Endogenous production and exogenous exposure to nitric oxide augment doxorubicin cytotoxicity for breast cancer cells but not cardiac myoblasts

Abstract

We studied the effect of nitric oxide (NO) on the anticancer activity of doxorubicin. When MCF-7 human breast cancer cells were exposed to an aqueous solution of NO delivered as a bolus 30 min prior to doxorubicin, the cytotoxic effect as measured in a clonogenic assay was increased (doxorubicin alone, 40% survival, doxorubicin plus NO, 5% survival). The NO donor diethylamine nitric oxide, but not inactivated donor, also yielded an increase in doxorubicin cytotoxicity. The sequence was important since the simultaneous application of NO with doxorubicin yielded only a small augmentation of effect, and the exposure of the cells to doxorubicin prior to the NO obliterated the augmentation. Prior depletion of glutathione by incubation of the cells for 24 h with d,l-buthionine-S,R-sulfoximine (BSO) further increased the cytotoxicity so that BSO plus NO plus doxorubicin killed all of the clones. MCF-7 cells transduced with inducible nitric oxide synthase gene (iNOS) through an adenoviral vector overexpressed iNOS and produced increased amounts of nitrite, an indicator of increased NO production. These iNOS transduced cells were more susceptible to doxorubicin than vector control or wild-type cells. Cell cycle progression of iNOS transduced cells was not different from controls. Likewise, iNOS transduction resulted in no change in cellular glutathione levels. For comparison, we examined the effect of iNOS transduction on the sensitivity of MCF-7 to edelfosine, a membrane-localizing anticancer drug without direct DNA interaction. Insertion of the iNOS had no effect on killing of the MCF-7 cells by this ether lipid class drug. We also tested the effect of iNOS transduction on doxorubicin sensitivity of H9c2 rat heart-derived myoblasts. We found no augmentation of cytotoxicity by NO, and this observation offers potential therapeutic tumor selectivity by using NO with doxorubicin. Therefore, we conclude that NO produced intracellularly by iNOS overexpression or delivered as a bolus sensitizes human breast cancer cells in culture to doxorubicin, but not to a cardiac cell line or to edelfosine. This augmentation is not due to a modulation of cell cycle distribution or measurable cellular glutathione resulting from the transduction.