Synergistic effects of isomorellin and forbesione with doxorubicin on apoptosis induction in human cholangiocarcinoma cell lines

BACKGROUND: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous studies, isomorellin and forbesione, caged xanthones isolated from Garcinia hanburyi, were found to induce cell cycle arrest and apoptosis in CCA cell lines. The subject of our inquiry is the synergistic effect(s) of these caged xanthones with doxorubicin on growth inhibition and apoptosis induction in human CCA cell lines. METHODS: KKU-100, KKU-M139 and KKU-M156 cell lines and Chang cells were treated with either isomorellin or forbesione alone or in combination with doxorubicin. Cell viability was determined using the sulforhodamine B assay. The combined effects of plant compounds with doxorubicin were analyzed using the isobologram and combination index method of Chou-Talalay. Apoptosis was determined by ethidium bromide/acridine orange staining. Protein expressions were determined by Western blot analysis. RESULTS: Isomorellin or forbesione alone inhibited the growth of these CCA cell lines in a dose-dependent manner and showed selective cytotoxicity against CCA cells but not against Chang cells. Isomorellin/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-M139 and KKU-M156 cells, while the forbesione/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-100 and KKU-M139 cells. The percentages of apoptotic cells were significantly higher in the combined treatments than in the respective single drug treatments. The combined treatments strongly enhanced the expression of Bax/Bcl-2, activated caspase-9 and caspase-3, while suppressing the expression of survivin, procaspase-9 and procaspase-3, compared with single drug treatments. The degree of suppression of NF-κB activation mediated by a decrease in the expression of NF-κB/p65, a reduction of the pIκB-α level and an increase in the IκB-α protein level, was significantly higher in the combined treatment groups than in the single drug treatment groups. The degree of suppression of MRP1 protein expression was also significantly higher in the combined treatment than in the single drug treatment groups. CONCLUSION: The combinations of isomorellin/doxorubicin and forbesione/doxorubicin showed significant synergistic effects on the growth inhibition and apoptosis induction in KKU-M156 and KKU-100 cells. Caged xanthones may be useful adjunct treatments with chemotherapy for Opisthorchis viverrini (OV)-associated CCA.