Qinghui Yang1, Chunhui Zhang2, Hong Wei1, Zenghui Meng1, Guangnan Li1, Yuanyuan Xu1, Yanjun Chen3. 1. Department of Cardiology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin, China. 2. Department of Gastrointestinal Medical Oncology, the Affiliated Tumor Hospital of Harbin Medical University, Harbin, China. 3. Department of Cardiology, Peking University Shenzhen Hospital, Shenzhen, China.
Abstract
BACKGROUND/AIMS: Cardiotoxicity is a predominant side-effect of nilotinib during chronic myeloid leukemia treatment. The underlying molecular mechanism remains unclear. The role of autophagy and mitochondrial signaling was investigated in nilotinib-treated cardiac H9C2 cells. METHODS: Cytotoxicity was assessed using Cell Death Detection kit. Immunoblot and immunofluorescence staining was performed, and cathepsin B and caspase3 activity was assessed in nilotinib-treated H9C2 cells with or without distinct pathway inhibitor or specific siRNA. RESULTS: Nilotinib time- and dose-dependently induced H9C2 apoptosis, which was not completely prevented by the pan caspase inhibitor z-VAD-fmk. Following nilotinib treatment, mitochondrial membrane potential decreased significantly accompanied with remarkable morphological changes. Nuclear translocation of mitochondrial apoptosis inducing factor (AIF) and increased p53 was detected in nilotinib-treated cells. AIF knockdown prevented nilotinib-induced increase of p53 and apoptosis. Additionally, increased cathepsin B activity was detected, and inhibition of cathepsin B by CA-074Me prevented nilotinib-induced apoptosis and nuclear translocation of AIF. Moreover, increased Atg5 and transition of LC3-I to LC3-II was revealed following nilotinib treatment. Increased cathepsin B activity and apoptosis by nilotinib was significantly prohibited by specific autophagy inhibitor bafilomycin A and Atg5 knockdown. CONCLUSION: Our findings demonstrate that nilotinib increases autophagy and cathepsin B activity, leading to mitochondrial AIF release and nuclear translocation, which is responsible for p53 and apoptosis induction in H9C2 cells.
BACKGROUND/AIMS: Cardiotoxicity is a predominant side-effect of nilotinib during chronic myeloid leukemia treatment. The underlying molecular mechanism remains unclear. The role of autophagy and mitochondrial signaling was investigated in nilotinib-treated cardiac H9C2 cells. METHODS: Cytotoxicity was assessed using Cell Death Detection kit. Immunoblot and immunofluorescence staining was performed, and cathepsin B and caspase3 activity was assessed in nilotinib-treated H9C2 cells with or without distinct pathway inhibitor or specific siRNA. RESULTS: Nilotinib time- and dose-dependently induced H9C2 apoptosis, which was not completely prevented by the pan caspase inhibitor z-VAD-fmk. Following nilotinib treatment, mitochondrial membrane potential decreased significantly accompanied with remarkable morphological changes. Nuclear translocation of mitochondrial apoptosis inducing factor (AIF) and increased p53 was detected in nilotinib-treated cells. AIF knockdown prevented nilotinib-induced increase of p53 and apoptosis. Additionally, increased cathepsin B activity was detected, and inhibition of cathepsin B by CA-074Me prevented nilotinib-induced apoptosis and nuclear translocation of AIF. Moreover, increased Atg5 and transition of LC3-I to LC3-II was revealed following nilotinib treatment. Increased cathepsin B activity and apoptosis by nilotinib was significantly prohibited by specific autophagy inhibitor bafilomycin A and Atg5 knockdown. CONCLUSION: Our findings demonstrate that nilotinib increases autophagy and cathepsin B activity, leading to mitochondrial AIF release and nuclear translocation, which is responsible for p53 and apoptosis induction in H9C2 cells.
Authors: Olja Mijanović; Ana Branković; Alexander N Panin; Solomiia Savchuk; Peter Timashev; Ilya Ulasov; Maciej S Lesniak Journal: Cancer Lett Date: 2019-02-20 Impact factor: 8.679