Objective: To investigate the cytotoxic activity and molecular mechanism(s) of two Thai noni juice (TNJ) products ethanolic extracts against cholangiocarcinoma (CCA) cell lines and non-cancerous cells, and to explore phenolic acid compositions of TNJ products. Methods: Phenolic acid profiles of TNJ Chiangrai (TNJ-Cr) and TNJ Buasri (TNJ-Bs) ethanolic extracts were determined by HPLC. The cytotoxicity of TNJ ethanolic extracts on cancer and non-cancerous cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue assays. Mechanism(s) underlying the anti-CCA activity of TNJ ethanolic extracts were determined by cell cycle, apoptosis, and reactive oxygen species (ROS) generation assays. The expression levels of proteins involved in apoptosis and ERK signaling were evaluated by Western blot analysis. Results: Phenolic acid profiles of both TNJ ethanolic extracts showed that the p-hydroxybenzoic, vanillic, and protocatechuic acids were the major phenolic acids in TNJ products. Cytotoxicity assays revealed that the TNJ-Cr and TNJ-Bs ethanolic extracts reduced viability of CCA cell lines through induction of apoptosis by up-regulation of p53 and Bax proapoptotic proteins. Both TNJ ethanolic extracts promoted ROS generation by activating the ERK1/2 signaling in well-differentiated CCA cells KKU-213B. Meanwhile, TNJ ethanolic extracts did not induce ROS production in poorly differentiated CCA cells KKU-100. Both TNJ ethanolic extracts showed no toxicity to human peripheral blood mononuclear cells. Conclusions: TNJ ethanolic extracts could inhibit CCA cell proliferation by inducing ROS generation and apoptosis and may be applicable for combination therapies in CCA treatment.

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- A nd 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.