In vitro and in vivo comparison between the effects of treatment with adenosine triphosphate and treatment with buthionine sulfoximine on chemosensitization and tumour growth of B16 melanoma

In this study we compare the effects of treatment with external sodium aden osine 5'-triphosphate (ATP) with the effects of L-buthionine-SR-sulfoximine (BSO) on B16 melanoma growth and on the modulation of the cytotoxic antime lanoma activity of cyclophosphamide (CY). We evaluated the in vitro effects of treatment with ATP or BSO on intracellular glutathione (GSH) levels, mi tochondrial membrane potential (Delta Psi(m)) and the proliferation rate of the B16F10 melanoma cell line. Compared with untreated cells, Delta Psi(m) , and GSH levels were already significantly decreased (25% and 57% reductio n, respectively) after the first hour of incubation in culture cells expose d to 3 mM ATP. After 24 and 48 h a major reduction was observed in Delta Ps i(m) (nearly 30%). GSH levels were also maximally depleted at 24h (approxim ately 75%) and partially recovered (up to 37% of levels of control) after A TP was removed from the medium. At 24 and 48 h, the proliferation rate was decreased 1.4- and 1.7-fold, respectively, compared with control cells. Tre atment with 50 mu M BSO produced a time-dependent decrease in GSH levels (0 .5, 21, 48 and 97.3% reduction at 1, 4, 8 and 24 h, respectively), but up t o 54% of the levels of control cells was recovered after BSO was removed fr om the medium. In contrast to ATP, neither Delta Psi(m) nor proliferation r ate was significantly modified in the first 24 h with BSO treatment. At 48 h, Delta Psi(m) was reduced by nearly 27%, and cell proliferation decreased 1.2-fold compared with controls. When the in vitro cytotoxic effect of low dose acrolein (an active metabolite of CY) in combination with BSO or ATP was analysed, a synergistic effect was found between BSO and acrolein, with a dose modification factor (DMF) of 1.98, but the antiproliferative effect s of acrolein plus ATP were only approximately additive (DMF=1.05). In addi tion, in in vivo studies differential effects were found between ATP and BS O. Specifically, whereas BSO alone significantly increased the survival tim e of mice bearing B16 melanoma liver metastases, and enhanced the cytotoxic effect of CY on this tumour model, no therapeutic benefits could be observ ed with AIP treatment, either alone or in combination with diethyl maleate (a GSH-depleting agent) and CY. In conclusion, our findings show that in ou r experimental system, both extracellular ATP and BSO have growth-inhibitor y properties against B16 melanoma in vitro. In vivo, however, only BSO prod uces a chemosensitizing effect, whereas ATP has not proved useful as a biol ogical modifier of chemotherapy. (C) 1999 Lippincott Williams & Wilkins.