| Summary: | A dissertation submitted in fulfilment of the requirements for the degree of Masters in Science in the School of Molecular and Cell Biology, University of the Witwatersrand
Johannesburg, 2013 === Cervical cancer is a gynaecological malignant disorder and is a common cause of death in women of the sub-Saharan Africa, striking nearly half a million of females each year worldwide. Cervical cancer is due to the persistence infection of human papillomavirus (HPV), a formidable virus that targets the cervix and is present in most cancers of the cervix. In South Africa, plants used to treat cancer are rare and there is a need for screening further plant extracts in order to identify potentially new anti-cancer drug discovery leads.
The purpose of this study was to screen Tulbaghia violacea (TV) and Agave palmeri (AG) for anti-cancer therapy in the cervical cancer cell lines HeLa and ME-180 and in the fibroblast cell line KMST-6. Staurosporine (ST) was used as a positive control. AG and TV crude plant extracts were screened for apoptosis induction, followed by elucidation of the role of Bax, Bcl-2, p53, Rb, RBBP and Mdm2 genes in cervical cancer. Plant extracts of TV and AG were time (24 hours) and dose (50, 100, 150 μg/ml) dependently screened against cervical cancer cell lines HeLa, ME-180 and in KMST-6 for anti-cancer activity using the thiazolyl blue test (MTT) assay. With an IC50 ~ 150 μg/ml, T. violacea extract exhibited significant cytotoxicity on both HeLa and ME-180 cancer cell lines, whilst A. palmeri was cytotoxic to ME-180 cells and 25nM ST as a positive control had a cytoxicity effect on all cell lines including the KMST-6, yet TV and AG had no cytotoxic effect on KMST-6.
The annexin-V/FITC detection assay was performed to evaluate the occurrence of apoptosis. Crude extracts of TV and AG together with ST induced significant apoptosis of HeLa, ME-180 and KMST-6 cells. The crude extracts were further analysed for DNA fragmentation, protein expression and gene expression by Western Blot and RT-PCR respectively, to investigate
whether these extracts have an effect on the on the expression of Bax, Bcl-2, p53, Rb, RBBP6, Mdm2 and the relationship between p53 and RBBP6. Morphological and biochemical changes were seen in this study. A further mixed response by several genes was observed following treatment with the two plant extracts, where RBBP6 was seen to be spliced in cancer cells while Bax was induced and Bcl-2 was inhibited, but the levels of p53 remained the same. Preliminary, the extracts of TV and AG induce cell death by down-regulating Bcl-2 and Mdm2. Quantitative RT-PCR showed that when p53 was silenced RBBP6 was up-regulated and vice versa. From these results it was deduced that RBBP6 gene interacts with p53 during cervical cancer development.
The anti-proliferative activity together with the characterization of p53, RBBP6 and Mdm2 and concentrations of these plant extract could be manipulated as diagnostic markers and potential therapeutic targets for cancer treatment; however, further studies on these plant extracts need to be performed to validate results obtained in this study.
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