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.