Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells.
OBJECTIVE
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-kappaB 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.


CONCLUSIONS
NQO1 may be important in sensitizing cells to anticancer drugs and inhibition of NQO1 may be a strategy for the treatment of CCA.
Buranrat
Benjaporn
B
Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Prawan
Auemduan
A
Kukongviriyapan
Upa
U
Kongpetch
Sarinya
S
Kukongviriyapan
Veerapol
V
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D001650:Bile Duct Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D001653:Bile Ducts, Intrahepatic / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D018281:Cholangiocarcinoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001728:Dicumarol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; C494811:BCL2L1 protein, human; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051020:bcl-X Protein; D003841:Deoxycytidine; D001728:Dicumarol; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D005978:Glutathione; D000093542:Gemcitabine