Cellular and Molecular Actions of Peloruside A, a Novel Anti-Cancer Agent

Abstract

The secondary metabolites peloruside, pateamine and mycalamide, isolated from a New Zealand marine sponge (Mycale sp.), are highly cytotoxic to mammalian cells. All three compounds activate apoptosis; although their biological targets differ. Peloruside stabilises microtubules in a similar fashion to the anti-cancer drug paclitaxel. Pateamine and mycalamide, however, are protein synthesis inhibitors, with anti-viral and anti-fungal properties, respectively. In the present study, we examined in more detail the ability of peloruside to stabilise microtubules. The concentration of peloruside required to induce microtubule formation (Ccrit) from highly purified tubulin in the presence of GTP was 10.8 µM. The Ccrit for paclitaxel under the same conditions was 4.3 µM. Binding competition assays using a fluorescent paclitaxel analogue demonstrated that peloruside bound to a site distinct to the taxoid binding site on β-tubulin. Laulimalide is the only other known microtubule stabilising agent that binds to a different site from paclitaxel. Using MALDI-TOF mass spectrometry to measure the presence of bound or free ligand, laulimalide was shown to displace peloruside from microtubules, indicating a similar tubulin binding site for these two compounds. These results were confirmed using a cell viability assay on cell lines with mutations at the taxoid binding site on β-tubulin rendering the cells resistant to paclitaxel and/or epothilone (another microtubule stabilising agent that binds to the taxoid binding site). Mutations at the taxoid binding site did not confer resistance to peloruside. As P-gp expression is a major cause of anti-cancer drug failure, it was important to determine whether peloruside was a substrate for this efflux pump. Using the MTT assay, cell lines overexpressing P-gp were found to be 25-fold more resistant to peloruside than the parental cells. Paclitaxel on the other hand, a known substrate for the P-gp pump, was 120- to 160-fold less potent in the two multi-drug resistant (MDR) cell lines used. The same cells were 15-fold or less resistant to epothilone B. The addition of the P-gp inhibitor verapamil increased the potency of paclitaxel greatly, but only had a moderate effect on peloruside and epothilone B, confirming the source of the resistance as increased P-gp expression. Mycalamide and pateamine, which are known protein synthesis inhibitors, initially activated caspase-8 and -9, respectively, in human myeloid leukaemic cells (HL-60). Peloruside and paclitaxel, however, failed to induce caspase-8 or -9 activity in these cells, and synchronisation of the cell cycle with 2 mM thymidine had no effect on this response. It was concluded from the overall results of this study that peloruside has good potential as a drug replacement to treat paclitaxel resistant cancers because of its moderate affinity for the P-gp efflux pump and its unique binding site on polymerised microtubules.