BackgroundCholangiocarcinoma (CCA) is highly resistant to most of the known chemotherapeutic treatments. NAD(P)H-quinone oxidoreductase 1 (NQO1) is an antioxidant/detoxifying enzyme recently recognized as an important contributor to chemoresistance in some human cancers. However, the contribution of NQO1 to chemotherapy resistance in CCA is unknown.MethodsTwo CCA cell lines, KKU-100 and KKU-M214, with high and low NQO1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents; 5-fluorouracil (5-FU), doxorubicin (Doxo), and gemcitabine (Gem). NQO1 and/or p53 expression in KKU-100 cells were knocked down by siRNA. NQO1 was over-expressed in KKU-M214 cells by transfection with pCMV6-XL5-NQO1 expression vector. CCA cells with modulated NQO1 and/or p53 expression were treated with chemotherapeutic agents, and the cytotoxicity was assessed by SRB assay. The mechanism of enhanced chemosensitivity was evaluated by Western blot analysis.ResultsWhen NQO1 was knocked down, KKU-100 cells became more susceptible to all chemotherapeutic agents. Conversely, with over-expression of NQO1 made KKU-M214 cells more resistant to chemotherapeutic agents. Western blot analysis suggested that enhanced chemosensitivity was probably due to the activation of p53-mediated cell death. Enhanced susceptibility to chemotherapeutic agents by NQO1 silencing was abolished by knockdown of p53.ConclusionsThese results suggest that inhibition of NQO1 could enhance the susceptibility of CCA to an array of chemotherapeutic agents.

Abstract. We previously reported that upregulation of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cholangiocarcinoma (CCA; a fatal bile duct cancer) was associated with poor prognosis. It was also demonstrated that the suppression of NQO1 was able to enhance the chemosensitivity of CCA cells. In the present study, in order to elucidate the biological role of NQO1 in CCA, the effects of small interfering RNA (siRNA)-mediated knockdown of NQO1 on cell proliferation, cell cycle and migration were determined in KKU-100 CCA cells, which notably expressed NQO1. The cell proliferation ability and cell cycle distribution were identified by clonogenic cell survival assay and flow cytometric analysis, respectively. Wound healing and Transwell migration assays were performed to evaluate cell migration. The molecules involved in cell proliferation and migration were determined by western blot analysis and reverse transcription-quantitative polymerase chain reaction analysis. The results demonstrated that NQO1 siRNA-mediated knockdown effectively impaired colony formation capacity, induced cell cycle arrest at the G 1 phase and suppressed migration of KKU-100 cells. CCA cells transfected with NQO1 siRNA exhibited increased expression levels of p21 and decreased cyclin D1 protein expression levels. Furthermore, the ratio of matrix metalloproteinase 9/tissue inhibitors of metalloproteinases 1 (TIMP1) mRNA expression level was decreased in the NQO1-knockdown cells. Therefore, the present study provided evidence supporting the biological role of NQO1 in the regulation of cell proliferation, cell cycle and migration of CCA cells. Therefore, NQO1 may prove to be a potential molecular target to enhance CCA treatment.

Cholangiocarcinoma (CCA), a malignancy of biliary duct with a very poor prognosis, is the leading cause of cancer death in countries of the Mekong subregion. Liver fluke infection is the main etiological factor, but genetic variation has been recognized as also important in conferring susceptibility to CCA risk. Nuclear factor (erythroid derived 2)-like 2 (NRF2) is a key transcription factor in detoxification and antioxidant defense. Emerging evidence has demonstrated that genetic polymorphisms in the NRF2 gene may be associated with cancer development. The objectives of this study were to investigate the association of NRF2 genetic polymorphism with CCA risk and to evaluate the influence of the NRF2 genotype on survival time of affected patients. Single nucleotide polymorphisms (SNPs) of the NRF2 gene, including rs6726395: A/G, rs2886161: C/T, rs1806649: C/T, and rs10183914: C/T, were analyzed using TaqMan ® SNP genotyping assays. Among 158 healthy northeastern Thai subjects, the allele frequencies were 41, 62, 94, and 92%, respectively. The correlation of NRF2 SNPs and CCA risk was analyzed in the 158 healthy subjects and 198 CCA patients, using unconditional logistic regression. The results showed that whereas the NRF2 SNPs were not associated with CCA risk (p>0.05), Kaplan-Meier analysis of 88 intrahepatic CCA patients showed median survival time with rs6726395 genotypes of GG and AA/AG to be 344±138 (95%CI: 73-615) days and 172±37 (95%CI: 100-244) days, respectively, (p<0.006). On multivariate Cox proportional hazard analysis, the GG genotype of rs6726395 was found to be associated with longer survival with a hazard ratio of 0.54 (95%CI: 0.31-0.94). In addition, non-papillary adenocarcinoma was associated with poor survival with a hazard ratio of 2.09 (95%CI: 1.16-3.75). The results suggest that the NRF2 rs6726395 polymorphism can be a potential prognostic biomarker for CCA patients.