Bufalin Induced Mitochondrial Dysfunction Promotes Apoptosis of Glioma Cells by Regulating Annexin A2 and DRP1 Proteins

Abstract

Background: Glioma is a common primary central nervous system tumor, and there is still a lack of effective therapeutic drugs that can effectively improve the survival rate of patients in clinical. Bufalin has a good effect in the treatment of various tumors, but the mechanism of how it promotes the apoptosis of glioma cells is still unclear. The aim of this study was to investigate the drug target of bufalin acting on glioma cells, and to clarify the mechanism of apoptosis. Methods: Cell viability and proliferation were evaluated by cck-8 and colony formation assay. Then, the cell cycle and apoptosis, intracellular ion homeostasis, oxidative stress levels and mitochondrial damage were detected after bufalin treatment. DARTS-PAGE technology combined with LC-MS/MS were employed to explore the drug targets of bufalin on U251 cells. Molecular docking and western blot were used to further identify the potential targets. siRNA of Annexin A2 and a DRP1 protein inhibitor Mdivi-1 were also used to verify the target of bufalin.Results: Bufalin upregulated the expression of cytochrome C, cleaved caspase 3, p-Chk1 and p-p53 proteins to cause U251 cell apoptosis and cycle arrest in S phase. Bufalin can also induce oxidative stress in U251 cells, destroy intracellular ion homeostasis, and cause mitochondrial damage. The expression of mitochondrial separation/fusion-related proteins in U251 cells was abnormal, Annexin A2 and DRP1 protein were translocated from cytoplasm to mitochondria, and MFN2 protein was released from mitochondria into cytoplasm after bufalin treatment, which disrupted the mitochondrial division/fusion balance of U251 cells. Conclusions: Our research indicated that bufalin can cause Annexin A2 and DRP1 oligomerization to be located on the surface of mitochondria, and disrupt the mitochondrial division/fusion balance to induce U251 cell apoptosis.