Effects of sinomenine on apoptosis of human hepatoma cell line SMMC-7721 and its molecular mechanism in vitro

Abstract

Evidence suggests that Sinomenine (SIN) has an excellent anticancer potential against hepatocellular carcinomas (HCCs), the apoptotic pathway genes affected by SIN and the mechanism of SIN that inhibits HCC are still unclear. Thus, the expression of apoptotic RNA and the early apoptosis in human HCC cells (SMMC-7721) caused by SIN need to be further investigated. In this study, to study the anti-tumor effect of SIN in vitro, human hepatoma cell line SMMC-7721 was treated with 0, 0.5, 1, 2 mM of SIN and 6.5 μM hydroxycamptothecin (HCPT) for 48 h. In addition, MTT assay, morphological observation and flow cytometry were used to detect the apoptosis of tumor cells, the changes of [Ca2+] and mitochondrial membrane potential (MMP) were detected by fluorescence microscopy. Moreover, the expression of tumor cell apoptosis-related genes was detected by real-time PCR, the anti-tumor mechanism of SIN was analyzed by bioinformatics methods including KEGG pathway graph was rendered by Pathview and heat map of three apoptosis pathways, including mitochondrial pathway, endoplasmic reticulum pathway (ER pathway), and death receptor pathway (DR pathway). In the results, significant inhibition of SMMC-7721 cell proliferation was found after SIN treatment. Under inverted microscopy, SIN-treated cells showed typical morphological alterations associated with apoptosis. A dose-dependent increase was observed in cytoplasmic Ca2+ in SIN-treated SMMC-7721 cells, while MMP levels decreased. The up-regulation and down-regulation of 47 apoptotic genes related to three apoptotic pathways were more clearly expressed by heat map analysis. Then the main pathways were analyzed, and the KEGG pathway graph was used to clearly express the expression of 92 apoptosis-related genes. Bid and Caspase-12 may be the main regulatory genes through mRNA expression. According to the previous changes in [Ca2+], SIN may affect the expression of caspase-12 through the change of [Ca2+], and then affect the expression of other related genes of Caspase family and Bcl-2 family. Finally, it affects the change of MMP of SMMC-7721 cell, leading to apoptosis of tumor cells.