3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Screening of South African medicinal plant Euphorbia tirucalli for anticancer properties(2015) Choene, Mpho SusanCancer is an enormous burden of disease, accounting for millions of deaths annually worldwide. Today, more people are dying from cancer than HIV/AIDS, malaria and tuberculosis combined. According to the American Cancer Society, it is expected that the global cancer burden will double by 2030 if preventative measures are not applied. Breast and gynaecological cancers remains a big scourge in developing countries, with breast cancer being the most common cancer and gynaecological cancers accounting for approximately 25% of all cancers in women in developing countries. Currently, the standard cancer treatments include surgery, radiation and chemotherapy. Adverse toxicities have been associated with these therapies and their effectiveness is also limited to drug resistance. The cost of treatment is another major burden. Limitations associated with these conventional cancer treatments have made discoveries of novel therapeutics which exhibit less toxicity and at a lowered cost of paramount importance. Medicinal plant extracts have recently attracted attention to modern medical science research with their non-lethal activity. Currently, up to 50% of the world drugs including chemotherapeutic drugs such as taxol and camptothecin are made from natural products or their derivatives. In this study we aimed to investigate the anti-cancer properties of the medicinal plant Euphorbia tirucalli. The crude extracts of E. tirucalli extracted using butanol; hexane and methanol solvents were screened for antiproliferative activity in breast (MCF-7 and MDA-MB231), ovarian (RMG-1) and cervical (SiHa) cancer cell lines. MTT assay and Real-Time Cell Analyzer (RTCA), xCELLigence were used to determine cytotoxicity of the extracts and calculate IC50. From MTT and xCELLigence results, we observed that E. tirucalli extracts exhibited dose-dependent inhibition of cell proliferation with RMG-1 and MCF-7 cells being more sensitive than MDA-MB231 and SiHa cells to all three extracts for an unclear reason. The butanol extract appeared to exhibit iv the most cytotoxicity with all cell lines reaching IC50 at low extract concentrations. Most therapies in anticancer treatment, such as chemotherapy, mainly induce cell death by causing either G0/G1 or G2/M cell cycle arrest and then inducing an apoptotic pathway. Therefore, cell cycle arrest and the induction of apoptosis in cancer cells become the major indicators of anticancer effects. Cells were stained with propidium iodide dye to determine if cells were arrested at G0/G1 or G2/M cell cycle stages while annexin V and PI staining were used to determine the type of cell death induced by the extracts. Cell cycle analyses revealed MCF-7, MDA-MB231 and SiHa cancer cells underwent arrest at G0/G1 following treatment with the plant extracts. Annexin V and PI staining revealed different proportions of apoptotic and necrotic populations. The extracts mainly induced apoptosis on MCF-7 and MDA-MB 231 cells, with the butanolic extracts inducing the most apoptosis. RMG-1 and SiHa cells had a high proportion of cells undergoing both late apoptosis and necrosis. The molecular mechanism of cell death induction was investigated using real time PCR and western blot. From the gene expression studies, p21 was observed to be over expressed in all cells following all treatments, in line with the observed cell cycle arrest at G0/G1. The extrinsic pathway of apoptosis was identified as the type of cell death induced with caspase 8 being overexpressed in MDA-MB 231 cells treated with butanol and hexane extracts. Upon further fractionation, flavonoids and especially isorhamnetin were identified as the active compounds in these extracts. Overall, the plant contains compounds that have some activity against cell proliferation and can be a promising tool to treat cancer cells. However, more work needs to be done to verify which compounds are mainly involved.Item Targeting retinoblastoma binding protein 6 (RBBP6) as an anti-ovarian cancer therapeutic strategy(2015-05-07) Ubanako, Philemon NjendeOvarian cancer is the most lethal gynaecological cancer. About 90% of ovarian cancers are epithelial (ovarian carcinomas), thought to arise from the ovarian surface epithelium. Diagnosed usually at clinically advanced stages, many patients show poor response to chemotherapy, with resistance and recurrent disease being prevalent. siRNA technology is currently being explored in clinical trials as a form of targeted therapeutic strategy in the disease. RBBP6 is a 250kD protein that enhances MDM2-mediated ubiquitination of p53 and also plays a role in cell cycle regulation and cell differentiation. It is upregulated in numerous cancers such as lung, oesophageal, colorectal and cervical cancer. RBBP6 suppresses p53 binding to DNA thereby inhibiting p53-dependent gene transcription. RBBP6 was knocked down using 30 nM siRNA in RMG-1 cells for 48 hours, after which the cells were treated with 50 nM paclitaxel and 0.5μM camptothecin for 24 hours. xCELLigence real time cell analysis was used to evaluate cell proliferation. qPCR and western blot were used to evaluate both gene expression and protein expressions respectively, of Bax, Bcl-2, MDM2, p53 and p21. Flow cytometry was used to determine the mode of cell death elicited apoptosis and also analyse changes in cell cycle progression. qPCR and Western blot analyses showed that RBBP6 expression reduced by approximately 57%. There was a significant upregulation of p53 and a significant downregulation of Bcl-2 in siRBBP6 transfected cells (p<0.05). Knockdown of RBBP6 resulted in a 37±5.8% cell death. There was a significant increase in cell death in paclitaxel and siRBBP6 co-treated cells (81.6±0.79%) as compared to cells treated with paclitaxel only (76.±1.14%). siRNA-mediated knock down of RBBP6 induces cell death in RMG-1 ovarian carcinoma cells. In addition, paclitaxel-induced cell death in RMG-1 cells is potentiated by RBBP6 siRNA transfection. A combination of chemotherapy with paclitaxel or camptothecin and RBBP6 siRNA could be a possible therapeutic strategy in combatting ovarian carcinomas.