Cellular Targets and Tumour Suppressive Mechanisms of Peloruside A
Peloruside A (PelA) is a novel secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. It is a potent microtubule-stabilizer and binds to a distinct site on β-tubulin compared to the widely used anti-cancer drug paclitaxel. PelA has clear potential benefits over paclitaxel, including increased solubility, reduced sensitivity to multiple drug resistance in cancer cell lines, and improved tolerability and efficacy in non-small cell lung cancer xenografts in mice. Using several established cancer cell lines, we investigated potential tumour suppressive effects of PelA. 1A9 human ovarian carcinoma cells treated with PelA were screened using several protease inhibitors to determine whether these inhibitors could protect against the induction of apoptosis. The greatest protection was conferred by the pan-caspase inhibitor zVAD-fmk, and subsequent biochemical assays suggested that caspases-8, - 9 and -3/7 were activated after prolonged treatment with PelA (> 24 h). These results indicate a predominant role for caspases in PelA-induced cell death. Additionally, decreased protein expression levels of stathmin and c-Myc, two proteins that have previously been shown to influence sensitivity to microtubule-targeting agents (MTAs), were observed after treatment of HL-60 cells with PelA, suggesting that they may also mediate some of PelA's cytotoxic effects. Flow cytometric analysis of 1A9 human ovarian carcinoma cells demonstrated that low concentrations of PelA could induce chromosome mis-segregation in up to 14% of cells. Cells treated with 40 nM PelA displayed a delayed mitotic entry (by ~ 1 h), but an otherwise fairly normal cell cycle progression profile. Western blot analyses of spindle activation checkpoint proteins found no change in expression levels of MAD2, BUBR1 or p55CDC at this drug concentration. In response to 100 nM PelA, cyclin B1 expression levels remained elevated, corresponding with the mitotic arrest that occurred at this concentration. The cell cycle kinetics of MAD2 and BUBR1 dissociation from p55CDC was investigated by co-immunoprecipitation. Despite a concentration-dependent increase in MAD2/p55CDC association by PelA, at 40 nM PelA the timing of MAD2/p55CDC dissociation was similar to that of controls. This contrasted with sustained MAD2/p55CDC complexes at 100 nM PelA. Changes in the levels of BUBR1 associated with p55CDC were observed in response to 40 nM and 100 nM PelA compared to controls. Dissociation of BUBR1/p55CDC complexes still occurred in the presence of 40 nM PelA, which contrasted with the sustained presence of BUBR1/p55CDC complexes at 100 nM PelA. These results suggested that at low concentrations of PelA, the spindle activation checkpoint is being silenced (or bypassed) despite the presence of erroneous microtubule-kinetochore attachments. Studies using the MCF7 human breast cancer cell line indicated that in addition to apoptosis, PelA induced cells to adopt a morphological and biochemical phenotype that is indicative of premature senescence (a permanent cell cycle arrest). The induction of premature senescence involved activation of the p53 and pRb tumour suppressor pathways, and also correlated with reduced clonogenicity. PelA was also screened for anti-angiogenic activity by assessing its ability to inhibit crucial endothelial cell functions. PelA potently inhibited human umbilical vein endothelial cell proliferation and migration, and also affected the number and quality of 3-dimensional capillary-like structures that could form on Matrigel™, a basement membrane matrix. Despite the acquired resistance and undesirable side effects (e.g. allergic reactions, neutropenia and neuropathy) associated with the use of MTAs in the clinic, tubulin still represents one of the most successful drug targets for cancer; thus, there is an ongoing need for new MTAs with improved therapeutic profiles. The work that we have presented here highlights the fact that PelA can induce multiple cell fates, some of which are potentially tumour suppressive. Additionally, the findings in this study further support its development as a candidate anti-cancer chemotherapeutic.