Role of glutathione in the in vitro synergism between 4-hydroperoxy-cyclophosphamide and cisplatin in leukemia cell lines.

To explain the sequence-dependent in vitro cytotoxic synergism between 4-hydroperoxycyclophosphamide (4-HC) and cisplatin in the K-562 human leukemia cell line, we have hypothesized that 4-HC decreases cellular glutathione (GSH) levels and that the resulting diminution of the cellular protective effect of GSH leads to the increased cytotoxicity of cisplatin. Exposure of K-562 cells to 4-HC resulted in a concentration- and time-dependent depletion of cellular GSH. To determine the effect of modulation of GSH levels on the toxicity of cisplatin, K-562 cells were exposed to buthionine sulfoximine (BSO) and/or GSH ethyl esters. Depletion of GSH to approximately 10% of control values by BSO potentiated the cytotoxicity of cisplatin, while rapid replenishment of GSH to within normal levels by GSH esters abolished the potentiation of BSO. Doubling cellular GSH by incubation with GSH esters protected against cisplatin cytotoxicity. Of importance, pretreatment of K-562 cells with BSO, in addition to increasing the cytotoxicity of 4-HC and cisplatin, abolished the synergism between the two drugs. The working hypothesis was also tested in two other cell lines in which the cytotoxic synergism between 4-HC and cisplatin was exhibited: the Raji cell line, a human lymphoblastic cell line, and the L1210-CPA cell line, a subclone of the murine L1210 leukemia with resistance to 4-HC. GSH levels in these two cell lines were not altered by incubation with concentrations of 4-HC at which the synergism was observed. In conclusion, the data for the K-562 cell line, indicating that (a) 4-HC depletes cellular GSH levels, (b) the lowering of cellular GSH levels enhances the toxicity of cisplatin, and (c) intact GSH stores are required for the synergism, strongly support the postulate that the cytotoxic synergism between 4-HC and cisplatin is modulated by GSH levels in this cell line. However, the lack of 4-HC-mediated depletion of GSH at concentrations of 4-HC resulting in cytotoxic synergism in the Raji and L1210-CPA cell line indicates that mechanisms other than modulation of GSH levels by 4-HC are responsible for the synergism in these cells.