Academic literature on the topic 'Synthetic progestin'

Our recent studies show that progestins induce vascular endothelial growth factor (VEGF) in breast cancer cells that express mutant p53 protein. Here, we show that natural and synthetic progestins also induce thrombospondin-1 (TSP-1) mRNA and protein in T47-D and BT-474 breast cancer cells. Antiprogestin RU-486 inhibits the induction of VEGF and TSP-1 by progestins, suggesting that this effect of progestin is mediated by the progesterone receptor (PR). Actinomycin-D, but not puromycin, also blocks progestin-dependent induction of TSP-1. A putative progestin-response element was identified in the human TSP-1 promoter, which is consistent with the hypothesis that a progestin–PR complex might directly regulate transcription of the TSP-1 gene in human cells. Conditioned medium from progestin-treated breast cancer cells stimulates endothelial cell proliferation in the absence though not in the presence of antibody to TSP-1, indicating that TSP-1 secreted by breast cancer cells could be pro-angiogenic. Since tumor cell-derived TSP-1 has the potential to promote angiogenesis in the tumor microenvironment, it could be a potential target for breast cancer therapy.

The role of progestins in combined hormone therapy is the inhibition of uterine epithelial cell proliferation. The Women’s Health Initiative study provided evidence for an increased risk of breast cancer in women treated with conjugated equine estrogens plus the synthetic progestin medroxyprogesterone acetate (MPA), compared with conjugated equine estrogens-only treatment. These findings continue to be discussed, and it remains to be clarified whether the results obtained for MPA in the Women’s Health Initiative study are directly applicable to other progestins used in hormone therapy. In this study we compared in a mouse model the effects of the synthetic progestins, MPA, and drospirenone in two major target organs: the uterus and mammary gland. As quantitative measures of progestin activity, we analyzed maintenance of pregnancy, ductal side branching in the mammary gland, and proliferation of mammary and uterine epithelial cells as well as target gene induction in both organs. The outcome of this study is that not all synthetic progestins exhibit the same effects. MPA demonstrated uterine activity and mitogenic activity in the mammary gland at the same doses. In contrast, drospirenone behaved similarly to the natural hormone, progesterone, and exhibited uterine activity at doses lower than those leading to considerable proliferative effects in the mammary gland. We hypothesize that the safety of combined hormone therapy in postmenopausal women may be associated with a dissociation between the uterine and mammary gland activities of the progestin component.

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.

The physiological role of ovarian progesterone in reproduction and the medical uses of synthetic progestins and antiprogestins are briefly reviewed. Although a number of progestins are in wide use, the search continues for better analogues that display fewer of the unwanted effects of the present compounds. The one antiprogestin approved for human use to date, RU486, exhibits considerable antiglucocortoid activity The classical bioassays currently in use for the development of these steroid analogues depend upon in vivo progestin effects. However, progestins induce measurable responses in several cell culture systems that could be used as alternative bioassays. Rabbit uterine stromal cells in primary culture are especially suitable for such use. Addition of progesterone to these cultures stimulates synthesis of a 42 kilodalton (42kD) protein that appears in the medium. The 42kD protein response is specific for a progesterone receptor-mediated event and it can be blocked by known progestins. Using this culture system as a bioassay, one rabbit yields enough cells to measure the effect of 30 test doses in triplicate. Thus, a culture system is described that could substitute for current in vivo bioassays. This culture bioassay would allow large scale screening for potential progestin or antiprogestin activity.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use.

Abstract Angiogenesis, the formation of new blood vessels, is essential for tumor expansion, and vascular endothelial growth factor (VEGF) is one of the most potent angiogenic growth factors known. We have previously shown that natural and synthetic progestins, including those used in hormone replacement therapy and oral contraception, induce the synthesis and secretion of VEGF in a subset of human breast cancer cells in a progesterone receptor-dependent manner. We now report that conditioned medium from progestin-treated breast tumor cells can induce the proliferation of endothelial cells in a paracrine manner and induce the proliferation of tumor epithelial cells in a paracrine and an autocrine manner. The use of an anti-VEGF antibody and SU-1498, an inhibitor of VEGF receptor-2 (VEGFR-2 or flk/kdr) tyrosine kinase activity, demonstrated that these effects involve interactions between VEGF and VEGFR-2. Also, blockage of progestin-induced VEGF by the antiprogestin RU-486 (mifepristone) eliminated VEGF-induced proliferative effects. The ability of VEGF to increase the proliferation of endothelial cells and tumor cells, including those that do not release VEGF in response to progestins, suggests that these effects are mediated by amplification of the progestin signal, which culminates in angiogenesis and tumor growth. These novel findings suggest that targeting the release of VEGF from tumor epithelial cells as well as blocking interactions between VEGF and VEGFR-2 on both endothelial and tumor epithelial cells may facilitate the development of new antiangiogenic therapies for progestin-dependent breast tumors. Furthermore, these data indicate that it would be useful to develop selective progesterone receptor modulators that prevent the release of angiogenic growth factors from breast cancer cells.

