Sensitization effect of l-2-oxothiazolidine-4-carboxylate on tumor cells to melphalan and the role of 5-oxo- l-prolinase in glutathione modulation in tumor cells

Abstract

5-Oxo- l-prolinase (5-OPase) (EC 3.5.2.9) links the synthesis and metabolism of glutathione (GSH) in the γ-glutamyl cycle. Previous studies showed that l-2-oxothiazolidine-4-carboxylate (OTZ), a 5-oxo- l-proline analog that is metabolized by 5-OPase, can preferentially decrease the cellular GSH levels in vivo in rat mammary tumors and sensitizes the tumors to the alkylating agent melphalan. The present study investigated the biochemical mechanism of this effect in a human breast cancer cell line, MCF7. We found that OTZ decreased the GSH levels in MCF7 cells. When the cells were treated with OTZ plus melphalan, the cytotoxicity of melphalan was increased as compared with that of melphalan alone, and this effect could be reversed by the addition of glutamate, which is the product of 5-OPase reaction and a critical substrate in GSH synthesis. We concluded that OTZ increases melphalan toxicity by limiting glutamate production from 5-OPase for GSH synthesis. We also observed that the expression of 5-OPase in the stably transfected MCF7 cells decreased the cellular GSH contents, sensitized the cells to melphalan toxicity, and diminished the sensitizing effect of OTZ. Furthermore, exposure to the GSH-depleting agent buthionine sulfoximine led to increased expression of 5-OPase in both MCF7 cells and the peripheral blood mononuclear cells of patients. These results indicate a critical interaction between cellular GSH levels and 5-OPase activity that could be important in GSH modulation in therapeutic settings.