Rapid Induction Of Chemoresistance In Melanoma Cells Exposed In Vitro To Temo-Zolomide Alone Or Combined With O6-Benzyl-Guanine

Abstract

Introduction Temozolomide (TMZ) an antitumor DNA methylating agent, is able to induce O6-methyl-guanine DNA adducts. Its activity is largely conditioned by a fully functional mismatch repair system (MMR). In contrast, the antineoplastic effects of the agent are suppressed by the DNA repair enzyme O6-alkylguanine-DNA-alkyl-transferase (OGAT), that removes methyl adducts. The OGAT inhibitor O6-benzyl-guanine (BG) abrogates resistance to TMZ of malignant cells expressing high OGAT levels (i.e. OGAT-dependent resistance, ODR), but not of MMR-deficient tumors, or of tumors showing other resistance mechanisms unrelated to OGAT (i.e. OGAT-independent resistance, OIR). Aim of the study To establish the onset of ODR or of OIR in melanoma cells treated in vitro with TMZ alone or associated with BG. Methods Three MMR-proficient melanoma cell clones with barely detectable OGAT levels and sensitive to TMZ, originated from 3 different parental lines, were treated with TMZ (50 μM), or TMZ (50 μM)+BG (5 μM) daily for 5 days, for 4 consecutive cycles, and tested for sensitivity to TMZ or TMZ+BG, at the end of each cycle. Results Dramatic increase of chemo-resistance to TMZ alone (i.e. ranging from more than 200% to more than 1200% increase of IC50 of TMZ) was detected in all 3 clones, after as early as 1 cycle of treatment with TMZ alone (i.e. TMZ sublines). This increase was maintained essentially constant for up to 4 treatment cycles. In all TMZ sublines OGAT expression was found to be increased, and BG partially reversed resistance to TMZ, thus suggesting that resistance was, at least in part, of ODR type. A similar phenomenon was observed when the 3 clones were treated with TMZ+BG (i.e. TMZ/BG sublines). However, in this case BG reduced TMZ resistance in only 2 of the 3 TMZ/BG sublines. Consistently with these results, OGAT levels were up-regulated in the 2 TMZ/BG sublines still sensitive to TMZ+BG, but not in the TMZ/BG subline in which the inhibitor failed to alleviate TMZ resistance. Conclusions Treatment of melanomas with TMZ, alone or with the OGAT inhibitor BG, shows an unexpectedly high capacity of inducing early anti-TMZ resistance of both ODR and OIR type. The elucidation of the molecular mechanisms underlying OIR requires further studies. However, the present results provide valuable information concerning the poor therapeutic response to triazenes observed in melanoma patients. Supported by the Italian Ministry of Health.