Original Article
Copyright ©2010 Baishideng. All rights reserved.
World J Gastroenterol. May 21, 2010; 16(19): 2362-2370
Published online May 21, 2010. doi: 10.3748/wjg.v16.i19.2362
Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells
Benjaporn Buranrat, Auemduan Prawan, Upa Kukongviriyapan, Sarinya Kongpetch, Veerapol Kukongviriyapan
Benjaporn Buranrat, Auemduan Prawan, Sarinya Kongpetch, Veerapol Kukongviriyapan, Department of Pharmacology, Faculty of Medicine, and Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen 40002, Thailand
Upa Kukongviriyapan, Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Author contributions: Buranrat B, Prawan A and Kukongviriyapan V designed the research; Buranrat B and Kongpetch S performed the research; Buranrat B, Prawan A, Kukongviriyapan U, Kongpetch S and Kukongviriyapan V analyzed the data; Buranrat B and Kukongviriyapan V wrote the paper; all authors gave final approval of the manuscript.
Supported by Thailand Research Fund, National Science and Technology Development Agency, research funding from Khon Kaen University; the Royal Golden Jubilee Ph.D. Program (to Kongpetch S); the Office of the Commission on Higher Education (to Buranrat B)
Correspondence to: Veerapol Kukongviriyapan, Associate Professor, Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. veerapol@kku.ac.th
Telephone: +66-43-348397  Fax: +66-43-348397
Received: January 29, 2010
Revised: March 5, 2010
Accepted: March 12, 2010
Published online: May 21, 2010

AIM: To investigate whether dicoumarol, a potent inhibitor of NAD(P)H quinone oxidoreductase-1 (NQO1), potentiates gemcitabine to induce cytotoxicity in cholangiocarcinoma cells (CCA) and the role of reactive oxygen generation in sensitizing the cells.

METHODS: Four human cell lines with different NQO1 activity were used; the human CCA cell lines, KKU-100, KKU-OCA17, KKU-M214, and Chang liver cells. NQO1 activity and mRNA expression were determined. The cells were pretreated with dicoumarol at relevant concentrations before treatment with gemcitabine. Cytotoxicity was determined by staining with fluorescent dyes. Oxidant formation was examined by assay of cellular glutathione levels and reactive oxygen species production by using dihydrofluorescein diacetate. Measurement of mitochondrial transmembrane potential was performed by using JC-1 fluorescent probe. Western blotting analysis was performed to determine levels of survival related proteins.

RESULTS: Dicoumarol markedly enhanced the cytotoxicity of gemcitabine in KKU-100 and KKU-OCA17, the high NQO1 activity and mRNA expressing cells, but not in the other cells with low NQO1 activity. Dicoumarol induced a marked decrease in cellular redox of glutathione in KKU-100 cells, in contrast to KKU-M214 cells. Dicoumarol at concentrations that inhibited NQO1 activity did not alter mitochondrial transmembrane potential and production of reactive oxygen species. Gemcitabine alone induced activation of NF-κB and Bcl-XL protein expression. However, gemcitabine and dicoumarol combination induced increased p53 and decreased Bcl-XL levels in KKU-100, but not in KKU-M214 cells.

CONCLUSION: NQO1 may be important in sensitizing cells to anticancer drugs and inhibition of NQO1 may be a strategy for the treatment of CCA.

Keywords: NAD(P)H quinone oxidoreductase-1, Dicoumarol, Cholangiocarcinoma, Chemotherapy, Oxidative stress