Mechanisms involved in the potentiation of melphalan by the bioreductive compound THNLA-1 in vitro

9-[3-(2-Nitro-1-imidazolyl)propylamino]-1,2,3,4-tetrahydroacridine hydrochl oride (THNLA-1) is a 2-nitroimidazole-based, weakly DNA-intercalating biore ductive agent that significantly potentiates the toxic effects of commonly used antitumor drugs such as melphalan (L-PAM) or cis-DDP in sensitive or r esistant cell lines in culture, as well as in solid tumors in mice, Potenti ation in vitro was observed when cells were preexposed to THNLA-1 under hyp oxic conditions before exposure to L-PAM under aerobic conditions. In this study we investigated possible mechanisms involved in the potentiation of L -PAM by THNLA-1 in V79 Chinese hamster cells. Limited depletion of glutathi one with buthionine sulfoximine or THNLA-1 under hypoxic pretreatment condi tions accounted for only 8.3% of the potentiation induced by THNLA-1. Howev er, DNA, RNA, and protein synthesis were inhibited in a synergistic way in cells preexposed to THNLA-1 under hypoxic conditions (2 h, 37 degrees C) an d then coexposed to various doses of L-PAM under aerobic conditions (1 h, 3 7 degrees C). Cell cycle analysis by flow cytometry showed a slow traverse through the S phase in the L-PAM-alone-treated cells. However, this phenome non was more prominent in the THNLA-1 plus L-PAM-treated cells. Under aerob ic co-incubation conditions with L-PAM, no difference was observed in the c ell cycle of L-PAM-alone-treated cells vs. THNLA-1 plus L-PAM-treated cells . Significantly increased apoptosis was observed in the hypoxia-pretreated cells with THNLA-1, 12 and 24 h posttreatment. Comet and alkaline elution a ssay analysis showed increased DNA cross-links in the hypoxia-pretreated ce lls with THNLA-1 compared to the L-PAM-alone-treated cells. Finally, potent ial lethal damage repair was totally suppressed only in the hypoxia-pretrea ted cells with THNLA-1. In conclusion, DNA damage and hindrance in its repa ir are the most important mechanisms in the potentiation of L-PAM by THNLA- 1, under hypoxic pretreatment conditions.