Browsing by Author "Perkins, Meghan Samantha"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Investigating the mechanism of action of hormones used in hormone replacement therapy via estrogen receptor subtypes and the influence of the progesterone receptor
    (Stellenbosch : Stellenbosch University, 2018-03) Perkins, Meghan Samantha; Africander, Donita; Louw-du Toit, Renate; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Estrogens and progestins used in conventional menopausal hormone therapy (HT) are associated with increased breast cancer risk. A diverse range of estrogens and progestins are available that mediate their effects primarily by binding to the estrogen receptor (ER) and progesterone receptor (PR), respectively. Although the link to breast cancer risk has not been shown for all estrogens and progestins, many women have turned to custom-compounded bioidentical hormone therapy (bHT) as it is claimed to not increase breast cancer risk. However, scientific evidence to support this claim is lacking. Estrogens and ERα are considered the main etiological factors driving breast cancer, while both ERα and the PR are required for progestin (medroxyprogesterone acetate (MPA)) effects on breast cancer cell proliferation. In this thesis, we investigated the activities of estrogens and progestins used in menopausal hormone therapies via the individual ER subtypes, and the role of ERα/PR crosstalk in mediating progestin-induced effects on gene expression, breast cancer cell proliferation and anchorage-independent growth. In the first part of the study, competitive whole cell bindings assays showed that bioidentical estradiol (bE2) and estriol (bE3) displayed similar binding affinities to the commercially available (natural) estradiol (E2) and estriol (E3) standards, while synthetic ethinylestradiol (EE) had a higher affinity for ERα, and natural E1 a lower affinity for ERβ. Furthermore, the bioidentical estrogens mimicked their respective natural estrogens and synthetic EE on transactivation and transrepression of gene expression, proliferation and anchorage-independent growth of the estrogen-sensitive MCF-7 BUS human breast cancer cell line. These assays showed that E3 and estrone (E1) are efficacious estrogens that do not antagonize E2. In the second part of this study, the estrogenic activities of selected progestins from different generations, MPA, norethisterone acetate (NET-A), levonorgestrel (LNG), gestodene (GES), nestorone (NES), nomegestrol acetate (NoMAC) and drospirenone (DRSP), were characterized relative to each other and natural progesterone (P4). Competitive binding assays revealed that only NET-A, LNG and GES could bind to ERα, while no progestin bound ERβ. Both transactivation and transrepression transcriptional assays showed that NETA, LNG and GES display estrogenic activity. In the third part of the study, the role of PR/ERα crosstalk in mediating the effects of MPA, NET and DRSP, relative to P4, on breast cancer cell proliferation, anchorage-independent growth and the expression of the ER-regulated trefoil factor 1 (pS2) and cathepsin D (CTSD) genes was investigated. All progestins could promote proliferation and anchorage-independent growth of MCF-7 BUS breast cancer cells to the same extent as P4 and E2 via a mechanism requiring both the PR and ERα, but DRSP was the least, and MPA the most potent for proliferation. Quantitative real-time RT-PCR (qPCR), chromatin immunoprecipitation (ChIP) and re-ChIP assays showed that only MPA and NET increased the expression of the pS2 and/or CTSD genes via a mechanism requiring co-recruitment of the PR and ERα to the promoter regions of these genes. In contrast, P4, MPA, NET and DRSP all caused recruitment of the PR/ERα complex to the PR-regulated oncogenes cyclin D1 and MYC. Taken together, the findings of this study suggest that there is no advantage in choosing bHT above conventional HT, and that while it is unlikely that the progestins used in this study will exert biological effects via ERα or ERβ in vivo, some progestins may increase breast cancer risk via a mechanism involving interplay between the PR and ERα.