Selman PJ, Mol JA, Rutteman GR, Rijnberk A. Progestin treatment in the dog. I. Effects on growth hormone, insulin-like growth factor I and glucose homeostasis. Eur J Endocrinol 1994;131:413–21. ISSN 0804–4643 The effects of two synthetic progestins, medroxyprogesterone acetate (MPA) and proligestone (PROL), on the release of growth hormone (GH) and glucose metabolism were studied in two groups of eight ovariohysterectomized dogs. Eight injections of long-acting progestins were administered at 3-week intervals. Recovery was studied in four dogs of each treatment group in the 6 months following cessation of progestin administration. Treatment with both MPA and PROL resulted in similar increases in plasma levels of GH and insulin-like growth factor I (IGF-I). The GH responses to both clonidine and growth hormone-releasing hormone became impaired. In neither treatment group did the elevated plasma GH levels decrease after administration of the synthetic somatostatin analogue SMS 201-995. The size and shape of the pituitary gland were not changed by progestin treatment. After cessation of progestin administration, basal plasma levels of GH and IGF-I did not return to pretreatment values. The GH response to growth hormone-releasing hormone remained impaired for at least 6 months after the last progestin administration. In both treatment groups, glucose homeostasis was sustained initially by increased insulin production. Prolonged treatment with MPA and PROL resulted in glucose intolerance. No amelioration was observed during the recovery period in either group. A small number of dogs developed diabetes mellitus. In more than 50% of the dogs in both treatment groups small mammary tumours developed. The recently discovered local production of GH probably played a role in mammary tumorigenesis. It is concluded that treatment with MPA and PROL results in similar increases in plasma levels of GH and IGF-I and leads to a similar degree of insulin resistance. The elevated GH levels can be neither stimulated nor inhibited, which is a feature compatible with autonomous secretion. These results are consistent with the recent finding of a progestin-induced ectopic production of GH in the mammary gland of the dog. JA Mol, Department of Clinical Sciences of Companion Animals, Utrecht University, PO Box 80. 154, 3508 TD Utrecht, The Netherlands

A commonly prescribed contraceptive, the synthetic progestin norethindrone (NET) inhibits ovulation in humans. However, ecotoxicological data are lacking. Preliminary tests produced an LC50 for NET of > 1.0 mg/L (96-hour, fathead minnow (FHM) and medaka) and a NOEC of 242.0 µg/L, a LOEC of 485.0 µg/L (7-day, growth for FHM and medaka). Reproductive testing revealed a LOEC for fecundity of 24.1 ng/L (21 days, medaka). Further testing confirmed the LOEC of 24.1 ng/L while defining a NOEC of 4.7 ng/L (28 days, medaka). Effect of NET in medaka life-cycle exposure at concentrations exceeding 4.7 ng/L was evident. Few females were present in the 24.7 ng/L exposure concentration, with none in the 104.6 ng/L. Egg production was significantly reduced at concentrations exceeding 4.7 ng/L. Additionally, weight, condition factor and somatic indices were significantly different in males exposed to concentrations exceeding 4.7 ng/L. For fecundity and sexual differentiation; the NOEC was 4.7 ng/L, the LOEC 24.6 ng/L; growth and somatic indices, the NOEC was more appropriately 0.9 ng/L, with effect evident at 4.7 ng/L. Sexual differentiation of the F1 population was similar to the F0. A defining result of this test was development of exceptionally large ovaries in NET- exposed female medaka, perhaps indicative of a threshold limit for exposure in these fish. Results of FHM life-cycle testing were similar, establishing a NOEC for fecundity of 0.9 ng/L, a LOEC of 4.8 ng/L. NET's inhibitory effect on gonadal development was obvious; GSI NOEC for males, 4.8 ng/L, and histological examination confirmed the presence of intersex development at elevated concentrations. Normal physical development and growth were impaired, generally at concentrations exceeding 24.1 ng/L. At exposure concentrations exceeding 4.8 ng/L, external sexual confirmation of fish was difficult; LOEC for finspot development in females, 4.8 ng/L. Sexual determination of the 97.1 ng/L exposure group was impossible; externally, all fish appeared male and internal examination revealed no gonadal development.

Thesis (Ph. D.)--University of Oregon, 2007.
Typescript. Includes vita and abstract. Includes results of four studies conducted at the University of Oregon. Includes bibliographical references (leaves 221-244). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (MSc)--Stellenbosch University, 2012.
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.
AFRIKAANSE OPSOMMING: Die sintetiese progestiene medroksieprogesteroon asetaat (MPA) en noretisteroon (Net) en derivate daarvan (noretisteroon enantaat (Net-EN) en noretisteroon asetaat (Net-A)), word op grootskaal gebruik as voorbehoedmiddels en in hormoonvervangingsterapie (HVT). Verskeie studies het al aangedui dat sintetiese progestiene immuunfunksie moduleer en die risiko vir seksuel oordraagbare infeksies verhoog. Daar is egter min bekend oor die molekulêre meganisme van aksie van MPA en Net, in die besonder die regulering van geenuitdrukking in die vroulike geslagskanaal in vergelyking met progesteroon (P4). In die eerste deel van hierdie tesis is die effek van P4, MPA en Net-A op die uitdrukking van endogene chemokiene gene, makrofaag inflammatoriese proteïen (MIP)-1α en MIP-1β, in 'n menslike vaginale epiteel sellyn (Vk2/E6E7) bestudeer. Kwantitatiewe intydse PKR (KPKR) het getoon dat beide P4 en MPA die TNF-α-geïnduseerde uitdrukking van beide die MIP-1α en MIP-1β mRNA uitdrukking op reguleer, terwyl Net-A geen effek getoon het nie. Met die gebruik van siRNA-tegnologie is daar bevind dat die effekte van P4 en MPA, bemiddel word deur die glukokortikoïd-reseptor (GR) op MIP-1α geen uitdrukking, maar nie op MIP-1β nie. In die tweede deel van die tesis, is ondersoek of die MIV-1-bykomstigheidsproteïen, virale proteïen R (Vpr), die aksie van die ligande op MIP 1α en MIP-1β geenuitdrukking kan moduleer. KPKR toon dat Vpr die uitwerking van P4 en MPA op die TNF-α-geïnduseerde uitdrukking van MIP 1α en MIP-1β kanselleer. Die verwydering van die GR met siRNA-tegnologie toon dat die GR 'n rol in die uitwerking van Vpr op die P4 en MPA-geïnduseerde uitdrukking van MIP-1α speel. Ter samevatting: hierdie resultate toon dat MPA en Net-A differensiële uitwerkings vertoon op chemokiene geenuitdrukking in 'n menslike vaginale epiteel sellyn, en dat Vpr hierdie uitwerkings moduleer van MPA gobonde aan die GR. Die resultate van hierdie tesis werp dus lig tot die uitwerking van sintetiese progestiene op die immuunreaksie in die vagina, sowel as hoe MIVinfeksie hierdie reaksies kan verander.

