Department of Biochemistry
Permanent URI for this community
Browse
Browsing Department of Biochemistry by browse.metadata.advisor "Africander, Donita"
Now showing 1 - 11 of 11
Results Per Page
Sort Options
- ItemCrosstalk between the androgen and estrogen receptors in breast cancer(Stellenbosch : Stellenbosch University, 2016-03) Ndlovu, Easter; Africander, Donita; Louw, Ann; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Hormone replacement therapy (HRT) is used by post-menopausal women to alleviate symptoms associated with decreased endogenous estrogen levels, such as hot flashes and vaginal dryness. Although HRT is considered an effective treatment method, results from clinical trials such as those conducted by the Women’s Health Initiative (WHI) and Million Women Study (MWS) revealed that HRT usage is associated with increased risk of invasive breast cancer. The mechanism whereby the hormones used in HRT contribute to breast cancer risk is not fully understood. These hormones elicit their biological effects by binding to intracellular steroid receptors such as the estrogen (ER) and progesterone receptor (PR). For many years it was thought that the ER is the main steroid receptor implicated in breast cancer biology, however, data in the literature suggests that crosstalk between steroid receptors play an important role in the development and progression of this disease. Recent evidence suggests that the androgen receptor (AR) can function as a tumour suppressor and thus has the potential of being a prognostic marker and therapeutic target in breast cancer. Given that the AR has been shown to inhibit the transactivation function of ERα, this raised the question of whether the AR would also inhibit the transactivation function of the ERβ subtype. Moreover, considering that ERs have both transactivation and transrepression functions, studies investigating the influence of the AR on the transrepression function of both ERα and ERβ are lacking. This study therefore investigated whether the AR, in the absence or presence of known AR agonists (the natural AR ligand 5α-dihydrotestosterone (DHT)), the androgenic progestins, medroxyprogesterone acetate (MPA) and norethisterone-acetate (NET-A)), could modulate the transcriptional activity of the ERβ. The ER- and AR-negative MDA-MB-231 breast cancer cell line, transiently transfected with expression vectors for either ERα or ERβ and the appropriate promoter-reporter construct, was used as model system. The results showed that the unliganded or liganded AR has no effect on the transactivation function of ERβ on a synthetic estrogen response element (ERE)-containing promoter but downregulates the ERβ-driven mRNA expression of the endogenous PR gene. This study also shows estradiol (E2)-induced transactivation on a synthetic ARE- and an endogenous androgen response element (ARE)-containing promoter via ERα. For transactivation via ERβ, similar results were only seen on the endogenous ARE-containing promoter. Moreover, we show for the first time that both the unliganded- and ligand-bound AR inhibits the transrepression function of ERα, while interestingly only the ligand-bound AR was able to inhibit the transrepression function of ERβ. No E2-induced cell proliferation was observed in the MDA-MB-231 cell line overexpressing either ERα or ERβ under the experimental conditions used. In conclusion, the results of this study are in agreement with previous evidence suggesting that the AR inhibits the activity of ERα. The precise physiological implications of these results remain to be determined. Finally, although the results from this study are preliminary and have certain limitations, it nonetheless highlights the fact that the role of the AR in breast cancer is a complex one. Thus, our findings may aid our understanding of crosstalk between the ER and AR signalling pathways, and how it contributes to the growth and survival of breast cancer cells.
