Blockage of endoplasmic reticulum stress attenuates nilotinib-induced cardiotoxicity by inhibition of the Akt-GSK3ß-Nox4 signaling.

Cardiotoxicity is a critical side-effect of nilotinib during treatment for cancer, such as chronic myeloid leukemia, while the potential signaling mechanisms remain unclear. The role of and the relationship between endoplasmic reticulum (ER) stress and mitochondrial dysfunction was investigated in nilotinib-induced cardiac H9C2 injury as a suitable cell model. Our results showed that ER stress was persistently induced in nilotinib-treated cells, evidenced by increase of GRP78, CHOP, ATF4 and XBP1 as well as phospho-PERKThr980. The results from 4-phenylbutyrate (PBA, an ER stress inhibitor) and SC79 (a specific Akt activator) suggested that ER stress increased activity of glycogen synthase kinase-3 beta (GSK3ß) that is reflected by decrease of phospho-GSK3ßSer9, through downregulation of phospho-AktSer473, and that prolonged ER stress and activated GSK3ß involved nilotinib-induced apoptosis. In addition, the data from JNK inhibition using SP600125 showed that over-activated JNK was responsible for Akt de-phosphorylation. Moreover, the abundance of NADPH oxidase (Nox4) was significantly increased following nilotinib treatment, which was prevented by SB216763 (a specific GSK3ß inhibitor). Additionally, mitochondrial dysfunction was indicated by reduced mitochondrial membrane potential (MMP) level and increased reactive oxygen species level. In nilotinib-treated cells, knockdown of Nox4 preserved MMP level, abrogated reactive oxygen species production, and decreased apoptosis. Accordingly, our data demonstrated that inhibition of ER stress may protect cardiomyocytes against nilotinib toxicity potentially through inactivation of Akt-GSK3ß-Nox4 signaling. These findings may provide an attractive therapeutic target for treatment of nilotinib-related cardiotoxicity.

Caspase-Independent Pathway is Related to Nilotinib Cytotoxicity in Cultured Cardiomyocytes.

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.