Doctoral Degrees (Biochemistry)

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Browsing Doctoral Degrees (Biochemistry) by browse.metadata.advisor "Africander, Donita"

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    The influence of sutherlandia frutescens on adrenal steroid hormones and downstream receptor interactions
    (Stellenbosch : Stellenbosch University, 2017-12) Sergeant, Catherine Anne; Swart, Amanda C.; Africander, Donita; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The study was aimed at: 1. linking anti-stress properties of Sutherlandia frutescens to its influence on adrenal steroid biosynthesis in terms of extracts, sutherlandioside (SU) compounds and gammaaminobutyric acid (GABA). 2. linking anti-inflammatory and anti-hypertension properties to steroid receptor interaction, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The influence of S. frutescens extracts, and SUs, on key steroidogenic enzymes was assayed in COS-1 cells and binding to adrenal P450-dependent enzymes was assayed in ovine adrenal mitochondrial and microsomal suspensions. The effects of the methanol extract and sutherlandioside B (SUB) were assayed on basal, forskolin and angiotensin II (Ang II) stimulated hormone levels in the adrenal H295R cell model. The effect of GABA on basal steroid production was investigated in H295R cells. Agonist activity of the methanolic extract and SUB, for both transactivation and transrepression of GR and MR mediated gene transcription, was subsequently investigated. S. frutescens extracts affected all the steroidogenic enzymes with inhibition being substrate specific. SUB had a greater effect on substrate binding to mitochondrial and microsomal P450 enzymes than sutherlandioside A (SUA). SUB inhibited the catalytic activity of CYP17A1, CYP11B2 and 3β-HSD2 while not affecting CYP21A2. In H295R cells, while the extract decreased total steroid production under basal and forskolin stimulated conditions, only cortisol and its precursor, deoxycortisol, and mineralocorticoid metabolites were decreased significantly in the presence of forskolin. A SU mixture did not influence steroid production, with only cortisol levels decreasing significantly with SUB decreasing cortisol, deoxycortisol, androstendione and 11-hydroxyandrostenedione significantly. The data show that the SU mixture was not able to elicit the same effects as SUB, suggesting that the compounds do not act synergistically. GABA significantly decreased adrenal steroid production in H295R cells with the greatest inhibitory effect observed in the production of adrenal androgens. S. frutescens extracts and SUB repressed NF-κB-driven gene expression without activating GREdriven gene expression while neither activated MR mediated gene transcription while both antagonized the effects of aldosterone via the MR. The results of this study provide evidence linking anti-stress, anti-inflammatory and antihypertensive properties of S. frutescens to the inhibition of steroidogenic enzymes and modulation of adrenal hormone biosynthesis. The findings suggesting that S. frutescens and SUB exhibit dissociated glucocorticoid characteristics underline potential therapeutic applications in the treatment of inflammation and hypertension.
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    Investigating the mechanism of action of hormones used in hormone replacement therapy via estrogen receptor subtypes and the influence of the progesterone receptor
    (Stellenbosch : Stellenbosch University, 2018-03) Perkins, Meghan Samantha; Africander, Donita; Louw-du Toit, Renate; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Estrogens and progestins used in conventional menopausal hormone therapy (HT) are associated with increased breast cancer risk. A diverse range of estrogens and progestins are available that mediate their effects primarily by binding to the estrogen receptor (ER) and progesterone receptor (PR), respectively. Although the link to breast cancer risk has not been shown for all estrogens and progestins, many women have turned to custom-compounded bioidentical hormone therapy (bHT) as it is claimed to not increase breast cancer risk. However, scientific evidence to support this claim is lacking. Estrogens and ERα are considered the main etiological factors driving breast cancer, while both ERα and the PR are required for progestin (medroxyprogesterone acetate (MPA)) effects on breast cancer cell proliferation. In this thesis, we investigated the activities of estrogens and progestins used in menopausal hormone therapies via the individual ER subtypes, and the role of ERα/PR crosstalk in mediating progestin-induced effects on gene expression, breast cancer cell proliferation and