ES-285, a novel marine anticancer agent: Investigation of mechanism of action using gene expression microarrays.

Abstract

Introduction: ES-285 is a novel anti-cancer agent originally isolated from the North Atlantic Clam, Spisula polynyma . Previous studies have shown that ES-285 has antiproliferative activity in a unique pattern across the NCI 60 cell line panel. Although the mechanism of this cellular response is unclear, ES-285 induces changes in cell morphology and the actin cytoskeleton, possibly though Rho signalling (Cuadros et al., Cancer Lett. 152 (1); 23-29). We have used cDNA gene expression microarrays to further investigate the mechanism of action of ES-285. . Methods: We have compared the cellular and molecular response to ES-285 with those of structurally-related compounds (safingol, sphingosine and ES-427, an inactive analogue of ES-285) together with compounds known to modulate Rho/ROCK signalling (FTI-277, GGTI-298 and Y-27632). Antiproliferative activity was assessed by MTT assay. Membrane integrity was investigated by following the release of LDH into culture medium after drug treatment. Effects on cell morphology and the actin cytoskeleton were visualized with confocal microscopy. Drug-induced changes in gene expression were measured using 22k cDNA microarrays ( www.crukdmf.icr.ac.uk ). . Results: ES-285 has antiproliferative activity against a panel of 10 cancer cell lines in vitro with IC50s 20-200nM. ES-285 causes minimal LDH release at 24 hours, but marked LDH release at 72 hours, when apoptotic cells are present. These effects are not seen with equimolar doses of ES-427. There was no correlation between constitutive actin stress fibre density of the different cell lines and their sensitivity to ES-285. However, in HT29 cells a concentration-dependent loss of stress fibres was seen, with abrogation of most fibres by 4 hours at a dose of 100nM (5xIC50). Loss of actin stress fibres was also seen with Y-27632 and GGTI-298 at 24 hours. No effect on stress fibres was seen with safingol, sphingosine or FTI-277. cDNA microarrays revealed that ES-285 treatment significantly alters the expression of a large number of genes at timepoints as early as 2 hours. This transcriptional response to ES-285 includes genes involved in apoptosis, cell cycle, signal transduction and the actin cytoskeleton. . Conclusions: ES-285 inhibits the proliferation of cancer cell lines in vitro at nanomolar concentrations. In HT29 colon cancer cells, treatment with ES-285 is associated with loss of actin stress fibres. The transcriptional response to ES-285 is distinct from, but overlaps with, that induced by structurally-related compounds and inhibitors of Rho/ROCK signalling. Further analysis and validation of these gene expression changes is being used to generate further hypotheses about the mechanism of action of ES-285 and to identify pharmacodynamic markers for use in a phase 1 clinical trial of ES-285 which is underway. . Acknowledgement: ES-285 and ES-427 were kindly supplied by PharmaMar S.A., Madrid, Spain.