Cytotoxic efficacy of 9-nitrocamptothecin in the treatment of human malignant melanoma cells in vitro.

Abstract

In a recent study, we showed that the plant alkaloid camptothecin (CPT) and its derivatives 9-nitro-CPT (9NC) and 9-amino-CPT (9AC) inhibit growth of both human melanocytes (MEL cells) and their malignant counterparts, malignant melanoma (BRO) cells in vitro. This growth inhibition was accompanied by an increase in the size of BRO cells followed by death, whereas cell size increase and death were not observed in MEL cells. In this study, we have extended those investigations to identify parameters and factors that can modulate the cytotoxic action of 9NC against BRO cells in culture. MEL cells treated with 9NC accumulate at the S/G2 boundary of the cell cycle and remain there for a prolonged period of time with only a small number of cells dying by apoptosis. The extent of accumulation correlates with the length of 9NC treatment and/or 9NC concentration in the cell culture. Furthermore, treatment with low 9NC concentrations for a prolonged time or treatment with high drug concentrations results in a fraction of MEL cells with hyperdiploidy. In contrast, 9NC-treated BRO cells are arrested in the S phase before they die by apoptosis. Interestingly, lower 9NC concentrations are more effective than higher concentrations in inducing apoptosis. Once 9NC initiates the process of apoptosis in BRO cells, these cells are irrevocably committed to it and continue to die even after removal of the drug from the culture. The drug effectiveness to induce apoptosis correlates with the stage of the S phase, in which it affects DNA replication, with late stages resulting in higher numbers of apoptotic cells. Finally, although various 9NC concentrations result in inhibition of BRO cell proliferation, higher 9NC concentrations produce more enlarged BRO cells as assessed by microscopy. Taken together, these observations provide useful information for clinical application of 9NC as a chemotherapeutic agent against malignant melanoma.