anchorage-independent growth. In the first part of the study, competitive whole cell bindings assays showed that bioidentical estradiol (bE2) and estriol (bE3) displayed similar binding affinities to the commercially available (natural) estradiol (E2) and estriol (E3) standards, while synthetic ethinylestradiol (EE) had a higher affinity for ERα, and natural E1 a lower affinity for ERβ. Furthermore, the bioidentical estrogens mimicked their respective natural estrogens and synthetic EE on transactivation and transrepression of gene expression, proliferation and anchorage-independent growth of the estrogen-sensitive MCF-7 BUS human breast cancer cell line. These assays showed that E3 and estrone (E1) are efficacious estrogens that do not antagonize E2. In the second part of this study, the estrogenic activities of selected progestins from different generations, MPA, norethisterone acetate (NET-A), levonorgestrel (LNG), gestodene (GES), nestorone (NES), nomegestrol acetate (NoMAC) and drospirenone (DRSP), were characterized relative to each other and natural progesterone (P4). Competitive binding assays revealed that only NET-A, LNG and GES could bind to ERα, while no progestin bound ERβ. Both transactivation and transrepression transcriptional assays showed that NETA, LNG and GES display estrogenic activity. In the third part of the study, the role of PR/ERα crosstalk in mediating the effects of MPA, NET and DRSP, relative to P4, on breast cancer cell proliferation, anchorage-independent growth and the expression of the ER-regulated trefoil factor 1 (pS2) and cathepsin D (CTSD) genes was investigated. All progestins could promote proliferation and anchorage-independent growth of MCF-7 BUS breast cancer cells to the same extent as P4 and E2 via a mechanism requiring both the PR and ERα, but DRSP was the least, and MPA the most potent for proliferation. Quantitative real-time RT-PCR (qPCR), chromatin immunoprecipitation (ChIP) and re-ChIP assays showed that only MPA and NET increased the expression of the pS2 and/or CTSD genes via a mechanism requiring co-recruitment of the PR and ERα to the promoter regions of these genes. In contrast, P4, MPA, NET and DRSP all caused recruitment of the PR/ERα complex to the PR-regulated oncogenes cyclin D1 and MYC. Taken together, the findings of this study suggest that there is no advantage in choosing bHT above conventional HT, and that while it is unlikely that the progestins used in this study will exert biological effects via ERα or ERβ in vivo, some progestins may increase breast cancer risk via a mechanism involving interplay between the PR and ERα.
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    Progestins and breast cancer : significance of progesterone receptor isoforms and their altered ratios
    (Stellenbosch : Stellenbosch University, 2021-03) Cartwright, Meghan Carni; Africander, Donita; Louw-du Toit, Renate; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Progestins used in menopausal hormonal therapy have been associated with increased incidence of breast cancer. While these synthetic ligands were designed, in four consecutive generations, to mimic the activity of natural progesterone (P4) via the progesterone receptor (PR), the precise mechanism whereby some progestins and/or their metabolites may cause an increase in breast cancer incidence is still mostly unknown. Whether the PR, existing as two isoforms, PR-A and PR-B, plays a role in mediating the effects of progestins on breast cancer is unclear. As the metabolism of a progestin can ultimately influence effects via the PR, ultrahigh performance supercritical fluid chromatography-tandem mass spectrometry was used to investigate the metabolism of P4 and selected progestins in three breast cancer cell lines in the first part of this thesis. Unlike P4 that was rapidly metabolised in all three cell lines, promegestone (R5020), gestodene (GES) and nomegestrol acetate (NOMAC) were not metabolised, while only drospirenone (DRSP) was metabolised in the MDA-MB-231 and T47D cells. Additionally, we showed that P4 metabolism occurred at a similar rate in the MDAMB- 231 and T47D cells, but faster than its metabolism in the MCF-7 BUS cells. In the second part of this study, transactivation and transrepression transcriptional assays showed that the activities of a selected panel of progestins from all four generations are not all similar to each other, P4 or R5020, via PR-A and PR-B. For transactivation, most progestins were more efficacious via PR-B, but more potent via PR-A. We also showed that an increase in PR-A density and excess PR-A relative to PR-B, resulted in decreased efficacies of all progestins for transactivation. While an increase in PR-A density resulted in an