Andrographolide inhibits hepatocellular carcinoma progression and programmed death ligand-1 expression by blocking STAT3 phosphorylation

Abstract: BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive malignancy with frequent recurrence and strong immune evasion. Programmed death ligand-1 (PD-L1) facilitates immune evasion by suppressing T-cell activity. Andrographolide (AD), a natural diterpenoid with anti-inflammatory, antiviral, and immunomodulatory properties, has demonstrated antitumor potential. MATERIALS AND METHODS: Subcutaneous HCC mouse models assessed the antitumor and immune-activating effects of AD. CCK-8 assay, colony-formation assay, apoptosis analyses, and western blot investigated AD’s impacts on Huh-7 cell proliferation, apoptosis, and cell cycle. Immunofluorescence staining and flow cytometry collectively measured PD-L1 expression after AD treatment. Molecular docking and cellular thermal shift assay detected the targeted binding between AD and STAT3. Finally, recombinant interleukin-6 (rIL-6), a STAT3 activator, was used to verify whether AD-mediated effects on Huh-7 cell viability and PD-L1 expression were STAT3-dependent. RESULTS: In vivo , AD significantly inhibited mouse tumor growth, decreased PD-L1 expression in tumor tissues, and enhanced T-cell infiltration. In vitro , through cell experiments, AD bound to STAT3 and inhibited tumor cell proliferation, induced apoptosis, and downregulated PD-L1 protein levels by suppressing the phosphorylation of STAT3. Treatment with rIL-6 significantly reversed AD’s antitumor effects. CONCLUSION: AD inhibits HCC progression and PD-L1 expression by blocking STAT3 phosphorylation, providing a theoretical foundation for developing AD-based therapeutic strategies against HCC.