Synergistic antitumor effect of oroxylin A and donafenib in hepatocellular carcinoma through tumor protein p53 signaling pathway activation

BACKGROUND The clinical application of donafenib in advanced hepatocellular carcinoma (HCC) is restricted by its limited therapeutic efficacy and a variety of treatment-associated adverse events. These factors collectively underscore the need for more effective and well-tolerated therapeutic strategies. AIM To investigate the effects and underlying mechanisms of oroxylin A in combination with donafenib on HCC through in vivo and in vitro studies. METHODS The antitumor efficacy of oroxylin A, donafenib, and their combination was assessed in xenograft mouse models and MHCC-97H/PLC-PRF-5 cell lines. Tumor growth was monitored using fluorescence live imaging. Cell viability, colony formation, and apoptosis were assessed using Cell Counting Kit-8, clonogenic, and flow cytometry assays, respectively. Molecular mechanisms were investigated by assessing the expression of tumor protein p53 (TP53) signaling-related regulators via quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry. Public datasets and Kaplan-Meier analysis were used to analyzed the relationship between their expression and patient survival. RESULTS The combination of oroxylin A and donafenib demonstrated superior anti-tumor efficacy in vivo compared to monotherapies, without inducing significant hepatorenal toxicity. The combination therapy demonstrated a stronger anti-proliferative and pro-apoptotic effects than two monotherapies in two HCC cell lines. Mechanistically, the drug combination synergistically activated the TP53 signaling pathway. Oroxylin A primarily targeted the cyclin-dependent kinase 9-murine double minute 2 (MDM2)/MDM4 axis to stabilize TP53, while donafenib suppressed the vascular endothelial growth factor receptor/B-rapidly accelerated fibrosarcoma proto-oncogene serine/threonine kinase axis to activate TP53. Clinical data has verified that the upregulation of key components of this pathway (TP53, MDM2, MDM4, and cyclin-dependent kinase 9) in patients with HCC is associated with a poor overall survival. CONCLUSION Oroxylin A and donafenib exert a synergistic anti-tumor effect in HCC by co-activating the TP53 signaling pathway through distinct but complementary molecular axes. This combination strategy presents a promising and viable therapeutic approach to overcome the limitations of donafenib monotherapy in the treatment of HCC.