Pharmaceuticals have routinely been detected in the environment resulting in a growing concern about whether these drugs could elicit effects on aquatic organisms. The concerns are centered on the highly conserved nature of mammalian therapeutic targets in fish. These pharmaceuticals are found at very low levels in the environment, which can result in sub-lethal effects in aquatic organisms. Therefore, 28 d early-life stage studies were conducted on six pharmaceuticals to assess their impacts on survival and growth fathead minnow larvae. Two pharmaceuticals tested, carbamazepine and fenofibrate, resulted in no alterations to survival and growth. However, amiodarone, clozapine, dexamethasone, and levonorgestrel (LNG) reduced survival at concentrations tested with LNG being the most potent at 462 ng/L. Survival was increased with amiodarone and clozapine; however LNG significantly decreased growth at 86 ng/L. Therefore, the most potent pharmaceutical tested was the synthetic progestin LNG with survival and growth impacts at concentrations less than 1 μg/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 d ELS exposure to LNG. Transcripts of 3β-HSD, 20β-HSD, and FSH were significantly down-regulated following 28 d exposure to both 16.3 and 86.9 ng/L LNG. Also, CYP19a expression was significantly down-regulated at 86.9 and 2392 ng/L LNG. Subsequently, a second study examined time periods that may be most sensitive (e.g., windows of sensitivity) for FHM larvae exposed to LNG. Larvae were exposed to a single concentration of LNG (i.e. LOECgrowth of 86.2 ng/L as determined in the 28 d ELS study) for different time periods starting with fertilized egg through 28 dph. Growth and mRNA expression of the four differentially expressed transcripts from the first study were measured. Regardless of the duration of exposure, LNG significantly decreased growth in fathead minnow larvae at day 28. For both 20β-HSD and CYP19a, mRNA expression was decreased following exposure to LNG; however, these transcripts returned to baseline levels after removal of LNG. 3β-HSD and FSH showed similar trends after exposure to LNG with 7-14 d and 14-28 d exposures exhibiting a decrease in expression; however, FSH expression returned to baseline once removed for LNG exposure. Based on these data, 3β-HSD was the only transcript to remain down regulated after LNG exposure. Together these data suggest LNG can negatively impact FHM larval survival and growth, with significant alterations in endocrine related responses. However, these changes in endocrine related responses may not directly correlate to the changes in growth demonstrated with LNG exposure to fathead minnows. Therefore, additional research is warranted to ascertain additional mechanisms, either endocrine related or non-endocrine functions, related to changes in growth of larval fathead minnows.

