School of Molecular & Cell Biology (ETDs)

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Browsing School of Molecular & Cell Biology (ETDs) by Author "Cronjé, Marianne J."

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    Insights into silver(I) phosphine complexes in targeting cell death and metastatic mechanisms in malignant cell lines
    (University of the Witwatersrand, Johannesburg, 2023-09) Roberts, Kim Elli; Engelbrecht, Zelinda; Cronjé, Marianne J.
    Cancer is the leading cause of death worldwide, with 18.1 million new cases and 9.6 million deaths reported annually. Cisplatin, a popular chemotherapeutic drug, exhibits certain limitations in terms of selectivity and efficacy. This emphasizes the necessity for novel therapeutic approaches in addressing a variety of cancer types. Multiple studies have shown that silver-based compounds suppress cancer cell proliferation and induce apoptosis. Thirteen novel silver(I) mono-dentate phosphine complexes were investigated for their anticancer effects on seven different human malignant cell lines; A375 non-pigmented melanoma, A549 lung adenocarcinoma, HEP-G2 hepatocellular carcinoma, HT-29 colorectal adenocarcinoma, MCF-7 and MDA-MB-231 breast adenocarcinoma, and SNO oesophageal squamous cell carcinoma. Two non-malignant human cell lines, HEK-293 embryonic kidney cells and MRHF foreskin fibroblast cells, were used to assess the selectivity of the complexes. Cisplatin and the efficient silver(I) phosphine complexes were selected for dose-response experiments to determine IC50 concentrations for the respective cell lines. On the basis of these screening results (chapter two), five difficult-to-treat cancer cell lines, and their most efficient complexes were selected for further investigation. Various cellular characteristics were investigated in chapter three (A549, HEP-G2, HT-29); these included morphological changes, ATP levels, GAPDH levels, Ptd-L-Ser externalization, mitochondrial membrane potential, oxidative stress levels, and the activity of a metabolic enzyme, cytochrome P450 isoform CYP1B1. The antimetastatic activity of the selected complexes was assessed by evaluating their ability to impede the migration of A549 cells. The fourth chapter examines the anticancer effect of selected complexes on hormone-dependent (MCF-7) versus triple-negative (MDA-MB-231) breast cells. Changes in morphology, Ptd-L-Ser externalization, alterations in mitochondrial membrane potential, oxidative stress levels, cytochrome c release, and DNA damage were studied. Furthermore, in chapter five, molecular docking simulations were used to determine whether the most potent silver(I) phosphine complex across all cell lines bonds to estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Seven of the thirteen silver(I) phosphine complexes significantly reduced cell viability in malignant cell lines while being less toxic to non-malignant cells. Complex 4 best targeted all cancer types, with IC50 values ranging from 5.75 to 10.80 µM across malignant cell lines. In the malignant treated cells, morphological changes, reactive oxygen species production, mitochondrial membrane depolarization, and Ptd-L-Ser externalization were observed. Complexes 1 and 4 repressed cell migration in the A549 cells. The presence of damaged nuclei, metabolically inactive mitochondria and cytochrome c translocation from the mitochondria’ intermembrane to the cytosol in MCF-7 cells were observed. These findings suggest that complexes 2, 4 and 7 induced apoptotic cell death. Furthermore, in silico computational predictions suggested a promising interaction between complex 4, and ER-α and ER-β. Overall, this study demonstrates the potential of silver(I) phosphine complexes as anticancer agents, with promising effects on various cancer cell lines.
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    Investigating the susceptibility of Enterobacter xiangfangensis Pb204 to various silver(I) phosphine compounds and identifying the molecular basis of resistance though comparative genomics
    (University of the Witwatersrand, Johannesburg, 2023-10) Ntuli, Phindile Lindiwe; Engelbrecht, Zelinda; Cronjé, Marianne J.
    In developing countries, the endemic of nosocomial infections is notably high, particularly in the ICU settings and neonatal infections, where it accounts for a prevalence of 15.5% with an increased mortality rate. ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) pathogens are highly virulent and resistant microbes that predominantly causes nosocomial infections in clinical settings. These pathogens adhere to the surface of hospital equipment and form biofilms, thereby increasing the susceptibility of patients to acquiring such infections during extended hospital stays. Recent studies have shown that Enterobacter xiangfangensis is the main clinical culprit in the Enterobacter cloacae complex, thus making it the main therapeutic target among the ESKAPE pathogens. This study evaluated the antimicrobial properties of various silver(I) phosphine compounds on E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T. The minimum inhibitory concentration (MIC) assay was done, though the microdilution technique coupled with iodonitrotetrazolium chloride (INT) dye. The antibiofilm activity of the silver(I) phosphine compounds on biofilm formation and degradation were evaluated using the crystal violet assay, and the metabolic activity of cells in biofilms was assessed using the XTT assay. Additionally, the molecular mechanism of silver resistance was investigated by measuring gene expression of E. xiangfangensis Pb204 cells when treated with the silver(I) phosphine compound UJ1, silver nitrate and silver sulfadiazine using RT-qPCR. The MIC of the silver(I) phosphine compounds was 0.125 mg/ml for both E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T. Moreover, the compound UJ1 showed a bactericidal effect against E. xiangfangensis LMG 27195𝑇. Furthermore, the silver(I) phosphine compounds have antibiofilm activity since they inhibit biofilm formation, disrupt matured biofilms, and reduce metabolic activity in E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T biofilms. The resistance in E. xiangfangensis Pb204 strain is due to the upregulation of the silver resistance pathway encoded on the integrative and conjugative element (ICE) element. Core and unique protein coding genes between E. xiangfangensis Pb204 and E. xiangfangensis LMG 27195T were identified though comparative genomics. Functional annotation of core and unique protein revealed that E. xiangfangensis Pb204 evolved to gain genetic material coding for inorganic ion transport and carbohydrate transport metabolism. Knowledge of these can be used in future studies in the utilization of silver metals as antimicrobial agents.