Philosophiae Doctor - PhD (Biodiversity and Conservation Biology)

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Browsing by Subject "Apoptosis"

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    Biochemical investigation of anti-cancer activity of Tulbaghia violacea
    (UWC, 2012) Saibu, Gbemisola Morounke; Meyer, Mervin; Katerere, David
    Natural products have been a source of many pharmaceutical drugs and a number of drugs that are currently used in the treatment of cancer are derivatives of compounds originally isolated from natural products. There is evidence that extracts of Tulbaghia violacea can be used to treat cancer. The activation of apoptosis in cancer cells is a target for the development of novel anti-cancer drugs since one of the characteristics of cancer cells is resistance to apoptosis due to the deregulation of biochemical pathways leading to apoptosis. In fact, many current anti-cancer drugs exert their effects through the activation of apoptosis. Previous studies showed that extracts of T.violacea induce apoptosis in cancer cells and one study reported on the isolation of a compound (methyl-ԃ-D-glucopyranoside), which is responsible for the pro-apoptotic activity of the T.violacea extract. Therefore the aim of this study was to investigate the anti-cancer activity of methyl-ԃ-Dglucopyranoside and extracts prepared from T.violacea. In this study the pro-apoptotic activity of methyl-ԃ-D-glucopyranoside and extracts prepared from T.violacea were investigated on a panel of human cancer cell lines, which included HepG2, MCF7, H157, HT29 and the non-cancerous cell line, KMST6. The induction of apoptosis was evaluated by flow cytometry using several bioassays which measures biochemical events (caspase activation, phosphatidylserine externalisation and reactive oxygen species (ROS) production that is associated with the induction of apoptosis. The results demonstrated that the effects of methyl--D-glucopyranoside on cultured cells are transient and that the cells recover from the effects of methyl--D-glucopyranoside. This suggested thatmethyl-ԃ-D-glucopyranoside is not the compound responsible for the pro-apoptotic bioactivity in the T.violacea extract. This study also showed that cytotoxic and pro-apoptotic bioactivity of the leaf-extract was significantly higher in comparison to the tuber-extract. The bioactivity of the organic solvent extracts (dichloromethane, hexane, methanol and 50% methanol/water) of T.violacea leaves was also significantly higher than water extracts of T.violacea leaves. A comparison of the different organic extracts prepared from the T.violacea leaves showed that the highest activity was observed for the dichloromethane and hexane extracts. In an effort to identify the bioactive compound(s) the dichloromethane extract was subjected to Versaflash® column chromatography. However, due to problems experienced with the solubility of the dichloromethane sub-fractions, these compounds could not be tested for their bioactivity. Palmitone (16-hentriacontanone) was identified as one of the major compounds present in the dichloromethane sub-fractions. This compound was previously shown to have anticonvulsant bioactivity but there is no evidence in the literature that it has anti-cancer or pro-apoptotic activities. Fingerprinting of the methanol extract showed the presence of long chain fatty acid derivatives, flavonoids and allicin derivatives in the methanol extract. Although, this study failed to isolate the pro-apoptotic bioactive compound(s) present in the extracts of T.violacea, it confirmed that extracts of this plant induce apoptosis in cultured human cancer cell lines.
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    A yeast 2-hybrid screen to identify and characterize interaction partners of the cancer associated protein Retinoblastoma binding protein 6
    (University of the Western Cape, 2009) Chibi, Moredreck; Pugh, David JR
    Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice, RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners, this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen. In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors V-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70). The results of this work suggest that, at least in the case of YB-1 and zBTB38, RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6's involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.