The Effects of Estradiol, Androgens and the Selective Estrogen Modulators: Ospemifene, Raloxifene and Tamoxifen on Explant Cultures of Human Breast Tissue

The impact of menopausal hormone therapy (MHT) on increasing the risk for breast cancer (BC) remains controversial. To understand MHT-elicited cellular breast effects and the potential risks, included with using this therapy, a further investigation into this controversy is the subject of this thesis. In this thesis, to study the effects of estrogen, progestin, androgens and selective estrogen receptor modulators (SERMs), a modified tissue explant culture system was used. The different types of human breast tissues (HBTs) used in this study were normal HBTs, obtained from reduction mammoplasties of premenopausal women (prem-HBTs) or postmenopausal (postm-HBTs) women and peritumoral HBTs (peritum-HBTs) which were obtained from surgeries on postmenopausal BC patients. The explants were cultured up to three weeks in the presence or absence of estradiol (E2), medroxyprogesterone acetate (MPA), testosterone (T), dihydrotestosterone (DHT) and SERMs - ospemifene (OSP), raloxifene (RAL) and tamoxifen (TAM). The cultured HBTs maintained morphological integrity and responded to hormonal treatment in vitro. E2, MPA or E2/MPA increased proliferative activity and was associated with increased cyclin-D1 and caused changes in the cell cycle inhibitors p21 and p27, whereas the androgens T and DHT inhibited proliferation and increased apoptosis in HBT epithelia and opposed E2-stimulated proliferation and cell survival. The postm-HBTs were more sensitive to E2 than prem-HBTs. The effects of OSP, RAL and TAM on HBT epithelium were antiproliferative. E2, androgens and SERMs were associated with marked changes in the proportions of epithelial cells expressing steroid hormone receptors: E2 increased ERα expressing cells and decreased androgen receptor (AR) positive cells, whereas T and DHT had opposite effects. The OSP, RAL and TAM, also decreased a proportion of ERα positive cells in HBT epithelium. At 100 nM, these compounds maintained the relative number of AR positive cells, present at control level, which may partly explain proliferative inhibition. In conclusion, the proliferative activity of E2, in the epithelium of postm-HBTs, is opposed by T and DHT, which suggests that the inclusion of androgens in MHT may decrease the risk for developing BC.Estradiolin, androgeenin ja selektiivisten estrogeenireseptorin muuntelijoiden (SERM), ospemifeenin, raloksifeenin ja tamoksifeenin vaikutus ihmisen rintakudokseen Vaihdevuosien hormonihoito (VHH) sisältää tyypillisesti estrogeenia, usein yhdistettynä keltarauhashormoniin tai androgeeniin. Hoidoilla on myönteinen vaikutus vaihdevuosioireisiin, luun tiheyteen ja rasva-aineenvaihduntaan, mutta VHH:n mahdollinen rintasyöpäriskiä lisäävä vaikutus on edelleen kiistanalainen ja aihetta koskevat tutkimusraportit ovat tuottaneet ristiriitaista tietoa. Tämän tutkimuksen tarkoituksena oli selvittää vaihdevuosien hormonihoidossa (VHH:ssa) käytettyjen hormonien ja muiden yhdisteiden vaikutusta ihmisen normaaliin maitorauhaskudokseen. Tutkimuksessa käytettiin kudosviljelymallia, jossa tutkittiin estrogeenin, keltarauhashormonin (medroksiprogesteroni-asetaatti, MPA), androgeenin (testosteroni, T ja dihydrotestosteroni, DHT) ja kolmen selektiivisen estrogeenireseptorin muuntelijan (SERM), ospemifeenin (OSP), raloksifeenin (RAL) ja tamoksifeenin (TAM) rintakudosvaikutuksia ihmisten rmaitorauhaskudokseen. Maitorauhaskudosta viljeltiin hormonien ja SERM- yhdisteiden kanssa 14-21 päivää. Tulokset osoittavat, että rintakudos säilyttää viljelyssä hormonivasteensa kuten E2:n aiheuttaman ER-spesifisten kohdegeenien (amfireguliini ja TFF1) sekä androgeenien aiheuttaman kohdegeenien (apolipoproteiini-D ja prostataspesifinen antigeeni, PSA) induktion. E2, MPA tai E2/MPA lisäsivät rintaepiteelisolujen proliferaatiota, kun taas androgeenit T ja DHT estivät sitä ja lisäsivät solukuolemaa. Ne myös vähensivät E2:n stimuloivaa vaikutusta proliferaatioon. Postmenopausaalisten naisten rintakudos oli yleisesti herkempi hormonihoidoille kuin premenopausaalisten naisten maitorauhasrintakudos. SERM-yhdisteet OSP, RAL ja TAM vähensivät maitorauhasen epiteelisolujen proliferaatiota pitoisuudesta riippuvalla tavalla. E2-, androgeeni- ja SERM-käsittelyihin liittyi voimakkaita muutoksia rintaepiteelien ER- ja ARreseptorien tasoissa ja vaikutusta välittävissä tekijöissä, mitkä selittänevät osaltaan yhdisteiden vaikutuksia. Yhteenvetona voi todeta, että androgeenit estävät maitorauhasen epiteelisolujen proliferaatiota sekä estrogeenin aiheuttamaa stimulaatiota ja siihen liittyvää rintasyöpäriskin lisääntymistä. Postmenopausaalisten naisten hormonihoitoihin suunnitellut SERM-yhdisteet OSP ja RAL estivät normaalin rintaepiteelin proliferaatiota lähes rintasyöpälääkkeenä käytettävään tamoksifeeniin verrattavalla tavalla. Tuloksilla on merkitystä vaihdevuosien hormonihoitojen kehitetyksessä.Siirretty Doriast

