Breast cancer remains the leading cause of cancer-related death in women worldwide. Furthermore, it has been demonstrated that the treatment-resistant ER-PR-HER2/neu- sub-type is more common among women of African descent, necessitating the search for novel chemotherapies for this form of the disease. The secondary metabolites produced by marine algae represent a rich source of structurally unique compounds with chemotherapeutic potential, particularly in South Africa, whose oceans allegedly host 15 % of the total number of species in the world. Indeed, a recent study reported the isolation of a range of novel compounds from South African red and brown algae of the Plocamium, Portiera and Sargassum genera which displayed cytotoxicity against oesophageal cancer cells in vitro. The molecular mechanisms mediating this toxicity were unknown, as was the effect of these and similar compounds on metastatic ER-PR-HER2/neu- breast cancer cell lines or breast cancer stem cells. The current study aimed to address these questions by screening a library of twenty-two novel marine algal compounds for the ability to inhibit MDA-MB-231 and Hs578T breast cancer cells, while having no adverse effects on non-cancerous MCF12A breast epithelial cells. While twelve of these were toxic in the micromolar range against breast cancer cells, only the polyhalogenated monoterpenes RU004 and RU007, and the tetraprenylated quinone sargaquinoic acid (SQA) were identified as hit compounds based on the criteria that their cytotoxicity was specific to breast cancer and not healthy breast cells in vitro. On the other hand, the halogenated monoterpene RU015 was found to be highly toxic to both breast cancer and non-cancerous breast cell lines, while the halogenated monoterpene stereoisomers RU017 and RU018 were non-toxic to either of these cell lines. The mode of action of RU004, RU007, RU015 and SQA, together with the previously characterized carotenoid fucoxanthin (FXN), was assessed in terms of the type of cell death induced and the effect on cell cycle distribution of these compounds. Flow cytometric analysis of the extent of Hoescht 33342 and propidium iodide staining along with PARP cleavage studies suggested that SQA induced apoptosis in MDA-MB-231 cells. On the other hand, the highly toxic compound RU015 appeared to induce necrosis as evidenced by 50 kDa PARP cleavage product in MDA-MB-231 cells. The flow cytometry profiles of MDA-MB-231 and Hst578T cells treated with the hit compounds RU004 and RU007 were suggestive of the induction of apoptosis by these compounds. Cell cycle analysis by flow cytometry with propidium iodide staining revealed that both SQA and FXN induced G0-G1 arrest together with an increase in the apoptotic sub-G0 population, which agreed with previous reports in the literature. The molecular mechanism of action of SQA and FXN were further investigated by the identification of specific signal transducer molecules involved in mediating their anti-cancer activities. SQA was found to require the activity of numerous caspases, including caspase-3, -6, -8, -9, -10 and -13, for its cytotoxicity and was demonstrated to decrease the level of the antiapoptotic protein Bcl-2. On the other hand, FXN was shown to require caspase-1, -2, -3, -9 and - 10 for its toxicity. This, together with the ability to decrease the levels of Bcl-2, pointed to the involvement of the intrinsic pathway in particular in mediating the activity of FXN. The screening of algal compounds against non-cancerous breast epithelial cells carried out in this study, together with the investigation into their mechanisms of action, represent one of the few reports in which characterization of algal metabolites goes beyond the initial cytotoxicity assays. Finally, in order to assess the potential anti-cancer stem cell activity of the marine algal compounds, a subset of these was screened using a mammosphere assay technique developed in this study. The cancer stem cell (CSC) theory proposes that cancers arise from and are maintained by a specific subpopulation of cells able to undergo asymmetric cell division and termed CSCs. These CSCs are capable of anchorage-independent growth in serum-free culture conditions, such as those in the mammosphere assay. Using this assay, the novel halogenated monoterpene stereoisomers RU017 and RU018 were demonstrated to possess putative anti- CSC activity as evidenced by their ability to completely eliminate mammosphere formation in vitro. Furthermore, since RU017 and RU018 were non-toxic to both breast cancer and healthy breast cells, it appeared that the activity of the compounds was potentially specific to the CSCs. The results require further validation, but represent the first report of selective anti-CSC activity.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:rhodes/vital:4143 |
| Date | January 2012 |
| Creators | De la Mare, Jo-Anne |
| Publisher | Rhodes University, Faculty of Science, Biochemistry, Microbiology and Biotechnology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Doctoral, PhD |
| Format | 186 leaves, pdf |
| Rights | De la Mare, Jo-Anne |
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