Isoliquiritigenin Induces Cytotoxicity in PC-12 Cells In Vitro.

Abstract

Isoliquiritigenin (ISL) has been reported to have a wide range of biological activities. This study evaluated the cytotoxic effect of ISL on norvegicus pheochromocytoma cell line (PC-12 cells) and its possible molecular mechanism. The cytotoxicity in vitro of ISL against PC-12 cells was investigated by MTT assay. The migration and invasion of PC-12 cells were performed by scratch test and transwell assay. Apoptosis was evaluated by microscopy and flow cytometry. The reactive oxygen species (ROS) and mitochondrial membrane potential were studied by fluorescent microscopy. DNA damage of PC-12 cells was analyzed by comet assay. The protein expression of caspase, Bcl-2 family member, autophagy-associated protein Beclin-1, and LC3 was detected by western blot. The autophagy of PC-12 cells was investigated by acridine orange (AO) and monodansylcadaverine (MDC) staining. The IC50 value of ISL against PC-12 cell is 17.8±1.8μM. ISL could suppress PC-12 cell migration and invasion. AO/ethidium bromide staining and flow cytometry suggested that ISL caused apoptosis of PC-12 cells. Significant DNA damages of PC-12 cells treated with ISL were observed in a comet assay. ISL inhibited the cell growth of PC-12 cells at S phase. Exposure of PC-12 cells to ISL increased the levels of cellular reactive oxygen species (ROS) and decreased the mitochondrial membrane potential. Additionally, ISL trigged the release of cytochrome c from the mitochondria to the cytoplasm. The expression levels of caspase-9, caspase-3, caspase-7, Bax, and Bim were upregulated, whereas the expression levels of Bcl-2 and Bcl-x were downregulated. AO and monodansylcadaverine (MDC) staining assay showed that ISL caused autophagy of PC-12 cells. The upregulation of protein Beclin-1 and LC3 was observed in PC-12 cells. Therefore, the results show that ISL induces apoptosis of PC-12 cells through ROS-mediated activation of the intrinsic mitochondria-cytochrome c-caspase protease mechanism and causes the autophagy of PC-12 cells. Graphical Abstract The in vitro cytotoxicity, apoptosis, comet assay, ROS, mitochondrial membrane potential, cell cycle arrest, autophagy, and western blot induced by ISL were investigated.