Cantharidin treatment inhibits hepatocellular carcinoma development by regulating the JAK2/STAT3 and PI3K/Akt pathways in an EphB4-dependent manner

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. The tyrosine kinase receptor EphB4 promotes oncogenesis and tumor development and progression. Its inhibition is regarded as an effective strategy for the treatment of solid tumors. In the present study, we identified cantharidin as a novel EphB4 inhibitor for HCC treatment and evaluated the underlying molecular pharmacological mechanisms of action. We observed increased expression levels of EphB4 in HCC patients and a positive correlation between EphB4 and p-JAK2 levels in HCC patient samples. Knockdown of EphB4 using small interfering RNA decreased the expression levels of p-JAK2 and p-STAT3 in HCC cells, suggesting JAK2/STAT3 being a novel downstream signaling target of EphB4. Cell viability experiments revealed that the anti-cancer effect of cantharidin was positively correlated with EphB4 expression levels in HCC cell lines. We confirmed the potent antiproliferative activity of cantharidin on HepG2 cells with high expression of EphB4 and tumor xenograft. Molecular docking assay, immunoblotting assay and quantitative Reverse transcription PCR assay indicated that cantharidin bound to EphB4, and thereby resulted in EphB4 suppression at mRNA and protein levels. Hep3B and SMMC-7721 cells were with low expression of EphB4. In EphB4^-/HepG2, EphB4⁺/HepG2, and EphB4⁺/Hep3B cells, EphB4 knockdown alleviated the cantharidin-induced decrease in cell viability and colony formation ability and increase in Apoptosis in HepG2 cells, while its overexpression exacerbated these effects in Hep3B cells and increased the Apoptosis of HepG2 cells. In nude mouse models, cantharidin suppressed tumor growth more effectively in EphB4⁺/SMMC-7721 xenografts than in wild-type SMMC-7721 xenografts. Underlying mechanistic study showed that by targeting EphB4, cantharidin blocked a novel target, the downstream JAK2/STAT3 pathway, and the previously known target, the PI3K/Akt signaling, resulting in intrinsic Apoptosis. These results indicated that cantharidin may be a potential candidate for HCC treatment by regulating the EphB4 signaling pathway.