Peanut testa extracts enhance anticancer effect of cisplatin against human cholangiocarcinoma cells via modulation of histone deacetylase inhibitory activity

Objective: To investigate the effect of combination treatments of cisplatin and KK4 and ICG15042 peanut testa extracts against cholangiocarcinoma cells in vitro. Methods: The growth inhibition, cell cycle arrest and apoptosis of cholangiocarcinoma cells were determined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry analysis, respectively. The levels of proteins involved in apoptosis were assessed using Western blotting assays. The caspase activity was assessed using a colorimetric caspase activity assay. Results: Cisplatin and peanut (KK4 and ICG15042) testa extracts inhibited the growth of cholangiocarcinoma cell lines (KKU- M214 and KKU-100 cells) in a dose- and time-dependent manner. The combination treatments reduced cell viability and induced apoptosis of cholangiocarcinoma cells more efficiently than singledrug treatments. Cancer cell death synergistically mediated by cisplatin and peanut testa extracts was observed in KKU-M214 cells (combination index < 1.0) but not in KKU-100 cells (combination index > 1.0). The combination treatments also increased the sub- G1 population and caused KKU-M214 cell cycle arrest at S and G2/ M phases, which were the combined effects of cisplatin (S phase arrest) and peanut testa extracts (G2/M phase arrest). In addition, pERK1/2, Ac-H3, Bcl-2 and proteins related to apoptosis, including Bax and caspases 3, 8, 9, exhibited enhanced expression in KKU- M214 cells. The combination treatments caused down-regulation of p53, whereas the expression of p21 was fairly constant when compared with cisplatin single drug treatment. Conclusions: Peanut testa extracts in combination with cisplatin synergistically reduce cell viability and induce apoptosis through stimulation of caspases 3, 8 and 9 in KKU-M214 cells.