Thesis (PhD)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: Although the progestins medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A) are widely used in reproductive therapy, the steroid receptors and their target genes involved in the actions of MPA and NET-A are not well understood. Surprisingly, it had not yet been investigated whether doses of MPA and NET-A used for contraception and HRT cause significant side effects through various target genes via the glucocorticoid receptor (GR). In this thesis results of in vitro studies showed that, MPA, like dexamethasone (dex) and prog, significantly repressed tumour necrosis factor (TN F)-stimulated IL-6 protein production, and IL-6 and IL-8 promoter reporter constructs at the transcriptional level in L929sA cells, via interference with nuclear factor KB (NFKB) and activator protein-1 (AP-1) transcription factors. Like dex and prog, MPA did not affect NFKB DNA-binding activity. Furthermore, unlike dex and prog, MPA did not inhibit mitogen-activated protein kinase (MAPK) activity. The antagonistic effects of the GR and progesterone receptor (PR) antagonist, RU486, as well as the MPAinduced nuclear translocation of the GR, strongly suggest that the actions of MPA in these cells are mediated at least in part via the GR. Although the mechanism was not investigated as extensively as for MPA, NET-A was shown to repress IL-8 promoter reporter activity very weakly relative to dex, MPA and prog in Hek293 cells stably transfected with the rat GR. Furthermore, NET-A, like MPA, dex and prog did not interfere with the DNA-binding activity of NFKB. Significant transactivation of a GRE-driven promoter reporter construct by MPA and dex in L929sA via endogenous GR and COS-1 cells via expressed rat GR, and by MPA, dex and prog in Hek293 cells via expressed rat GR was also observed. In contrast, NET-A, unlike MPA, dex and prog showed no transactivation in Hek293 cells. MPA, NET-A and prog were shown to compete with dex for binding to the endogenous human GR in human lung carcinoma A549 cells. Similarly, MPA and NET-A were shown to compete with dex for binding to expressed rat GR in COS-1 cells. MPA displayed a higher relative binding affinity than NET-A for the GR in both systems, and a higher relative binding affinity than prog in A549 cells. Equilibrium dissociation constants (Ki values) for MPA (Ki = 10.8 ± 1.1 nM), NET-A (Ki = 270 ± 1.3 nM) and prog (Ki = 215 ± 1.1 nM) towards the human GR in A549 cells were also established. Furthermore, dose-response curves showed that MPA displays significantly greater GC agonist potency and efficacy than NET-A and prog for both transactivation of a synthetic GRE-reporter construct and transrepression of a synthetic IL-8 reporter construct via expressed rat GR in Hek293 cells, as NET-A showed no transactivation and very weak partial agonist activity for transrepression. Based on these observations, MPA behaves as a GR agonist whereas NET-A is proposed to be a weak antagonist. These results show that MPA and NET-A are not alike and not the same as prog in their mechanism of action via the GR, which may have serious health implications in vivo. Such insights may provide women and their clinicians with more information to facilitate the selection of contraception or reproductive therapy regimes with fewer side effects.
AFRIKAANSE OPSOMMING: Alhoewel MPA en NET-A algemeen gebruik word in hormoontherapie, is dit nie duidelik watter steroïedreseptore en teikengene betrokke is by die werking van MPA en NET-A nie. Verrassend is dat geen studie nog gedoen is om te bepaal of die dosisse van MPA en NET-A wat gebruik word in voorbehoeding en hormoonvervangingsterapie (HVT), newe-effekte veroorsaak deur die glukokortikoïedreseptor (GR) en verskeie teikengene nie. In hierdie tesis is in L929sA selle aangetoon dat MPA, net soos deksametasoon (dex) en prog, TNF-gestimuleerde IL-6 produksie onderdruk, en dat IL-6 en IL-8 promoter-rapporteerderkonstrukte op transkripsionele vlak onderdruk word deur middel van inmenging met NF-KB en AP-1 transkripsie-faktore. Net soos dex en prog het MPA nie die DNA-bindingsaktiwiteit van NF-KB beïnvloed nie. Anders as dex en prog het MPA egter nie MAPK aktiwiteit onderdruk nie. Die antagonistiese effekte van RU486, asook die MPA-geïnduseerde translokasie van die GR na die selkern, dui sterk daarop dat die effekte van MPA in hierdie selle ten minste gedeeltelik deur die GR geskied. Alhoewel die meganisme vir NET -A nie so breedvoerig bestudeer is as dié van MPA nie, is tog aangetoon dat, in Hek293 selle wat stabiel getransfekteer is met die rot GR, die onderdrukking van die IL-8 promoter deur NET-A baie swakker is as met dex, prog en MPA. Verder is daar ook gevind dat NET-A, net soos MPA, dex en prog, nie kon inmeng met die DNA-bindingsaktiwiteit van NF-KB nie. Beduidende transaktivering van 'n GRE-bevattende promoterrapporteerderkonstruk deur MPA en dex in L929sA en COS-1 selle, en deur MPA, dex en prog in Hek293 selle, is ook gevind. Daarteenoor het NET-A, anders as MPA, dex en prog, geen transaktivering in Hek293 selle getoon nie. Verder moes die relatiewe bindingsaffiniteit (ewewigs-dissosiasiekonstantes) van MPA, NET-A en prog vir die GR, asook die relatiewe sterkte en effektiwiteit vir transaktivering en transonderdrukking van verskeie teikengene deur die GR, ook bepaal word. Daar is gevind dat MPA, NET-A en prog meeding met dex vir binding aan die endogene GR in mens longkarsinoom A549 selle. Soortgelyk hieraan is ook gevind dat MPA en NET-A meeding met dex vir binding aan rot GR wat in COS-1 selle uitgedruk is. MPA het in beide sisteme 'n hoër relatiewe bindingsaffiniteit vir die GR getoon as NET-A, asook 'n hoër relatiewe bindingsaffiniteit as prog in A549 selle. Ewewigs-dissosiasiekonstantes (Ki waardes) vir MPA (Ki = 10.8 ± 1.1 nM), NET- A (Ki = 270 ± 1.3 nM) en prog (Ki = 215 ± 1.1 nM) vir die mens GR in A549 selle is ook bereken. Dosisrespons-grafieke het ook aangedui dat MPA 'n beduidend beter GC sterkte en effektiwiteit as NET-A en prog het, vir beide transaktivering van 'n sintetiese GRE-rapporteerderkonstruk en transonderdrukking van 'n sintetiese IL-8 rapporteerderkonstruk via rot GR wat uitgedruk is in Hek293 selle. Dit kon afgelei word aangesien NET-A geen transaktivering en slegs baie swak gedeeltelike agonisaktiwiteit vir transonderdrukking getoon het. Op grond van hierdie waarnemings tree MPA op as 'n GR agonis, terwyl dit lyk asof NET-A 'n swak antagonis is. Hierdie resultate dui aan dat MPA en NET-A nie dieselfde is nie, en ook nie dieselfde meganisme van werking deur die GR het as prog nie. Dit kan ernstige gesondheidsimplikasies inhou in vivo. Hierdie insigte kan dus meer inligting aan vroue en kliniese personeel verskaf om sodoende die keuse van voorbehoeding of voortplantingsterapie met minder newe-effekte te vergemaklik.