2-Hydroxy-3-Methylanthraquinone Suppresses Hepatocellular Carcinoma Progression by Blocking Annexin A5-Mediated Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling.

Hepatocellular carcinoma (HCC), the most common subtype of primary liver cancer, remains a major cause of cancer-related mortality worldwide. Although 2-hydroxy-3-methylanthraquinone (HMA), a natural anthraquinone compound, has demonstrated antitumor activity in various malignancies, its specific role and underlying mechanisms in HCC are not fully understood. This study aimed to evaluate the antitumor effects and molecular mechanisms of HMA in HCC. Human HCC cell lines were treated with HMA, and cell proliferation and apoptosis were assessed using Cell Counting Kit-8 and flow cytometry assays, respectively. A heterotopic xenograft tumor model was established in nude mice to evaluate in vivo tumor growth and weight. Immunohistochemical staining for Ki67 and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to assess tumor cell proliferation and apoptosis. Network pharmacology analysis was conducted to predict potential targets of HMA in HCC. Quantitative real-time polymerase chain reaction and Western blotting were used to evaluate mRNA and protein expression levels. Cell migration and invasion were assessed using wound healing and transwell assays. Our data revealed that HMA significantly suppressed cell proliferation, induced apoptosis, and inhibited migration and invasion in both HCC cells and tumor tissues. Mechanistically, HMA downregulated Annexin A5 (ANXA5) expression and inhibited activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Silencing of ANXA5 replicated the inhibitory effects of HMA and further enhanced its pro-apoptotic and anti-invasive activities. Conversely, overexpression of ANXA5 restored PI3K/AKT signaling activity and reversed the inhibitory effects of HMA on HepG2 cell proliferation, migration, and invasion. These reversal effects were abolished by treatment with LY294002, a selective PI3K inhibitor. In summary, HMA suppresses the progression of HCC by targeting ANXA5 and inhibiting the PI3K/AKT signaling pathway, highlighting its potential as a novel therapeutic agent for HCC.