Identification of novel marine algal compounds with differential anti-cancer activity: towards a cancer stem-cell specific chemotherapy
- Authors: De la Mare, Jo-Anne
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells -- Research , Chemotherapy , Algae -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4143 , http://hdl.handle.net/10962/d1016250
- Description: 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.
- Full Text:
- Date Issued: 2012
- Authors: De la Mare, Jo-Anne
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells -- Research , Chemotherapy , Algae -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4143 , http://hdl.handle.net/10962/d1016250
- Description: 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.
- Full Text:
- Date Issued: 2012
Analysis of the anti-cancer activity of novel indigenous algal compounds in breast cancer: towards the development of a model for screening anti-cancer stem cell activity
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
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