Effects of estradiol and medroxyprogesterone acetate on morphology, proliferation and apoptosis of human breast tissue in organ cultures

Background: Human breast tissue undergoes phases of proliferation, differentiation and regression regulated by changes of the levels of circulating sex hormones during the menstrual cycle or aging. Ovarian hormones also likely play a key role in the etiology and biology of breast cancer. Reports concerning the proliferative effects of steroid hormones on the normal epithelium of human breast have been conflicting. Some studies have shown that steroid hormones may predispose breast epithelial cells to malignant changes by stimulating their proliferation, which is known to be regulated tightly by stromal cells. The aim of this study was to investigate the effects of 17 beta-estradiol and medroxyprogesterone acetate on proliferation, apoptosis, expression of differentiation markers and steroid hormone receptors in breast epithelium using an in vitro model of freshly isolated human breast tissue, in which a proper interaction of breast epithelium and stroma has been maintained. Methods: Human breast tissues were obtained from women undergoing surgery for breast tumours. Peritumoral tissues were excised and explants were cultured for 3 weeks in medium supplemented with E-2 or MPA or with E-2+ MPA. Endpoints included histopathological, histomorphometric and immunohistochemical assessment of the breast explants. Results: Culture of breast explants for 14 or 21 days with steroid hormones increased proliferative activity and the thickness of acinar and ductal epithelium. E-2-treatment led to hyperplastic epithelial morphology, MPA to hypersecretory single-layered epithelium and E-2+ MPA to multilayered but organised epithelium. The proliferative response to E-2 in comparison to control ( p < 0.001) was more pronounced than to MPA ( p < 0.05) or E-2+ MPA ( p < 0.05) at 7 and 14 days for Ki-67 and PCNA. E-2 treatment also decreased the proportion of apoptotic cells after 7 ( p < 0.01) and 14 ( p < 0.01) days. In addition, the relative number of ER alpha, ER beta and PR positive epithelial cells was decreased by all hormonal treatments. Conclusion: Organ culture system provides a model for studying the direct effects of steroid hormones and their analogues on postmenopausal human breast tissue

Effects of estradiol and medroxyprogesterone acetate on expression of the cell cycle proteins cyclin D1, p21 and p27 in cultured human breast tissues