- ItemCrosstalk between the androgen receptor and progesterone receptor isoforms in breast and prostate cancer(Stellenbosch : Stellenbosch Univesity, 2018-03) Cabral, Angelique Aida; Africander, Donita; Storbeck, Karl-Heinz; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Breast and prostate cancer growth and survival are dependent on signalling via the estrogen receptor (ER) and androgen receptor (AR), respectively. However, other steroid receptors such as the progesterone receptor (PR), are also implicated in both cancers, and emerging evidence suggests considerable crosstalk between these steroid receptors in breast cancer. Investigations into similar crosstalk mechanisms are lacking in prostate cancer. As the AR and PR are likely co-expressed in a subset of breast and prostate cancers, it is surprising that no studies have investigated crosstalk between the AR and PR in these cancers. Both these receptors can activate transcription by binding to DNA on a classical response element, termed either the progesterone response element (PRE) when the PR is bound, or the classical androgen response element (ARE) when the AR is bound. However, the AR can also bind to an AR-selective ARE as well as to the ER binding site, termed the estrogen response element (ERE). Whether the PR isoforms, PRA and PRB, can similarly activate the AR-selective ARE and ERE is not known. In this study, we investigated whether the PR isoforms, in the absence and presence of known PR agonists (synthetic promegestone (R5020), natural progesterone (P4), and synthetic progestin medroxyprogesterone acetate (MPA)), could modulate the transactivation function of the AR via the above-mentioned response elements in the MDA-MB-231 breast cancer and PC3 prostate cancer cell lines. The cells were transiently transfected with the expression vectors for the AR and/or PR isoforms, together with the applicable promoter-reporter constructs. The general trend observed was that both the unliganded and liganded PR isoforms augmented AR activity in a cell line-, ligand- and/or promoter-specific manner. Specifically, we showed that PRB, both in the absence and presence of PR ligands, generally upregulated AR transactivation on the various response elements in the breast and prostate cancer cells. While AR transactivation function was also increased by PRA on the selective ARE and ERE, PRA decreased AR-mediated transactivation on the classical ARE. We also provide novel evidence that both PR isoforms mimic AR activity on the selective ARE and the ERE in both cell lines, which may provide a mechanism through which the PR mediates oncogenic effects in both cancers. We did not observe cell proliferation in the presence of 5α-dihydrotestosterone (DHT) in either cell line transfected with the AR under the experimental conditions used in this study. In summary, even though the results from this study are preliminary, we are the first to show that the transactivation function of the AR is generally enhanced in the presence of the PR isoforms in both breast and prostate cancer. These findings support a potential crosstalk mechanism between the AR and PR isoforms in these cancers. Although the precise physiological implications of these results require further investigation, our findings contribute to the understanding of crosstalk between steroid receptors, particularly the AR and the PR isoforms, and how this may influence breast and prostate cancer cell growth.
- ItemThe 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.
- ItemInvestigating progesterone and estrogen receptor crosstalk in breast cancer(Stellenbosch : Stellenbosch University, 2017-03) Van der Meer, Yolandi; Africander, Donita; Louw-du Toit, Renate; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Progestins are synthetic compounds designed to mimic the natural hormone progesterone (Prog), and are widely used in hormone replacement therapy (HRT) and contraception. These compounds can be divided into four generations, with newer generations increasing in progesterone receptor (PR) specificity. Although progestins have many therapeutic benefits, a number of undesirable side-effects, such as increased risk of breast cancer, have been reported. As a result, many postmenopausal women have sought alternatives for HRT, such as compounded bio-identical hormones like bio-identical Prog (bProg), claimed not to increase breast cancer risk. Progestins, Prog and bProg (collectively referred to as progestogens) elicit their biological effects primarily by binding to the PR, which exists as two predominant isoforms, PR-A and PR-B, with PR-B being the more transcriptionally active and proliferative isoform in breast cancer. Emerging evidence suggest that the PR plays an important role in breast cancer development and progression, and that there is crosstalk between the PR and estrogen receptor (ER)-α, a major etiological factor in breast cancer biology. Moreover, it has been shown that ER-α is required for PR-B-mediated effects of medroxyprogesterone acetate (MPA) on activation of gene expression and breast cancer cell proliferation. The latter raised the questions of whether ER-α is needed for PR-B-mediated effects of other progestins, and whether the ERβ subtype would also be required. Given that PR-B has both transactivation and transrepression functions, this study used transactivation and transrepression transcriptional assays to investigate the PR-B-mediated agonist efficacies and potencies of Prog, bProg and select progestins from different generations (MPA, norethisterone acetate (NET-A), levonorgestrel (LNG), gestodene (GES) and drospirenone (DRSP)), and whether these were modulated by ERα and/or ERβ. Furthermore, the effects of the progestogens on breast cancer cell proliferation were evaluated in the absence and presence of ERα- and ERβ-specific antagonists. Results showed that progestins mostly displayed similar agonist efficacies and potencies for transactivation and transrepression via PR-B. The exception was first generation MPA that was less efficacious for transactivation and least potent for transrepression, and third generation GES that was more potent for transactivation. This study is the first to show that ERα and ERβ differentially decreased PR-B-mediated agonist efficacies of progestogens for transactivation and transrepression. However, the ERα-specific antagonist had no effect on progestogen-induced expression of the endogenous PR-B regulated c-myc gene or repression of the interleukin (IL)-8 gene in the T47D breast cancer cell line, while the ERβ-specific antagonist had no effect on progestogen-induced c-myc gene expression, and appeared to abolish repression of the IL-8 gene. Additionally, we showed that all progestogens, except NET-A and DRSP, displayed similar proliferative efficacies and potencies for cell proliferation. Interestingly, while the ERα-specific antagonist had no effect on progestogen-induced cell proliferation, increased cell proliferation by LNG- and GES was enhanced by the ERβ-specific antagonist. Taken together, the results from this study, although having limitations, emphasizes the complexity of crosstalk between the PR and ER subtypes in breast cancer. Although the physiological implications of these results have to be evaluated, our findings may assist us in our understanding of crosstalk between PR-B and the ER subtypes, and how it may be contributing to progestin-induced breast cancer cell growth.