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NETA)), are used by millions of women as contraceptives and in hormone replacement therapy (HRT). Although both progestins are widely used, very little is known about their mechanism of action at the molecular level. In this thesis, the differential regulation of gene expression and molecular mechanism of action via different steroid receptors by these synthetic progestons, as compared to progesterone (Prog) was investigated in human cell lines. In the first part of the study, the effect of Prog, MPA and NET-A on the expression of endogenous cytokine genes was investigated in two epithelial cell lines of the human female genital tract, Ect1/E6E7 (an ectocervical cell line) and Vk2/E6E7 (a vaginal cell line). Quantitative realtime RT-PCR (QPCR) showed ligand-specific and cell-specific regulation of the interleukin (IL)-6, IL-8 and RANTES (Regulated-upon-Activation, Normal T cell Expressed and Secreted) genes with Prog, MPA and NET-A. Moreover, the repression of the TNF -induced RANTES gene by MPA in the Ect1/E6E7 cell line was found to be mediated by the androgen receptor (AR). The second part of the study focused on elucidating the androgenic activities of these two progestins, in comparison to Prog. Competitive binding in whole cells revealed that Prog, MPA and NET-A have a similar binding affinity for the hAR as the natural androgen dihydrotestosterone (DHT). Both transactivation and transrepression transcriptional assays demonstrate that, unlike Prog, MPA and NET-A are efficacious AR agonists, with activities comparable to DHT. Using a mammalian two-hydrid assay, it was shown that MPA and NET-A exert their androgenic actions by different mechanisms. NET-A, like DHT and other well-characterised androgens, induces the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C-interaction) of the AR independent of promoter-context, while MPA does this in a promoterdependent manner. In the third part of this study, competitive binding revealed that MPA and NET-A have a similar binding affinity to each other, but about a 100-fold lower affinity than Prog for the human mineralocorticoid receptor (hMR), while RU486 has an even lower affinity for the hMR. Promoter-reporter assays showed that MPA, NET-A and RU486 are all antagonists of the hMR, but unlike Prog, they have weak antagonistic activity. However, on the endogenous MR-regulated Orm-1 (a-glycolytic protein or orosomucoid-1) gene expressed in a rat cardiomyocyte cell line, NET-A and RU486, but not MPA, has similar antagonistic activity as Prog. This study is the first to show that, NET-A and RU486, but not MPA, can dissociate between transrepression and transactivation via the hMR. Taken together, these results show that natural Prog and the synthetic progestins, MPA and NET-A display differential promoter-, cell- and receptor-specific effects on gene expression. Furthermore they may have important implications for cervicovaginal immune function, cardiovascular and other physiological functions.
AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan (noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), word deur miljoene vroue gebruik as voorbehoedmiddels en vir hormoon vervangingsterapie (HVT). Tenspyte daarvan dat beide hierdie progestiene algemeen gebruik word, is min bekend oor hulle meganisme van werking op molekulêre vlak. In hierdie proefskrif word die differensiële regulering van geenuitdrukking asook die molekulêre meganisme van werking deur middel van steroïedreseptore van beide hierdie sintetiese progestiene, ondersoek, en vergelyk met progesteroon (Prog), in menslike sellyne. In die eerste deel van die studie is die effek van Prog, MPA en NET-A op die uitdrukking van endogene sitokinien gene ondersoek in twee epiteel sellyne van die menslike vroulike geslagskanaal, Ect1/E6E7 (‘n ektoservikale sellyn) en Vk2/E6E7 (‘n vaginale sellyn). Kwantitatiewe intydse RT-PKR het ligand-spesifieke en selspesifieke regulering van interleukien (IL)-6, IL-8 en RANTES (Regulering-na- Aktivering, Normale T-sel Uitgedrukte en Afgeskei) gene getoon met Prog, MPA en NET-A. Verder is gevind dat die onderdrukking van die TNF- - geïnduseerde RANTES geen deur MPA in die Ect1/E6E7 sellyn bemiddel word deur die androgeen reseptor (AR). Die tweede deel van die studie het gefokus op die toeligting van die androgeniese aktiwiteit van die twee progestiene in vergelyking met Prog. Kompeterende binding in volselle het getoon dat Prog, MPA en NET-A ‘n soortelyke bindings affiniteit vir die menslike AR as die natuurlike androgeen dehidrotestosteroon (DHT) vir die menslike AR het. Beide transaktiverings en transonderdrukkings transkripsionele analieses toon dat, anders as Prog, MPA en NET-A effektiewe AR agoniste is met aktiwiteite wat vergelykbaar is met die van DHT. Deur die gebruik van ‘n soogdier twee-hibried toets, kon gewys word dat MPA en NET-A hul androgeniese effekte uitoefen deur verskillende meganismes. NET-A, soos DHT en ander goed gekarakteriseerde androgene, induseer die ligand-afhanklike interaksie tussen die NH2- en COOH-terminale domeine (N/C-interaksie) van die AR, onafhanklik van die promoter-konteks. MPA, aan die ander kant, doen dit op ‘n promoter-afhanklike manier. In die derde deel van die studie het kompeterende binding getoon dat MPA en NETA soortelyke relatiewe bindings affiniteite vir die menslike mineralokortikoïed reseptor (hMR) het, maar dat hierdie affiniteit ongeveer 100-voud laer is as die van Prog en dat die affiniteit van RU486 vir hMR selfs nog laer is. Promoter-rapporteerder toetse het getoon dat MPA, NET-A en RU486 almal antagoniste van die hMR is, maar anders as Prog, is hierdie ‘n swak antagonistiese aktiwiteit. Nietemin, op die endogene MR-gereguleerde Orm-1 ( -glikolitiese proteïen of orosomukoïed-1) geen, uitgedruk in ‘n rot kardiomiosiet sellyn, het NET-A en RU486, maar nie MPA nie, ‘n soortgelyke antagonistiese aktiwiteit as Prog. Hierdie studie is die eerste om te wys dat NET-A en RU486, maar nie MPA nie, kan onderskei tussen transrepressie en transaktivering deur middel van die hMR. Samevattend toon die resultate dat natuurlike Prog en die sintetiese progestiene, MPA en NET-A, ‘n differentiële promoter-, sel- en reseptor-spesifieke effek op geenuitdrukking het. Verder mag die resultate belangrike implikasies vir servikovaginale immuunfunksie, asook kardiovaskulêre en ander fisiologiese funksies, inhou.

