IDENTIFICATION OF GLUTATHIONE-S-TRANSFERASE AS A DETERMINANT OF 4-HYDROPEROXYCYCLOPHOSPHAMIDE RESISTANCE IN HUMAN BREAST-CANCER CELLS

Aldehyde dehydrogenase (ALDH) is well known for its involvement in the resistance of tumor cells to cyclophosphamide (CPA) and its activated derivatives, such as 4-hydroperoxy-CPA (4HC). The role of other drug- metabolizing enzymes such as glutathione S-transferase (GST) in CPA re sistance is, however, less certain. In the present study of a human br east cancer cell line (MCF-7) exhibiting about 6-fold resistance to 4H C (MCF/HC), cellular levels of glutathione (GSH) were increased 1.4-fo ld, while cytosolic GST and ALDH activities were increased 2.7- and 7. 2-fold, respectively, relative to the MCF-7 parental line. No signific ant changes in glutathione peroxidase and NADPH cytochrome P450 reduct ase activity, and no increase in microsomal GST and GST pi mRNAs were found in the resistant cells. Treatment with the ALDH substrate octana l sensitized the cells to the cytotoxic effects of 4HC to a modest ext ent in both MCF-7 and MCF/HC cells [dose modification factor (DMF) of 1.4 and 1.6, respectively]. Depletion of GSH by treatment with the GSH synthesis inhibitor buthionine sulfoximine (BSO) enhanced the cytotox ic effect of 4HC to a similar extent in both cell lines. By contrast, ethacrynic acid, which inhibited GST activity by > 85% in MCF-7 and MC F/HC cell extracts without depletion of GSH, sensitized the resistant but not the parental cells to 4HC cytotoxicity, indicating the importa nce of GST as a determinant of 4HC resistance in these cells. This con clusion is supported by the observation that in MCF/HC cells, ethacryn ic acid in combination with BSO increased the DMF 3-fold higher than d id BSO or EA alone, while in the parental MCF-7 cells ethacrynic acid with BSO had no significant chemosensitization effect over BSO alone. These studies establish that in addition to ALDH, GST overexpression c an contribute to acquired resistance of tumor cells to IHC and, furthe rmore, suggest that modulators that target the GSH/GST system could be useful in overcoming CPA resistance in the clinic.