- ItemInvestigating progestin-mediated crosstalk between the androgen receptor and estrogen receptor subtypes in breast cancer cell lines(Stellenbosch : Stellenbosch University, 2019-02) Brink, Danielle; Africander, Donita; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Estrogen and the estrogen receptor alpha (ERα) are traditionally considered as the main etiological factors in breast cancer. However, other members of the steroid receptor family, including ERβ and the androgen receptor (AR) have also been shown to play a role. It is known that ERα drives breast cancer cell proliferation, while ERβ antagonizes ERα-mediated effects. Moreover, the AR, which is expressed in the majority of ER-positive breast cancer tumours, has been shown to inhibit the transcriptional activity of ERα and increase ERβ expression when activated by the potent natural androgen, 5α-dihydrotestosterone (DHT). Together, this suggests that the AR is associated with a good prognosis in ER-positive breast cancer. The question that arises is whether all agonists binding to the AR would elicit similar effects. This is particularly relevant to progestins used by millions of women in contraception and menopausal hormone therapy (HT), as a number of progestins are known to bind to the AR, with some displaying androgenic properties similar to DHT and others displaying anti-androgenic properties. For example, progestins like medroxyprogesterone acetate (MPA), norethisterone acetate (NET-A), and levonorgestrel (LNG) have been shown to be as potent and efficacious as the natural androgen DHT, while others like nestorone (NES) and nomegesterol acetate (NOMAC) display anti-androgenic properties similar to the natural progestogen, progesterone (P4). It is noteworthy that MPA, NET-A and LNG have all been associated with an increased risk of breast cancer. However, the underlying mechanisms whereby these progestins contribute to increased breast cancer risk has not been established. In this study, our main aim was to investigate whether androgenic progestins, unlike anti-androgenic progestins, would elicit similar effects as DHT and the synthetic androgen, mibolerone (Mib), on ERβ and ERα expression in human breast cancer cell lines. First however, we used mammalian two-hybrid assays to investigate the ability of the progestins to induce the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C interaction) of the AR, and showed that progestins elicit different conformations in the receptor. Western blot analysis showed that unlike the androgens that increased AR protein levels, the progestins did not influence AR protein levels in the MCF-7 BUS or MDA-MB-453 breast cancer cells. Quantitative real-time PCR (qPCR) showed that like the androgens, MPA, NET-A and LNG all increased ERβ mRNA expression in the MDA-MB-453 cell line, while P4, NES and NOMAC did not. Moreover, by using the AR antagonist, hydroxyflutamide, we showed that these effects were mediated by the AR. Although these results suggest another mechanism by which the AR may inhibit breast cancer cell growth, the results should be interpreted with caution as we show that the ARmediated effects of the androgens and androgenic progestins, in fact, increased proliferation of the MCF-7 BUS and T47D breast cancer cell lines. Unlike, Mib and DHT, which decreased ERα mRNA expression via the AR, MPA, NET-A and LNG had no effect on ERα expression. Although the precise physiological implications of these preliminary results remain to be determined, our findings highlight the fact that the role of progestins in breast cancer is not straightforward. Moreover, these findings contribute to our understanding of crosstalk between the AR and ER subtypes in breast cancer.
