Biological Activity of Mycale Marine Sponge Secondary Metabolites, Including Peloruside A, a Novel Microtubule Stabilising Compound

Abstract

The three Mycale sp. marine sponge secondary metabolites mycalamide, pateamine and peloruside, were examined for cytotoxic and immunosuppressive activity. Each of the compounds potently inhibited the proliferation of a number of cells lines (measured by trypan blue dye exclusion and MTT assay) due, at least in part, to the induction of apoptotic cell death as evidenced by DNA laddering, phosphatidylserine exposure and nuclear fragmentation. None of the sponge metabolites selectively inhibited the production of interleukin-2 from mixed lymphocyte reaction-stimulated lymphocytes in the absence of cytotoxicity, thereby indicating that the compounds are not selectively immunosuppressive. Although mycalamide and pateamine were more potent inducers of apoptosis in 32D cells transformed with either the ras or bcr/abl oncogene than untransformed parental 32D cells, the increased susceptibility of the transformed cells was also observed in response to the other anti-proliferative compounds cytosine-β-D-arabinofuranoside, puromycin, and cycloheximide. The molecular mechanism of action of peloruside was examined in more detail. Despite sharing structural similarity with bryostatin, a protein kinase C activator, peloruside did not exhibit protein kinase C-dependent biological activity. Unlike bryostatin, peloruside was not to synergise with interleukin-2 or ionomycin to stimulate proliferation of splenocytes, and was too structurally dissimilar to compete with [20(n)-3H]-phorbol 12, 13-dibutyrate for binding to protein kinase C in HL-60 cells. Peloruside's ability to induce arrest in the G2/M phase of the cell cycle in H44l cells was similar to paclitaxel, a microtubule polymerising compound. Peloruside and paclitaxel also shared the ability to cause β-tubulin to translocate from the soluble to the particulate HL-60 cell fraction, and to cause purified tubulin to polymerise in vitro. Abnormally polymerised microtubule bundles and multiple microtubule asters were observed in both peloruside and paclitaxel treated H44l cells by β-tubulin immunostaining. Disruption of microtubule dynamics for 48 h by either peloruside, paclitaxel, or colchicine exposure induced multiple micronuceli in H441 and K562 cells. In addition, unusual rod-like fibres were observed in peloruside-treated H441 cells, which did not stain for β-tubulin or actin. The identity of these fibres is unknown, although they were also observed in H441 cells exposed to either paclitaxel or colchicine. 2-Dimensional electrophoresis of proteins extracted from HL-60 cells exposed to 1 μM peloruside for 5 h revealed a mobility shift in a 25 kDa protein identified using the peptide mass fingerprint obtained by MALDI-TOF mass spectrometry, and protein sequence database searching, as a lymphocyte specific Rho-GDP dissociation inhibitor. This mobility shift was not observed in response to paclitaxel treatment. Modification of a 37 kDa protein (most likely to be an α-tubulin isoform) to a more acidic position on the 2-dimensional gel was observed in response to both peloruside and paclitaxel exposure. Thus, peloruside shares the microtubule stabilising properties of paclitaxel, making it a promising candidate for further development as an anti-cancer compound.