Bufalin inhibits hepatocellular carcinoma progression by blocking EGFR-mediated RAS-RAF-MEK-ERK pathway activation

Background: Hepatocellular carcinoma (HCC) remains one of the most challenging malignancies with persistently dismal long-term survival outcomes despite multidisciplinary advances in diagnostic and therapeutic strategies. Cinobufacini preparations have garnered increasing attention as adjunctive therapeutic agents in integrated management strategies for HCC. Bufalin (BF), the active ingredient in Cinobufacini, has garnered substantial attention due to its potent antitumor effects. However, the precise molecular mechanisms underlying its antitumor actions remain incompletely characterized.

Methods: A clinical retrospective cohort analysis was conducted to establish the definitive clinical benefit of Cinobufacini in improving treatment outcomes among HCC patients. Building upon these clinical insights, a multi-dimensional approach was implemented to elucidate the anti-HCC molecular mechanisms mediated by the bioactive component BF of Cinobufacini.

Results: Western medical treatment combined with Cinobufacini shows an improving trend in the overall survival (OS) and progression free survival (PFS) of HCC patients. Moreover, our exploratory analysis suggests a potential dose-response relationship where longer cumulative exposure to Cinobufacini appears to be associated with improved clinical outcomes. In vitro experiments demonstrated that BF significantly inhibited cell viability and proliferation, and induced Apoptosis in HepG2 and HCCLM3. Network pharmacology analysis identified 20 core targets, and molecular docking revealed high-affinity binding between BF and key proteins, including EGFR, GRB2, Src, and MAPK1. HCC tissue microarrays confirmed the overexpression of EGFR and GRB2 in HCC tissues. Further mechanistic investigations revealed that BF suppressed the EGFR-mediated Ras/Raf/MEK/ERK pathway activation in HepG2 and HCCLM3. BF intervention significantly reduced tumor volumes in C57BL/6 mouse subcutaneous HCC xenograft and BALB/c Nude mouse orthotopic HCC xenograft models. Moreover, BF inhibited the phosphorylation levels of EGFR, Raf, MEK, and ERK in tumor tissues, further corroborating its inhibitory effects on the Ras/Raf/MEK/ERK signaling pathway.

Conclusions: Our observational data suggest a potential association between Cinobufacini use and favorable trends in OS and PFS among HCC patients. BF exerts its antitumor effects against HCC by interfering with the EGFR-mediated Ras/Raf/MEK/ERK signaling pathway. These findings not only elucidate the molecular mechanisms underlying the antitumor actions of BF but also highlight the potential of Cinobufacini preparations as a valuable therapeutic option for HCC.

Bufalin; Cinobufacini preparations; EGFR; Hepatocellular carcinoma; RAS/RAF/MEK/ERK.