- ItemInvestigating the influence of the progesterone receptor isoforms on androgen receptor activity in breast and prostate cancer cell lines(Stellenbosch : Stellenbosch University, 2021-04) Rademeyer, Anina Zenda; Africander, Donita; Storbeck, Karl-Heinz; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Breast cancer growth and survival are primarily dependent on signalling of estrogens via the estrogen receptor (ER), while for prostate cancer it is dependent on androgens acting via the androgen receptor (AR). However, other steroid receptors such as the progesterone receptor (PR) have also been found in both breast and prostate cancer tumours. Results from a previous study in our laboratory, have suggested crosstalk between the AR and both the PR isoforms, PRA and PRB, in both breast and prostate cancer cell lines. This study also showed that, in addition to transactivation on an androgen response element (ARE), the AR could also transactivate an estrogen response element (ERE), which is the binding site for the ER. As the aforementioned study used synthetic ARE- and ERE-containing promoters in reporter assays, the aim of this study was to assess whether similar responses would also be observed on endogenous AR- and ER-regulated genes in the MDA-MB-231 breast cancer cell line. As a proof of concept, a preliminary experiment was also performed in the PC3 prostate cancer cell line. Whether the AR and PRA or PRB occur in a molecular complex was also investigated using co-immunoprecipitation (Co-IP) assays. Realtime quantitative PCR (qPCR) results showed that both PRA and PRB, modulate the activity of the AR on the expression of the endogenous AR-regulated gene, prostate specific antigen (PSA) and on the ER-regulated genes, cathepsin-D (CTSD) and PR. More specifically, both the unliganded and progesterone (P4)-liganded PR, further increased the dihydrotestosterone (DHT)-induced expression of the AR-regulated PSA gene in the MDA-MB-231 breast cancer cell line. While neither the unliganded PR isoforms, nor liganded PRB, modulated the AR-mediated downregulation of the ER-regulated CTSD gene by DHT, the P4-activated PRA, when expressed in excess, reversed this downregulation. Ligand-activated AR also decreased PR mRNA expression, while both the unliganded and liganded PRA and PRB reversed this decrease. Preliminary results in the PC3 prostate cancer cell line show that ligand activated AR downregulated the expression of the AR-regulated TMPRSS2 gene. Unliganded PRA, expressed at equivalent levels to the AR, lifted this decrease, while when either unliganded or liganded PRA and PRB was present in excess relative to AR, TMRPSS2 mRNA expression was increased. CTSD mRNA expression was increased in MDA-MB-231 breast cancer cells transfected with PRA, suggesting that PRA can activate transcription of an ER-regulated gene. In contrast, PRB decreased expression of an ER-regulated gene, as indicated by the decreased CTSD mRNA expression in MDA-MB-231 cells transfected with PRB. Under the experimental conditions used in this study, we could not show a molecular interaction between the AR and PRA or PRB in COS-1 or MDA-MB-231 cells. However, collectively, the realtime qPCR results support previous findings with promoter-reporters showing crosstalk between the AR and PR in breast cancer, and contribute to our knowledge of steroid receptor crosstalk in breast cancer.
- ItemInvestigating 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.
