Abstract
The effects of the diatomic radical, nitric oxide (NO), on melphalan-induced cytotoxicity in Chinese hamster V79 and human MCF-7 breast cancer cells were studied using clonogenic assays. NO delivered by the NO-releasing agent (C2H5)2N[N(O)NO]- Na+ (DEA/NO; 1 mM) resulted in enhancement of melphalan-mediated toxicity in Chinese hamster V79 lung fibroblasts and human breast cancer (MCF-7) cells by 3.6- and 4.3-fold, respectively, at the IC50 level. Nitrite/nitrate and diethylamine, the ultimate end products of DEA/NO decomposition, had little effect on melphalan cytotoxicity, which suggests that NO was responsible for the sensitization. Whereas maximal sensitization of melphalan cytotoxicity by DEA/NO was observed for simultaneous exposure of DEA/NO and melphalan, cells pretreated with DEA/NO were sensitized to melphalan for several hours after NO exposure. Reversing the order of treatment also resulted in a time-dependent enhancement in melphalan cytotoxicity. To explore possible mechanisms of NO enhancement of melphalan cytotoxicity, the effects of DEA/NO on three factors that might influence melphalan toxicity were examined, namely NO-mediated cell cycle perturbations, intracellular glutathione (GSH) levels and melphalan uptake. NO pretreatment resulted in a delayed entry into S phase and a G2/M block for both V79 and MCF-7 cells; however, cell cycle redistribution for V79 cells occurred after the cells returned to a level of cell survival, consistent with treatment with melphalan alone. After 15 min exposure of V79 cells to DEA/NO (1 mM), GSH levels were reduced to 40% of control values; however, GSH levels recovered fully after 1 h and were elevated 2 h after DEA/NO incubation. In contrast, DEA/NO (1 mM) incubation did not reduce GSH levels significantly in MCF-7 cells (approximately 10%). Melphalan uptake was increased by 33% after DEA/NO exposure in V79 cells. From these results enhancement of melphalan cytotoxicity mediated by NO appears to be complex and may involve several pathways, including possibly alteration of the repair of melphalan-induced lesions. Our observations may give insights for improving tumour kill with melphalan using either exogenous or possibly endogenous sources of NO.
Highlights
Melphalan is a widely used chemotherapeutic agent for a variety of tumour types and is thought to exert its cytotoxic effect by alkylating and cross-linking DNA (Calabresi and Parks, 1985; Colvin and Chabner, 1990)
Because of the recent availability of chemical agents that can release nitric oxide (NO) (Maragos et al, 1991) and our recent findings that NO can enhance the cytotoxicity of BCNU (Laval and Wink, 1994), we have studied the effect of NO on the cytotoxicity of melphalan in Chinese hamster V79 lung fibroblasts and human breast cancer (MCF-7) cells
DEA/NO treatment alone resulted in a 50% reduction in survival for V79 cells
Summary
Melphalan is a widely used chemotherapeutic agent for a variety of tumour types and is thought to exert its cytotoxic effect by alkylating and cross-linking DNA (Calabresi and Parks, 1985; Colvin and Chabner, 1990). Melphalan has shown efficacy alone or in combination with other chemotherapeutic agents, there remains a need to explore avenues to broaden its spectrum and effectiveness. Intracellular glutathione (GSH) levels have been shown to modulate melphalan cytotoxicity, and depletion of GSH by buthionine sulphoximine (BSO) has been shown to enhance melphalan cytotoxicity significantly (Green et al, 1984; Ozols et al, 1987) Based on these preclinical findings, phase I clinical trials have been completed recently establishing a safe BSO dose to use in conjunction with melphalan (O'Dwyer et al, 1996)
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