Thesis (MSc)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: The aim of this thesis was to define the interactions of the androgen receptor (AR) with an analog of a non-steroidal plant compound, Compound A (CpdA), as well as two synthetic progestins, medroxyprogesterone acetate (MPA) and norethindrone acetate (NET-A). The data presented indicates that CpdA has antiandrogenic properties, as it represses androgen-induced activation of both specific and non-specific androgen-responsive reporter constructs. It was found that CpdA exerts these effects by a mechanism other than competition with androgen for binding to the ligand-binding domain (LBD) of the receptor. On the other hand, it is demonstrated that both MPA and NET-A compete with androgen for binding to the AR and induce partial agonist activity via the receptor. Using mammalian two-hybrid assays it was revealed that CpdA, similar to anti-androgenic compounds that are able to compete with androgens for binding to the receptor, represses the androgen-induced interaction between the NH2- and COOH-terminals of the AR (N/C-interaction) without competing for binding to the LBD. Furthermore, it was shown that CpdA slightly represses the androgen-dependent recruitment of steroid receptor co-activator 1 (SRC1) to the activation function (AF2) domain of the AR. When the effects of MPA and NET-A on the N/C-interaction were studied, intriguing results were obtained. NET-A, as expected, induced this AR agonist-induced interaction. MPA, however, repressed this AR agonist-induced interaction, an effect previously associated with anti-androgenic activity, despite displaying partial agonist activity in transctivation experiments. On the other hand, both MPA and NET-A induced the interaction between SRC1 and the AF2 domain. In additional experiments with CpdA, it was found that CpdA did not affect the recruitment of SRC1 to the AF1 domain of the receptor; neither did it influence the constitutive activity of the NH2-terminal domain. The anti-androgenic activities of CpdA were confirmed by the toxic effect that this compound had on the androgen-dependent lymph node carcinoma of the prostate (LNCaP) cell-line as well as its ability to repress the androgen-induced expression of the prostate specific antigen (PSA) protein. Taken together, the results presented in this thesis, in combination with the knowledge available on AR function, contribute to an improved understanding of AR function. Furthermore, the importance of defining the precise mechanism by which individual compounds exert their effects is highlighted. In this regard it is demonstrated that two compounds (MPA and NET-A) that display partial agonist activity, can exert their effects via different mechanisms at the molecular level. Detecting such differences in the molecular mechanisms of action could facilitate the improved design of progestins as well as aid clinicians and their patients in selecting the best method of contraception. Lastly, the insights gained into the mechanisms of the anti-androgenic action of CpdA could be useful in therapeutic drug design for diseases, such as prostate cancer, that have an androgen-dependent etiology.
AFRIKAANSE OPSOMMING: Die doel van hierdie tesis was om die interaksies van die androgeen reseptor (AR) met ‘n analoog van ‘n nie-steroiediese plant verbinding, Verbinding A (VbgA), sowel as met twee sintetiese progestogene, medroksiprogesteroon asetaat (MPA) en noretiendroon asetaat (NET-A), te definieer. Die data verskaf dui daarop dat VbgA anti-androgeniese eienskappe besit deurdat dit androgeen-gei'nduseerde aktivering van beide spesifieke- en nie-spesifieke androgeen-responsiewe rapporteerderkonstrukte onderdruk. VbgA veroorsaak hierdie effekte deur ‘n meganisme wat nie kompetisie met androgeen vir binding aan die ligand-bindingsdomein (LBD) van die reseptor behels nie. In teenstelling hiermee word getoon dat beide MPA en NET-A kompeteer met androgeen vir binding aan die AR en gedeeltelike agonistiese aktiwiteit induseer via hierdie reseptor. Deur gebruik to maak van ‘n soogdier twee-hibried essai word getoon dat VbgA, soos ander anti-androgeniese verbindings wat kompeteer met androgeen vir binding aan die reseptor, die androgeen-gei'nduseerde interaksies tussen die NH2- en COOH-terminale van die AR (N/C-interaksie) onderdruk, sonder om te kompeteer vir binding aan die LBD. Daarby is dit bewys dat VbgA die androgeenafhanklike werwing van steroied reseptor ko-aktiveerde 1 (SRC1) na die aktiverings funksie (AF2) domein van die AR gedeeltelik onderdruk. Die studie van die effekte van MPA en NET-A op die N/C-interaksie het interessante resultate opgelewer. NETA, soos verwag, het hierdie AR agonis-gei'nduseerde interaksie geinduseer. MPA, aan die ander kant, het hierdie AR agonis-gei'nduseerde interaksie onderdruk, ‘n effek wat tevore met anti-androgeniese aktiwiteit geassosieer is, al het die transaktiveringseksperimente daarop gedui dat MPA ‘n AR agonis is. Aan die ander kant, het beide MPA en NET-A die interaksie tussen SRC1 en die AF2 domein geinduseer. In addisionele eksperimente met VbgA is gevind dat VbgA geen effek het op die werwing van SRC1 na die AF1 domein van die reseptor nie en ook geen invloed het op die konstitutiewe aktiwiteit van die NHh-terminaal domein nie. VbgA se antiandrogeniese eienskappe is bevestig deur die toksiese effekte op die androgeenafhanklike limfknoop karsinoom van die prostaat (LNCaP) sellyn sowel as deur sy vermoe om die androgen-gei'nduseerde uitdrukking van die prostaat spesifieke antigeen (PSA) protei'en te onderdruk. Die resultate aangebied in hierdie tesis, in kombinasie met die beskikbare kennis oor AR funksie, dra by tot ‘n verbeterde kennis van AR funksionering. Verder word die belang van die definiering van die meganisme waardeur individuele verbindings hulle effekte veroorsaak, getoon. In hierdie verband is getoon dat twee verbindings (MPA en NET-A), wat gedeeltelike agonistiese aktiwiteit besit, hulle effekte via verskillende meganismes op die molekulere vlak veroorsaak. Deur hierdie verskille in die molekulere meganismes van aksie uit te wys, kan beter progestogene ontwikkel word, en verder sal dit vir dokters en hul pasiente help om die beste voorbehoedmiddel te kies. Laastens, die insig wat verkry is ten opsigte van die meganismes van anti-androgeniese aktiwiteit van VbgA mag nuttig wees in die ontwerp van terapeutiese middels vir die behandeling van siektetoestande met androgeen-afhanklikke etiologie (bv. prostaatkanker).