- ItemInvestigating the role of inflammation, progestins and steroid receptors in breast cancer(Stellenbosch : Stellenbosch University, 2019-04) Eksteen, Anishka; Africander, Donita; Verhoog, Nicolette J. D.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Progestins are used by women all over the world in menopausal hormone therapy (HT) and contraception. Numerous clinical trials have, however, reported that some progestins are associated with an increased risk for developing breast cancer. Although multiple progestins with different chemical structures and biological activities have been synthesised, not all progestins have been evaluated for increased breast cancer risk. Obesity-related inflammation is also strongly associated with increased breast cancer risk, particularly amongst postmenopausal women. This increased inflammatory state is characterised by enhanced production of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNFα). Both these inflammatory mediators have been implicated in breast cancer development and progression. However, the exact mechanism whereby inflammation and progestins contribute to breast cancer risk is yet to be established. The aim of this study was thus to investigate the effects of different progestins, in the absence and presence of IL-6 or TNFα, on the proliferation, apoptosis, migration and invasion of breast cancer cells. Given that some progestins can interact with multiple steroid receptors, all of which are known to play important roles in breast cancer biology, the first part of the study investigated effects on steroid receptor expression in the T47D breast cancer cell line. Western blot analysis showed that all the progestins decreased the expression of the estrogen receptor (ER)-subtypes, progesterone receptor (PR)-isoforms and the androgen receptor (AR), while no effects were observed on the expression of the glucocorticoid receptor (GR). This study also showed that IL-6 has no effect on steroid receptor expression levels, while TNFα increased the expression of the GR. Results from this study also show that the selected progestins differentially increased cell survival of T47D and MCF-7 BUS breast cancer cells, while all progestins and both inflammatory mediators increased the migration of T47D breast cancer cells. IL-6 had no effect on cell survival of either cell line, while TNFα decreased the survival of the MCF-7 BUS cells. In addition, the progestins medroxyprogesterone acetate (MPA) and drospirenone (DRSP), as well as IL-6, appeared to increase invasion of the MDA-MB-231 breast cancer cell line. In contrast, TNFα appeared to decrease invasion of the MDA-MB-231 cells. This study is the first to show that none of the progestin-mediated effects on steroid receptor expression, proliferation, apoptosis and migration were modulated by either IL-6 or TNFα. On the other hand, TNFα appeared to decrease the progestin-induced effects on the invasion of the MDA MB-231 breast cancer cell line. Taken together, the results show that even though the progestins and the pro inflammatory cytokines may differentially affect breast cancer growth and survival, all the selected progestins, as well as IL-6 and TNFα, increase migration of T47D breast cancer cells. These results suggest that all these progestins and the inflammatory mediators may possibly increase the risk of developing metastatic breast cancer. Finally, the results obtained in this study indicate that the effects of the progestins on steroid receptor expression and hallmarks of cancer, were not exacerbated by the addition of the inflammatory mediators, suggesting that the combination of inflammation and progestins used in HT does not further increase breast cancer risk.
- ItemProgestins 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.
- ItemRegulation of chemokine gene expression by synthetic progestins in a human vaginal epithelial cell line(Stellenbosch : Stellenbosch University, 2012-03) Noeth, Dewald Johan; Africander, Donita; Hapgood, Janet P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The synthetic progestins, medroxyprogesterone acetate (MPA) and norethisterone (Net) and its derivatives (norethisterone enanthate (Net-EN) and norethisterone acetate (Net-A)), are widely used as contraceptives and in hormone replacement therapy (HRT). Several studies have indicated that synthetic progestins modulate immune function and increase the risk of sexually transmitted infections. However, little is known about the molecular mechanism of action of MPA and Net, in particular their regulation of gene expression in the female genital tract, as compared to progesterone (P4). In the first part of this thesis, the effect of P4, MPA and Net-A on the expression of the endogenous chemokine genes, macrophage inflammatory protein (MIP)-1α and MIP-1β, was investigated in a human vaginal epithelial cell line (Vk2/E6E7). Quantitative realtime PCR (QPCR) showed that both P4 and MPA upregulated the TNF-α-induced expression of MIP-1α and MIP-1β mRNA, while Net-A had no effect. Using siRNA technology, it was found that the responses to P4 and MPA on the MIP-1α gene, but not the MIP-1β gene, are mediated via the glucocorticoid receptor (GR). In the second part of the thesis, it was investigated whether the HIV-1 accessory protein, viral protein R (Vpr), could modulate the action of ligands on MIP-1α and MIP-1β gene expression. QPCR showed that Vpr abrogates the effects of P4 and MPA on the TNF-α induced expression of MIP-1α and MIP-1β. Silencing the GR with siRNA technology showed that the GR plays a role in the effect of Vpr on the P4 and MPA-induced expression of MIP-1α. Taken together, these results show that MPA and Net-A display differential effects on chemokine gene expression in a human vaginal epithelial cell line. Furthermore, this study shows that Vpr modulates the effects of MPA bound to the GR. Thus, the results of this thesis provide insight into the effect of synthetic progestins on the immune response in the vagina, and possibly how HIV-infection may alter these responses.
- ItemA 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.