Abstract
Dicumarol is a naturally occurring anticoagulant derived from coumarin that induces cytotoxicity and oxidative stress in human pancreatic cancer cells (Cullen, J. J., Hinkhouse, M. M., Grady, M., Gaut, A. W., Liu, J., Zhang, Y., Weydert, C. J. D., Domann, F. E., and Oberley, L. W. (2003) Cancer Res. 63, 5513-5520). Although dicumarol has been used as an inhibitor of the two-electron reductase NAD(P)H:quinone oxidoreductase (NQO1), dicumarol is also thought to affect quinone-mediated electron transfer reactions in the mitochondria, leading to the production of superoxide (O2*-) and hydrogen peroxide (H(2)O(2)). We hypothesized that mitochondrial production of reactive oxygen species mediates the increased susceptibility of pancreatic cancer cells to dicumarol-induced metabolic oxidative stress. Dicumarol decreased clonogenic survival equally in both MDA-MB-468 NQO1(-) and MDA-MB-468 NQO1+ breast cancer cells. Dicumarol decreased clonogenic survival in the transformed fibroblast cell line IMRSV-90 compared with the IMR-90 cell line. Dicumarol, with the addition of mitochondrial electron transport chain blockers, decreased clonogenic cell survival in human pancreatic cancer cells and increased superoxide levels. Dicumarol with the mitochondrial electron transport chain blocker antimycin A decreased clonogenic survival and increased superoxide levels in cells with functional mitochondria but had little effect on cancer cells without functional mitochondria. Overexpression of manganese superoxide dismutase and mitochondrial-targeted catalase with adenoviral vectors reversed the dicumarol-induced cytotoxicity and reversed fluorescence of the oxidation-sensitive probe. We conclude mitochondrial production of reactive oxygen species mediates the increased susceptibility of cancer cells to dicumarol-induced cytotoxicity.
Highlights
Dicumarol (3,3Ј-methylenebis[4-hydroxycoumarin]) is a naturally occurring anticoagulant derived from coumarin that is obtained from the sweet clover (Melilotus alba) (1, 2)
To determine the role of NQO1 in dicumarol-induced cytotoxicity in our model system, clonogenic cell survival, cell viability, and hydroethidine fluorescence were determined in MDA-MB-468 breast cancer cells, and the isogenic cell line MDA-MB-468 was stably transfected with constitutive NQO1 after treatment with dicumarol 50 M for 4 and 24 h (Fig. 1)
There was a trend toward increased cell viability (Fig. 1A) and clonogenic survival (Fig. 1B) after treatment with dicumarol 50 M for 24 h in the NQO1ϩ cell line, which may be attributed to the antioxidant properties of NQO1 (30)
Summary
Mn-SOD, manganese superoxide dismutase; METC, mitochondrial electron transport chain; mitCAT, mitochondrially targeted catalase; AntA, antimycin A; Myx, myxothiazol; O2. , superoxide anion radical; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; DCFDA, carboxymethyl dichlorofluorescein (DCF) diacetate; m.o.i., multiplicity of infection; pHPA, para-hydroxyphenyl acetic acid; ROS, reactive oxygen species. Dicumarol-induced cytotoxicity of MIA PaCa-2 cells was accompanied by a dose-dependent and time-dependent increase in apoptosis as measured by flow cytometry and diaminobenzidine staining, which was associated with cytochrome c release from mitochondria and poly(ADP-ribose) polymerase cleavage (18). These results demonstrated that dicumarol treatment in these human tumor cells caused cytotoxicity as well as activation of mitochondrial signaling and pathways thought to be involved with cell death. We demonstrated dicumarol-induced cytotoxicity, and increased superoxide levels were enhanced with the addition of mitochondrial electron transport chain (METC) blockers in cancer cell lines. Overexpression of Mn-SOD and mitochondrial-targeted catalase with adenoviral vectors reversed the dicumarol-induced cytotoxicity and decreased ROS levels
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