Browsing by Author "Manunu, Bayanika "

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    Determination of the mechanism of synergy of SQ109 with rifampicin and isoniazid in Mycobacterium smegmatis
    (Stellenbosch : Stellenbosch University, 2015-03) Manunu, Bayanika; Williams, Monique Joy ; Friedrich, Sven; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
    ENGLISH ABSTRACT : Multidrug resistance tuberculosis (MDR-TB) is a serious concern in the public health environment globally and the understanding of its mechanisms may help to prevent the emergence and spread of resistant strains of Mycobacterium tuberculosis (Mtb). Several compounds are being tested in clinical trials and SQ109 was identified as a promising new anti-TB drug because of its bactericidal activity against Mtb and demonstrated synergistic activity with the fist-line TB drugs. This study focussed on the mechanism of synergy of SQ109 with rifampicin (RIF) and isoniazid (INH) in Mycobacterium smegmatis (Msmeg). The influence of SQ109 on efflux in Msmeg was evaluated using two approaches. Firstly, accumulation and efflux of ethidium bromide (EtBr) was monitored using a semi-automated fluorometric assay and secondly efflux and accumulation of RIF in Msmeg was assessed using tandem mass spectrometry. Although SQ109 resulted in a slight decrease in EtBr efflux by Msmeg in some of the assays performed, this decrease was not consistently seen. SQ109 appeared to have no significant influence on the efflux or accumulation of RIF in Msmeg, suggesting that it does not act to inhibit efflux in this organism. Six spontaneous SQ109-resistant mutants were generated in Msmeg and bactericidal activity of SQ109, RIF and INH against wild-type and mutant strains of Msmeg was assessed. The minimum inhibitory concentrations (MICs) for all three drugs increased in the mutant strains compared to the wild-type. Drug-drug interaction studies performed on one of the SQ109-resistant mutants revealed a change from synergy to additivity for both SQ109/RIF and SQ109/INH combinations, suggesting that identification of the genes harbouring mutations in these strains would shed light on the mechanism of synergy of SQ109 with RIF and INH. Sanger sequencing revealed that none of the SQ109-resistant mutants harboured mutations in MSMEG_0250 (mmpL3 homologue), a gene previously implicated in SQ109 resistance in M. tuberculosis. Preliminary whole genome sequencing data for six SQ109-resistant mutants identified SNPs in 10 genes, however the role of these genes in SQ109 resistance and synergy with RIF and INH in Msmeg remains to be verified.
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    Evaluation of the BCL-2, PI3K, PARP-1, and HSP90 pathways in breast, lung and glial cell lines for identification of candidate genes as therapeutic targets for overcoming radioresistance.
    (Stellenbosch : Stellenbosch University, 2021-11) Manunu, Bayanika ; Akudugu, John M.; Serafin, Antonio M. ; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Cancer remains a major cause of mortality globally. This disease can be treated with surgery, chemotherapy or radiotherapy. Radiotherapy is an important modality to treat many types of cancers. About half of patients presenting with cancer are treated with radiotherapy throughout the duration of their disease. The effectiveness of radiotherapy is attributed to the capacity of radiation to cause damage to DNA which subsequently leads to cell death. Although radiotherapy is an effective cancer treatment, a large number of patients subsequently experience radio resistance and recurrence of their cancers. A number of molecular signalling pathways contribute to cellular resistance against radiotherapy, such as DNA damage repair and PI3K/Akt/mTOR pathways. Identifying signalling pathway-related genes associated with radio resistance of cancers may be helpful in designing targeted therapeutic strategies which could enhance the efficacy of radiotherapy for human cancers. Therefore, novel therapeutic radio sensitisers are needed in order to overcome radioresistant cancers and to improve the outcome of therapy. The main objective of this study was to identify the Bcl-2, PI3K, PARP-1 and Hsp90 pathway related gene families as candidate genes associated to radio resistance of human breast, lung and glial cancer cells following exposure to radiation, so as to establish potential gene targets that may be inhibited in order to sensitise radioresistant cells, and guide the development of more potent therapeutic approaches. This study found that the lung cancer cell line (A549) and the glioblastoma cell line (G28) were the most radioresistant cancer cells based on the gene function-specific numerical difference of the 12 highly expressed (upregulation and downregulation) genes from each cell line. This resistance was attributed to BAG1, MGMT, FOXO1 and DNAJA1 as candidate radio resistance related genes involved in apoptosis, DNA repair, PI3K and Hsp90 pathways, respectively. Furthermore, pre-treatment of A549 and G28 cells with small molecule inhibitors, Thioflavin S (against BAG1), O6-Benzylguanine (against MGMT), AS1842856 (against FOXO1), and 116-9e (against DNAJA1), singly resulted in modest radio sensitisation in A549 and G28 cells, modest radio sensitisation in A549 cells only, moderate radio sensitisation in both cell lines, and radioprotection in both cell lines, respectively. This array of radio modulation was observed at 2 Gy (fractional dose in conventional radiotherapy), indicating that might inform the design of radiotherapy when these target inhibitors are considered as adjuvants. These findings suggest that targeting radio resistance-related genes (BAG1, MGMT, and FOXO1) could potentially be effective in the treatment of radioresistant cancers, in particular, lung cancer and glioblastoma multiforme. However, validation of the current in vitro findings in a larger panel of cell lines is needed, and it would be instructive to perform research experiments at a preclinical level using in vivo models.