Owing to the crucial role of Src in cancer metastasis, interruption of Src and its signaling has been considered a promising strategy for cancer metastasis treatment. Cucurbitacin B, a dietary triterpenoid, has been shown to possess anti-proliferative and apoptosis-inducing activities in cholangiocarcinoma (CCA) cells via suppressing the activation of FAK which is a main downstream Src effector. We hypothesized that cucurbitacin B might act as a Src suppressant which conferring anti-metastasis effect against CCA cells. To investigate this, the role of Src in regulating metastasis behavior of CCA cells and the effect of cucurbitacin B on Src-mediated metastatic phenotype of these cells were determined. The results showed that activation of Src significantly enhanced the migratory and invasive abilities of CCA cells. Molecular analysis revealed that Src-facilitated metastasis behavior of CCA cells occurred by modifying expression of a wide range of metastasis-related genes in the cells. Consistent with gene expression results, activation of Src significantly induced the protein expression of 2 important metastasis-associated molecules, MMP-9 and VEGF. Cucurbitacin B markedly suppressed activation of Src and its key effector, FAK. As a consequence, the alteration of expression profiles of metastasis-associated genes induced by Src activator in CCA cells was diminished by cucurbitacin B treatment. The compound also down-regulated Src-induced expression of MMP-9 and VEGF proteins in the cells. Moreover, molecular docking analysis revealed that cucurbitacin B could interact with Src kinase domain and possibly restrain the kinase from being activated by hindering the ATP binding. In conclusion, cucurbitacin B exhibited anti-metastatic property in CCA cells via negatively influencing Src and Src-related oncogenic signaling. This compound may therefore be a potential therapeutic drug for further development as an anti-Src agent for treatment of metastatic CCA.

Objective: Cholangiocarcinoma (CCA) is a malignant tumor with aggressive metastatic property resulted from dysregulation of metastasis-regulated signaling pathways. The aim of this study was to investigate the effect of cucurbitacin B on metastatic behavior of CCA cells through modulation of focal adhesion kinase (FAK) protein. Methods: KKU-452 cells were treated with a specific FAK inhibitor, FAK inhibitor-14, or cucurbitacin B at various concentrations for 24 h. Cell viability was assessed by sulforhodamine B assay. The migratory and invasive abilities of the cells were investigated using wound healing and transwell invasion assays, respectively. The fibronectin-coated plate was used for adhesion assay. The effects of the test compounds on FAK activation and the expression of metastasis-associated proteins were determined by Western blot analysis. The amount of MMP-9 was evaluated using a commercial ELISA Kit. Results: FAK inhibitor-14 and cucurbitacin B at concentrations which minimally affected KKU-452 cell viability could suppress FAK activation, evidently by decreased level of phospho-FAK protein after exposure to the compound. At these conditions, cucurbitacin B suppressed metastatic behavior including migration, invasion and adhesion abilities of CCA cells similar to FAK inhibitor-14. Further molecular studies demonstrated that FAK inhibitor-14 and cucurbitacin B downregulated the expression of metastasis-associated proteins including MMP-9, ICAM-1 and VEGF. Consequently, exposure to cucurbitacin B inhibited the production of MMP-9 enzyme in CCA cells similar to FAK inhibitor-14 treatment. Conclusion: FAK participated in regulation of metastatic behavior of KKU-452 CCA cells. Cucurbitacin B suppressed FAK activation in the cells which was associated with inhibition of metastasis essential steps and their related metastatic proteins. The compound may be developed as a novel therapeutic agent for CCA metastasis therapy.