Antagonism of the x(-/c) cystine/glutamate antiporter by sulfasalazine inhibits proliferation of human breast carcinoma cells

Breast cancer is the leading cause of death in women aged 18–54 in the United States. Its etiology most likely reflects contribution of numerous factors. Our study demonstrates that malignant progression of breast cancer is associated with increased reliance on the x_c− cystine/glutamate antiporter. In addition, we provide evidence suggesting that the anti-inflammatory drug, sulfasalazine (SASP), selectively antagonizes the x_c− antiporter, and inhibits proliferation of breast epithelial cells. The structure activity relationship studies have identified various moieties of SASP which may be involved in antagonism of the x_c− antiporter. Suppression of cystine uptake by SASP led to depletion of intracellular glutathione (GSH) in a time- and concentrated-dependent manner. In addition, we provide evidence which suggest that SASP may be an inhibitor of drug-efflux pump, multidrug associated resistance protein (MRP). Importantly, SASP, in conjunction with a potent MRP-antagonist MK-571, exerted an additive effect in the intracellular uptake of 3H-methotrexate, a well-characterized substrate for MRP. As MRP and GSH have been shown to play major roles in drug-efflux and drug-metabolism of various potential anti cancer drugs, we investigated the effect of SASP-mediated GSH reduction on the sensitivity of tumor cells to growth suppressive effects of doxorubicin (DOX). It was observed that SASP enhanced the potency of DOX in the less invasive estrogen receptor (ER) positive cell line, MCF-7. The degree of enhancement was significantly higher in highly invasive ER-negative cell line, MDA-MB-231. Identification of new roles of SASP as GSH-depletor and inhibitor of MRP may lead to its utilization for treatment of various types of drug-resistant tumors. Unlike in other model systems, in which nuclear factor kappa B (NFκB) and GSH-S-transferase have been identified as targets for SASP, in breast cancer cell lines SASP did not exhibit an effect on these proteins. Alternate pathways for providing cystine, such as metabolism of methionine, may underlie the cytostatic effect rather than lethal effect of SASP in breast carcinoma cell lines. Thus, the project has identified a new marker, the x_c− antiporter, of tumor progression and revealed potential role of SASP in the treatment of cancer.