H2Valdien derivatives induce apoptosis and cell cycle arrest in HepG2 hepatocellular carcinoma cells via a p53-dependent pathway.

Abstract

Abstract Objective: In this study, two human hepatocellular carcinoma cell lines, HepG2 (p53+/+) and Hep3B (p53-/-), were used with different p53 mutation statuses. The role of p53 in the induction of cytotoxicity by H2Valdien derivatives was investigated, as well as how p53 regulates GADD45a and p21. To examine whether the mechanism of action of H2Valdien derivatives on cell cycle arrest and apoptosis in human hepatocellular carcinoma cells is related to p53 deficiency. Methods: Cell viability was analyzed using the CCK-8 assay, and RNA sequencing was used for differential gene expression and enrichment analyses. The expression of apoptosis and cell cycle related proteins was analyzed by western blotting. DAPI and TUNEL staining techniques were employed to effectively visualize the nuclear morphology and apoptotic properties of the cells under investigation. Cell proliferation ability was assessed using colony formation assays, and mitochondrial membrane potential (MMP) was detected by JC-1 staining. Cell cycle progression was assessed by flow cytometry. Results: H2Valdien derivatives (5, 10, 20, and 40 mg/L) inhibited the proliferation of HepG2 and Hep3B cells and the formation of cell colonies in a dose-dependent manner, and decreased the MMP of HepG2 cells, but had no effect on the MMP of Hep3B cells. H2Valdien derivatives upregulated cleaved caspase-9, cleaved PARP, and Bax in HepG2 cells but not in Hep3B cells. RNA sequencing analysis revealed that H2Valdien derivatives increased p53, p21, and GADD45A expression, and western blotting and flow cytometry confirmed this finding. Despite the induction of p21 and GADD45a in Hep3B cells, there was no change in related proteins associated with drug concentration. Conclusion: GADD45a and p21 expression is regulated by H2Valdien derivatives in a p53-dependent manner, and p53 has a pro-apoptotic impact on H2Valdien derivative-induced toxicity. H2Valdien derivative-induced apoptosis and cycle hinder are reduced by p53 deletion.