Estrogen and progesterone are key regulators of normal breast epithelial cell proliferation and differentiation. They are also involved in the initiation and progression of breast tumorigenesis. Several experimental studies have demonstrated that steroid hormones affect cell cycle proteins associated with tumor initiation and progression. Hormone replacement therapy (HT) is widely used to alleviate climacteric symptoms among postmenopausal women. Little is known, however, about cell cycle protein regulation during hormonal treatment of human breast tissue (HBT). In this study we aimed to evaluate the effects of 17 beta-estradiol (E-2) and medroxyprogesterone acetate (MPA) on cultured HBTs representing samples from reduction mammoplasty of premenopausal (pre-HBT) and postmenopausal (postm-HBT) women, and from peritumoral tissue (peritum-HBT) after breast tumor surgery among postmenopausal patients. Explants of HBT were cultured for 14 days in medium supplemented with E-2, MPA or E-2 + MPA. Expression of cyclin D1, p21 and p27 was assessed by immunohistochemical staining of explants cultured for 2 and 14 days. Further, Ki-67 staining was performed to evaluate correlation between proliferation and cell cycle regulatory protein expression. Our results showed that HBTs studied were positive for ER alpha, ER beta and PR (>= 10% of the cells stained). The level of p21 was lower (p < 0.001) in pre-HBT than in postm-HBT, whereas p27 levels were higher (p < 0.05) in pre-HBT than in postm-and peritum-HBT. The level of Ki-67 positive cells was higher in pre-HBT than in post-HBT. Interestingly, level of p21 positive cells showed an opposite pattern. Treatment with E-2 increased the relative number of cyclin D1-staining cells and decreased that of p27-staining cells in postm-HBT (p < 0.05), but not in pre-HBT. All hormone regimens (E-2, MPA, E-2 + MPA) increased the number of p21-positive cells in postm-HBTs at 14 days and E-2 even at 2 days. In pre-HBT p21 staining was increased (p < 0.05) in explants cultured with E-2 for 14 days but no response was observed in cyclin D1 and p27. The number of cyclin D1-staining cells was clearly higher (p < 0.05) in peritum-HBT than in non-tumorous pre-or postm-HBT, but the response cyclin D1 to all hormonal treatments in peritum-HBT was the same as in postm-HBT. Moreover, we found that E-2, MPA, E-2 + MPA in vitro increased numbers of Ki-67 positive cells in post-HBTs at 14 days and E-2 also in pre-HBT. Stimulated proliferation rate was associated with increase of cyclin D1 and p21-positive cells and reduced numbers of p27, especially in post-HBTs. Taken together, our results suggest that cell cycle regulatory proteins are more sensitive to exogenous hormone treatment in postm-HBT than in pre-HBT

Androgens Inhibit the Stimulatory Action of 17β-Estradiol on Normal Human Breast Tissue in Explant Cultures.

Background: The data concerning the effects and safety of androgen in human breast tissue are conflicting. Objective: Our aim was to analyze the effects of androgens on normal human breast tissue (HBT). Approach: We cultured explants of HBT (obtained from reduction mammoplasty operations of postmenopausal women) with or without testosterone (T) and 5α-dihydrotestosterone (DHT) or in combination with 17β-estradiol (E(2)) for 7 and 14 d to study the effects of androgens on proliferation, apoptosis, target gene expression, and steroid receptors. The androgen receptor (AR) and estrogen receptor (ER) dependences of the effects were studied with the antihormones bicalutamide and fulvestrant, respectively.Results:The hormone responsiveness of cultured breast tissue was assessed by assaying apolipoprotein-D and prostate-specific antigen expression increased by androgens and amphiregulin and trefoil factor-1 expression induced by E(2) treatment. T and DHT reduced proliferation and increased apoptosis in breast epithelium, the effects of which were reversed by bicalutamide. In combination with E(2), they suppressed E(2)-stimulated proliferation and cell survival. DHT also inhibited basal (P < 0.05) and E(2)-induced expression of cyclin-D1 mRNA (P < 0.05). Immunohistochemistry showed that T (P < 0.05) and DHT (P < 0.05) increased the relative number of AR-positive cells, whereas ERα-positive (P < 0.001) cell numbers were strongly decreased. The percentage of ERβ-positive cells remained unchanged. E(2) treatment increased ERα-positive (P < 0.01) cells, whereas AR- (P < 0.05) and ERβ-expressing (P < 0.001) cells diminished. These effects were repressed in combination cultures of E(2) with T and DHT. Conclusion: T and DHT inhibited proliferation and increased apoptosis in the epithelium of cultured normal HBT and opposed E(2)-stimulated proliferation and cell survival in an AR-dependent manner. These effects were associated with changes in the proportions of ERα- and AR-positive epithelial cells