beta-escin and Bile-Duct-Neoplasms

To develop a safe and effective agent for cholangiocarcinoma (CCA) chemotherapy.. A drug combination experiment was conducted to determine the effects of β-escin in combination with chemotherapy on CCA cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of β-escin and common chemotherapeutics on the proliferation of human CCA cells (QBC939, Sk-ChA-1, and MZ-ChA-1). Immunocytochemistry was used to detect the expression of P-glycoprotein (P-gp) protein. Luciferase reporter assay was used to detect the activation of the Wnt/β-catenin pathway. The protein levels of P-gp, pS9-GSK3β, pT216-GSK3β, GSK3β, β-catenin, and p-β-catenin were further confirmed by western blotting.. The drug sensitivity of QBC939 and QBC939/5-fluorouracil (5-FU) cells to 5-FU, vincristine sulfate (VCR), or mitomycin C was significantly enhanced by β-escin compared with either agent alone (P<0.05). In addition, the combination of β-escin (20 μmol/L) with 5-FU and VCR was synergic with a combination index<1. Further investigation found that the mRNA and protein expression of P-gp was down-regulated by β-escin. Moreover, β-escin induced GSK3β phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of β-catenin. Interestingly, activation of the GSK3β/β-catenin pathway induced by Wnt3a resulted in up-regulation of P-gp, which was effectively abolished by β-escin, indicating that β-escin down-regulated P-gp expression in a GSK3β-dependent manner.. β-escin was a potent reverser of P-gp-dependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3β/β-catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA.

Topics: Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B; beta Catenin; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Escin; Fluorouracil; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mitomycin; Phosphorylation; RNA, Messenger; Signal Transduction; Transfection; Vincristine

The study aimed to evaluate the effects of β-escin on human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1) and to explore its mechanisms. Cell growth, cell cycle and apoptosis were investigated, respectively, by MTT assay, single PI and FITC/PI double-staining flow cytometry, and fluorescence microscopy. The protein expression was determined by western blotting. The study revealed that β-escin inhibited cholangiocarcinoma cell growth in a dose- and time-dependent manner, and the cell cycle of QBC939 and Sk-ChA-1 cells was arrested in the G2/M phase, and MZ-ChA-1 cells in G1 phase. Apoptosis of the three cholangiocarcinoma cell lines induced by β-escin was associated with the collapse of the mitochondrial membrane potential and the activation of caspase-3. The apoptotic effect of β-escin was suppressed by pancaspase inhibitor z-VAD-fmk. Molecular dissection revealed that the antiapoptotic protein bcl-2 was down-regulated after cholangiocarcinoma cell lines were treated with β-escin, while the protein levels of bax and p53 were unchanged. Apoptosis was accompanied by an increase in reactive oxygen species (ROS). These results suggest that β-escin induces apoptosis of cholangiocarcinoma cells through an intrinsic mitochondrial caspase-dependent pathway, and the increase in the bax/bcl-2 ratio and ROS may play important roles in β-escin-induced apoptosis of cholangiocarcinoma cells.

Topics: Aesculus; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Escin; Humans; Membrane Potential, Mitochondrial; Mitochondria; Nuclear Proteins; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Tumor Protein p73; Tumor Suppressor Proteins