Zhuo Yu1, Hai Feng2, Yunhui Zhuo3, Man Li4, Xiaojun Zhu3, Lingying Huang3, Xin Zhang4, Zhenhua Zhou4, Chao Zheng3, Yun Jiang3, Fan Le3, Dae-Yeul Yu5, Alfred Szelok Cheng6, Xuehua Sun7, Yueqiu Gao8,9. 1. Liver Disease Department, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No.528. Zhangheng Road, Pudong New District, Shanghai, People's Republic of China. zhuoyu@shutcm.edu.cn. 2. Department of pharmacology, School of Pharmacy, Harbin Medical University, Harbin, People's Republic of China. 3. Liver Disease Department, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No.528. Zhangheng Road, Pudong New District, Shanghai, People's Republic of China. 4. Laboratory of Cellular Immunity, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, People's Republic of China. 5. Disease Model Research Laboratory, Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Republic of Korea. 6. School of Biomedical Sciences, State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, SAR, People's Republic of China. 7. Liver Disease Department, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No.528. Zhangheng Road, Pudong New District, Shanghai, People's Republic of China. susan_sxh@shutcm.edu.cn. 8. Liver Disease Department, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No.528. Zhangheng Road, Pudong New District, Shanghai, People's Republic of China. gaoyueqiu@shutcm.edu.cn. 9. Laboratory of Cellular Immunity, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, People's Republic of China. gaoyueqiu@shutcm.edu.cn.
Abstract
PURPOSE: Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), which has a male predominance, lacks effective therapeutic options. Previously, the cardiac glycoside analogue bufalin has been found to inhibit HBV infection and HCC development. As yet, however, its molecular role in HBV-associated HCC has remained obscure. METHODS: Colony formation and soft agar assays, xenograft and orthotopic mouse models and HBV X protein (HBx) transgenic mice with exposure to diethylnitrosamine were used to evaluate the effect of bufalin on HBV-associated HCC growth and tumorigenicity. HBx-induced oncogenic signaling regulated by bufalin was assessed using PCR array, chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, transcription and protein expression assays. Synergistic HCC therapeutic effects were examined using combinations of bufalin and sorafenib. RESULTS: We found that bufalin exerted a more profound effect on inhibiting the proliferation of HBV-associated HCC cells than of non HBV-associated HCC cells. Bufalin significantly inhibited HBx-induced malignant transfromation in vitro and tumorigenicity in vivo. Androgen receptor (AR) signaling was found to be a target of bufalin resistance to HBV-associated hepatocarcinogenesis. We also found that bufalin induced both AR dephosphorylation and cell cycle-related kinase (CCRK) degradation to inhibit β-catenin/TCF signaling, which subsequently led to cell cycle arrest via cyclin D1 down-regulation and p21 up-regulation, resulting in HCC regression. Furthermore, we found that bufalin reduced > 60% diethylnitrosamine-induced hepatocarcinogenesis in HBx transgenic mice, and improved the sensitivity of refractory HBV-associated HCC cells to sorafenib treatment. CONCLUSION: Our results indicate that bufalin acts as a potential anti-HCC therapeutic candidate to block HBx-induced AR/CCRK/β-catenin signaling by targeting AR and CCRK, which may provide a novel strategy for the treatment of HBV-associated HCC.
PURPOSE: Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), which has a male predominance, lacks effective therapeutic options. Previously, the cardiac glycoside analogue bufalin has been found to inhibit HBV infection and HCC development. As yet, however, its molecular role in HBV-associated HCC has remained obscure. METHODS: Colony formation and soft agar assays, xenograft and orthotopic mouse models and HBV X protein (HBx) transgenic mice with exposure to diethylnitrosamine were used to evaluate the effect of bufalin on HBV-associated HCC growth and tumorigenicity. HBx-induced oncogenic signaling regulated by bufalin was assessed using PCR array, chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, transcription and protein expression assays. Synergistic HCC therapeutic effects were examined using combinations of bufalin and sorafenib. RESULTS: We found that bufalin exerted a more profound effect on inhibiting the proliferation of HBV-associated HCC cells than of non HBV-associated HCC cells. Bufalin significantly inhibited HBx-induced malignant transfromation in vitro and tumorigenicity in vivo. Androgen receptor (AR) signaling was found to be a target of bufalin resistance to HBV-associated hepatocarcinogenesis. We also found that bufalin induced both AR dephosphorylation and cell cycle-related kinase (CCRK) degradation to inhibit β-catenin/TCF signaling, which subsequently led to cell cycle arrest via cyclin D1 down-regulation and p21 up-regulation, resulting in HCC regression. Furthermore, we found that bufalin reduced > 60% diethylnitrosamine-induced hepatocarcinogenesis in HBxtransgenic mice, and improved the sensitivity of refractory HBV-associated HCC cells to sorafenib treatment. CONCLUSION: Our results indicate that bufalin acts as a potential anti-HCC therapeutic candidate to block HBx-induced AR/CCRK/β-catenin signaling by targeting AR and CCRK, which may provide a novel strategy for the treatment of HBV-associated HCC.
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