increase in the activity of all progestins for transrepression, the activity of only a few progestins were influenced by excess expression of PR-A relative to PR-B. Realtime PCR showed progestin- and gene-specific regulation of endogenous genes known to play a role in breast cancer in T47D breast cancer cells. While the response of some progestins on the selected genes were PR-B mediated, some progestin effects were not mediated by either PR-A or PR-B. In the third part of this thesis, investigations into the effects of the progestins on proliferation, apoptosis, anchorageindependent growth, migration and invasion showed that these processes are differentially influenced by P4 and the selected progestins, and that the responses are also differentially mediated by PR-A or PR-B. Excess expression of PR-A resulted in both positive and/or negative ligand-independent, as well as progestin-induced, effects on these cancer hallmarks. Taken together, the findings of this thesis emphasize the fact that progestins do not always mimic the activities of P4 or each other. The results further highlight the complexity of progestin action via the PR, underscoring the importance of distinguishing progestin activities via PR-A and PR-B, and also considering the PR-A:PR-B ratio when investigating the mechanisms of progestins and the PR in breast cancer. Finally, our results suggest that a progestin such as medroxyprogesterone acetate (MPA) acting via PR-A and/or PR-B may indeed increase breast cancer risk, while others like DRSP may not.
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    A study of the molecular mechanism of progestin-induced regulation of IL-12 and IL-10 and implications for HIV pathogenesis
    (Stellenbosch : Stellenbosch University, 2013-03) Louw, Renate; Africander, Donita; Hapgood, Janet P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)), designed to mimic the actions of the endogenous hormone progesterone (Prog), are extensively used by women as contraceptives and in hormone replacement therapy (HRT). A number of reports have indicated that these synthetic progestins affect immune function in the female genital tract thereby increasing the risk of acquiring sexual transmitted infections. Despite these findings, very little is known about their mechanism of action at the cellular level, in particular their steroid receptor-mediated effects on cytokine gene expression. In the first part of this thesis, the effect of Prog, MPA and NET-A on the expression of the endogenous pro-inflammatory cytokine gene, interleukin (IL)-12p40, and anti-inflammatory cytokine gene, IL-10, was investigated in a human ectocervical epithelial cell line, Ect1/E6E7. Quantitative realtime PCR (qPCR) showed that all three ligands significantly upregulated the tumor necrosis factor alpha (TNF )-induced IL-12p40 gene expression, while IL-10 gene expression was downregulated. Moreover, by reducing the glucocorticoid receptor (GR) levels with siRNA, these effects were shown to be mediated by the GR. A more detailed investigation into the molecular mechanism of the progestogen-induced upregulation of IL-12p40 gene expression, using chromatin immunoprecipitation (ChIP), siRNA, co-immunoprecipitation and re-ChIP analyses, showed that the progestogen-bound GR is recruited to the CCAAT enhancer binding protein (C/EBP)- regulatory element of the IL-12p40 promoter, most likely via an interaction with the transcription factor C/EBP . Similar experiments for the progestogen-induced downregulation of IL-10 gene expression showed that the progestogen-bound GR is recruited to the signal transducer and activator of transcription (STAT)-3 regulatory element of the IL-10 promoter, most likely via an interaction with the transcription factor STAT-3. The second part of this study elucidated the influence of the HIV-1 accessory viral protein R (Vpr) on progestogen-induced regulation of IL-12p40, IL-12p35 and IL-10 in the Ect1/E6E7 cell line. Results showed that in these cells, the overexpression of Vpr significantly modulated the effects of Prog, MPA and NET-A on the mRNA expression of IL- 12p40 and IL-10, while only the NET-A effect was modulated on IL-12p35. Moreover, reducing the GR protein levels by siRNA suggested that the GR is required by Vpr to mediate its effects. Taken together, these results show that Prog, MPA and NET-A promote the pro-inflammatory milieu in the ectocervical environment, and that during HIV-1 infections, this milieu is modulated. Furthermore, the results suggest that the use of MPA or NET in vivo may cause chronic inflammation of the ectocervical environment, which may have important implications for ectocervical immune function, and hence susceptibility to infections such as HIV-1.