H-ras transformation increases susceptibility of a myeloid cell line to the cytostatic and cytotoxic effects of peloruside A, a novel natural marine compound

Abstract

Marine organisms have proven a rich source of natural, bioactive compounds. Mycale sp, a marine sponge growing in New Zealand coastal waters, is one of those organisms. A novel cytotoxic compound, peloruside A, was isolated from this sponge just recently. Preliminary experiments have shown peloruside to be cytotoxic, with an IC50 value in the nanomolar range. The cytoxicity of peloruside and its mechanism of action were further examined in this thesis. The murine myeloid 32D cell line and its H-ras-transformed derivative 32D-ras were used. Since the ras gene is a commonly mutated proto-oncogene, the 32D-ras cell line was used as a model malignant cell line to test peloruside's potential for anti-cancer treatment in humans. To be worthy of further development, peloruside would have to target malignant cells, selectively inducing cell death in these cells, without affecting normal cells. Furthermore, the toxin would have to induce this cell death by apoptosis, since apoptosis, unlike necrosis, does not cause an inflammatory reaction with consequent harmful side-effects. In this study, peloruside inhibited growth in both 32D and 32D-ras cells (cytostatic effect). MTT cell proliferation assays indicated that 32D cells were more susceptible to metabolic repression by peloruside than 32D-ras cells. Trypan blue (TB) exclusion assays that measure the actual number of viable cells demonstrated, however, that there was no significant difference in growth inhibition between the two cell lines. 32D-ras cells on the other hand, proved to be more susceptible to induction of cell death by peloruside (cytotoxic effect). Using TB assays to measure the% of viable cells, it was shown that the dose and time of exposure altered the selectivity of peloruside on 32D-ras cells. The viability of untransformed 32D cells was also affected by peloruside, but to a lesser extent. Further flow cytometric analysis by Annexin-V-FLUOS binding experiments and propidium iodide/DNA staining showed that at 1.6 J.lM, peloruside induces cell death specifically by apoptosis in at least a fraction of the cells. However, at this high concentration, peloruside induced significant necrosis as well. The exact proportion of cells dying by either process could not be determined.