This research explored the mechanism of ganoderic acid X(GAX) on human hepatocellular carcinoma cell models(HepG2, HuH6) and nonobese diabetic-severe combined immune deficient(NOD-SCID) mouse subcutaneous tumor models using proteomics, aiming to provide a basis for the clinical application of GAX. CCK-8 assay was employed to evaluate the effect of GAX on the viability of HepG2 and HuH6 cells. EdU assay was used to assess the effect of GAX on cell proliferation. Scratch assay was used to examine the effect of GAX on cell migration ability. Hoechst 33258 staining was used to investigate the effect of GAX on cell apoptosis. Moreover, a NOD-SCID mouse subcutaneous tumor model was established to analyze the tumor volume and weight in control group and GAX low-, medium-, and high-dose groups(5, 10, and 20 mg·kg~(-1)). HE staining was conducted to evaluate the drug toxicity of GAX. Additionally, HepG2 cells in the control group and the GAX high-dose group were subjected to label-free proteomics analysis to identify differential proteins and enrich relevant signaling pathways. CYTO-ID® staining was performed to detect autophagy, and Western blot was conducted to measure the expression levels of relevant proteins. In vitro results demonstrated that GAX dose-depen-dently inhibited proliferation, migration, and induced apoptosis in HepG2 and HuH6 cells. In vivo studies showed that GAX significantly inhibited tumor volume and weight without causing significant damage to major organs(heart, liver, spleen, lung, and kidney) in mice. Label-free proteomics analysis revealed that GAX participated in multiple signaling pathways during the treatment of hepatocellular carcinoma, with a high enrichment in the autophagy pathway. CYTO-ID® staining and Western blot results showed that GAX induced autophagy, upregulated the expression of Beclin-1, ATG5, and LC3-Ⅱ proteins, and downregulated the expression of p62 protein. This study suggests that GAX inhibits the proliferation, migration, and induces apoptosis of hepatocellular carcinoma cells by inducing autophagy, thereby significantly inhibiting tumor growth. GAX represents a promising adjuvant therapy for cancer treatment.
Keywords: autophagy; ganoderic acid X; hepatoblastoma; proteomics.