Background: Natural progesterone is currently unavailable for the treatment of endometrial hyperplasia. The first line of treatment for this condition is with the synthetic progestins Medroxyprogesterone Acetate (MPA) and Levonorgestrel (LNG). However, these hormones frequently trigger side effects which occur as a result of adverse gene and protein regulation. The current unavailability of natural progesterone is down to its poor bioavailability which is due to the low aqueous solubility and extensive first-pass metabolism of this compound. Aims: The first aim of this thesis is to compare natural progesterone with MPA and LNG in their regulation of a subset of genes and proteins in the endometrium whilst also assessing the selected proteins in biopsies from patients with endometrial hyperplasia before and after progestin therapy. The second aim is to study progesterone metabolism within the gastrointestinal tract and liver in order to direct the production of progesterone amorphous solid dispersions. Methodology: During this thesis both microbiological and pharmacological laboratory techniques were used. For microbiology investigations immunohistochemistry, polymerase chain reaction, InCell Analysis and enzyme-linked immunosorbent assay were the key research methods. For pharmacological research the main methods used were stability assays analysed by tandem mass spectrometry and high performance liquid chromatography, solvent emulsion evaporation, powder x-ray diffraction and scanning electron microscopy. Conclusions: Amorphous solid dispersions are an attractive option for the future production of a natural progesterone oral formulation with enhanced solubility. Such a formulation can be targeted for delivery to the distal small intestine where progesterone metabolism is reduced compared to other intestinal regions. This formulation would be of particular benefit to patients with endometrial hyperplasia as the most commonly used progestins to treat this disease were shown to differentially regulate genes and proteins in the human endometrium compared to the natural product.

