抗真菌藥物咪康唑 (Miconazole) 在人類膀胱癌細胞中活化NRF2之化學預防效果
蔡德甫1#、陳栢均2、楊尚哲2、陳宏恩1、林宜佳1、仇光宇1、林致凡2、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
Miconazole, an anti-fungal agent, exerts bladder cancer chemoprevention by NRF2 activation
Te-Fu Tsai 1#, Po-Chun Chen2, Shan-Che Yang2, Hung-En Chen1, Yi-Chia Lin1, Kuang-Yu Chou1, Ji-Fan Lin2, and Thomas I-Sheng Hwang1,3,4,5
Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital1, Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital2, Department of Urology, Taipei Medical University3, Division of Urology, School of Medicine, Fu-Jen Catholic University4, Taipei, Taiwan.
Background: Bladder cancer (BC) with increasing incidence is a common urological cancer worldwide. Nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, a transcription factor plays critical roles in chemoprevention, inflammation and aging. NRF2 has recently been proposed as a novel target for cancer chemoprevention. Miconazole (MIC), an antifungal agent, displays anti-tumorigenic activity in various types of cancers, including BC. MIC has been demonstrated to induce apoptosis through the death receptor 5-dependent pathways in human BC cells. Herein, we investigated the effect of MIC on NRF2 activation and characterized its underlying molecular mechanism.
Materials and Methods: Human BC cell lines (5637 and T24) were used to evaluate the effects of MIC in this study. The activation of p62-Keap1-NRF2-pathway was examined by using Western blot, qPCR, and immunofluorescence staining. The BC cells treated with different concentrations of MIC were subjected to evaluate the ROS production by flow cytometry. To define the regulatory role of ROS production in NRF2 activation, the BC cells pretreated with ROS scavengers including N-acetyl-L-cysteine (NAC), catalase, and diphenyleneiodonium (DPI) were performed with Western blot to investigate NRF2 activation.
Results: We demonstrate in this study that miconazole dramatically increases NRF2 activation in bladder cancer cells, in a dose- and time-dependent manner. Interestingly, levels of expression of p62, a noncanonical pathway that mediates NRF2 activation, appeared to increase in accordance with NRF2. We also investigated levels of the negative regulator kelch-like ECH-associated protein 1 (KEAP1), which is involved in NRF2 activation. As expected, a decrease in KEAP1 expression was found after miconazole exposure. Confirmation of NRF2 nuclear translocation was monitored by immunofluorescence. Miconazole-induced generation of reactive oxygen species (ROS) promoted NRF2 activation. Pretreatment of bladder cancer cells with ROS scavengers abolished NRF2 expression and nuclear translocation, indicating that miconazole activates the noncanonical p62-KEAP1-NRF2 pathway, which is regulated by ROS production.
Conclusions: Our data provided the first that MIC is a novel NRF2 activator. Furthermore, the present study elucidates the mechanisms through which miconazole stimulates NRF2 to exert cancer chemopreventive effects.
Keywords: Bladder cancer cells, Miconazole, P62, KEAP-1, NRF2, ROS.