were treated with various concentrations (6, 12, 25, 50 and 100 μM) of MIC for 24 h each. Cell viability was determined by MTT assay. The apoptosis mechanism was detected by sub-G1 population, DNA fragmentation and reactive oxygen species (ROS) generation, and the mitochondrial membrane potentials were evaluated by flow cytometry. SDS-PAGE/Western blot assay was used to detect apoptotic proteins (PARP, caspase-3/-8/-9), Bcl-2 family proteins and cell-cycle-regulated proteins.
Results: The study showed that MIC elicited cytotoxic effects on human T24 and TSGH-8301 bladder cancer cells in a dose- and time-dependent manner with IC50 ~48.3 ± 0.35 μM and 47.8 ± 0.27 μM, respectively, as determined by MTT assay. MIC increased the sub-G1 population, DNA fragmentation formation, activation of caspase-3/-8/-9 and cleavage of poly (ADPribose) polymerase (PARP), and provoked apoptosis in both bladder cancer cell lines. Western blot analysis revealed increases of p21 and p27 protein levels, along with decreases of Cyclin E1, CDK2 and CDK4 expressed in MIC-treated T24 and TSGH-8301 cells, which is related to the G0/G1 phase of the cell cycle. Meanwhile, there was an induced increase of cleaved Bax proteins and a decrease of Bcl-2 proteins, up-regulation of the DR5 death receptor and ROS generation subsequent to reduced mitochondrial membrane potential at higher concentrations (>50 μM) of MIC for 24 h, in both the T24 and the TSGH-8301 bladder cancer cells.
Conclusions: These results indicate that MIC triggered apoptosis via G0/G1 arrest, leading to the activation of the TRAIL death receptor, then activation of caspase-3/-8/-9 and the ROS-dependent mitochondrial pathway in human bladder cancer cells. This suggests that MIC may be a potential anti-bladder cancer agent in humans.