Includes bibliographical references (pages 127-171).
The endocervical mucosae of the female reproductive tract (FRT) not only serve as a physical barrier against microbial infection, but they also express a wide variety of immune mediators. The endocervical epithelial cells express a distinct profile of immune-regulators, which is higher than vaginal and ectocervical epithelial cells. Constant cytokine production would ensure rapid responses to infections and maintenance of the sterility of the upper genital tract. However, overproduction of cytokines could inhibit normal reproductive processes and stimulate excess growth and cell proliferation. The synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)) are synthetic steroidal hormones designed to elicit progestational effects similar to those of the endogenous hormone progesterone (P4). They are extensively used as contraceptives and in hormone replacement therapy (HRT). Numerous studies, however, have reported that synthetic progestins affect immune function, increase the risk of sexually transmitted infections (STIs) and also change the morphology of the cervicovaginal mucosa. Despite these findings little is known about the molecular mechanisms of action of MPA and NET, in particular their differential effects on gene expression.

The Women’s Health Initiative (WHI) clinical trial was the first randomised, double blind, placebo-controlled disease prevention trial to demonstrate epidemiological evidence of a casual link between the use of combined hormone replacement therapy (cHRT) comprising conjugated equine estrogens (CEE) and the synthetic progestin medroxyprogesterone acetate (MPA) and increased breast cancer risk in post-menopausal women. Since the first WHI report in 2002, other observational studies have demonstrated that it is the addition of the synthetic progestin in the cHRT that is associated with an increase in breast cancer risk. The focus of this thesis was to investigate the following hypothesis formulated in the Dame Roma Mitchell Cancer Research Laboratory, which proposed that MPA possesses antagonistic actions on androgen receptor (AR)-signalling and thereby can disrupt the protective effect of androgens in the breast, thus leading to an increased risk of breast cancer. Androgens have been associated with a growth restrictive role in breast tissue in both humans and animals and are now emerging as key hormonal pathways involved in the pathogenesis of breast cancer. The objectives of this thesis were firstly to determine the relationship between the use of cHRT containing MPA and breast cancer incidence in Australian women, and secondly to perform biological studies to investigate the effect of AR-action in breast epithelial cells. Initial findings described in this thesis led to the identification of a positive association between the use of cHRT preparations containing MPA and breast cancer incidence in Australian women. Subsequent biological based studies were undertaken with non-malignant breast tissues samples from pre- and post- menopausal women in an ex vivo breast explant tissue culture experimental model and the oestrogen receptor (ER), progesterone receptor (PR) and AR positive ZR-75-1 breast cancer cell line to investigate the actions of MPA on AR-signalling and cancer-related intracellular signalling pathways. Collectively these studies demonstrated that the actions of MPA can impede the anti-proliferative actions of DHT in both human postmenopausal non-malignant and malignant breast epithelial cells via AR-mediated actions. Furthermore, the combined actions of DHT and MPA were also shown to de-regulate cancer-related intracellular pathways compared to individual hormone treatments. The findings described in this thesis provide novel results indicating that MPA may promote the development of breast cancer in post-menopausal women taking cHRT via AR-mediated actions and that use of this form of hormone therapy remains a major public health concern.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2012

This chapter discusses both benign breast lesions and those that associated with an increased risk of breast cancer development, as well as the aetiology, clinical and pathologic prognosis, and hormonal treatment of breast cancers. Clinical observations in women suggest that hormones play a role in the aetiology of benign lesions. In postmenopausal women receiving oestrogens (with or without progestins) for more than 8 years, the prevalence of benign breast lesions is increased 1.7-fold; whilst the anti-oestrogen, tamoxifen, is associated with a 28 % reduction in prevalence of benign breast lesions. Cyclic and non-cyclic breast pain, nipple discharge and breast lumps are all clinical features of benign breast lesions. A practical classification, based primarily on degree of proliferation, distinguishes benign breast lesions with no associated increase in breast cancer risk from those with a small or moderate (i.e. 1.1–2.0- fold), or high risk (higher than 2.0-fold). Women considered at high risk of developing breast cancer can be treated with selective oestrogen receptor modulators (SERMS) including tamoxifen and raloxifene, which have been shown to decrease breast cancer risk by approximately 50 % when compared to placebo. The aetiology of breast cancer includes the accumulation of mutations of key genes involved in cell proliferation, DNA repair, vasculogenesis, invasion, metastasis, and apoptosis; dietary, environmental, and lifestyle factors also play a key role. The majority of risk factors for breast cancer relate to the duration or intensity of a woman’s exposure to endogenous or exogenous oestrogens; mitogenic and mutagenic effects of oestradiol probably act in concert to initiate and promote the breast cancer development. In this chapter, a variety of established and newer methods for classifying established breast cancers and predicting their prognosis and response to hormonal treatment strategies are discussed. The mechanisms (blockade of oestrogen synthesis or function) of hormonal treatments are described, as is the development of resistance to these treatments. An overview of the clinical efficacy of different hormonal treatments for breast cancer is given, as are recommended approaches to hormonal treatment of breast cancer in pre- and post-menopausal women, and in advanced disease states.

Objective: The aim of this study was to epidemiologically analyze the hormone replacement therapy (HRT) and the emergence of breast cancer. Methodology: This is an article review from the databases such as LILACS, SciELO, Bireme, and Medscape, utilizing the keywords: menopause, HRT, breast cancer and complications, employing the use of connectors when necessary. This review aims to elucidate what has been published in the last years about the usage of hormone therapy and the emergence of breast cancer since there are disagreements between the literature. Results: The analysis displayed positive effects during the usage of HRT, such as maintenance of bone density, prevention of fractures, and cardiovascular events in patients with no previous changes in this system, and also showed us a strong relationship between HRT and the incidence of breast cancer in menopausal women with a focus into the imposing time of use ratio. Meanwhile, this development risk of breast CA can be reduced in the long run with the withdrawal of the previously initiated therapy. The progesterone HRT has been shown to have lower risks in association with estrogen than when compared with the association of synthetic progestins and estrogen. Associations have similar results for oral and skin HRT. In patients using the postmenopausal hormone therapy, the risk of mortality from breast cancer was reduced in patients with exposure for a maximum of 5 years, more than 5–10 years, or more than 10 years. Conclusion: In view of the exposure, it is considered that the HRT is more beneficial than malefic to the life and health of women. Meanwhile, the risk of breast CA goes up while the HRT time stretches over the years. Thus, it is necessary to individually evaluate the benefits and risks to better identify the therapy that should be utilized.