The aim of this study is the identification of direct endothelial regulation by the androgens testosterone (T) and dihydrotestosterone (DHT). We tested the effects of T and DHT on nitric oxide (NO) synthesis and on tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) expression in human endothelial cells and in ovariectomized (OVX) rats. The results showed that at physiological concentrations T and DHT increase endothelial synthesis of NO. This depends on a rapid recruitment of the extracellular-related kinase (ERK) 1/2 and of the phosphatidylinositol 3-OH kinase (PI3K)/Akt cascades, resulting in endothelial nitric oxide synthase (eNOS) Ser(1177)-phosphorylation. In addition, a later increase of eNOS expression is found. With supra-physiological amounts of T or DHT the induction of NO synthesis is lost. A concentration-related increase of t-PA expression starting from physiological concentrations of T or DHT is found, whereas PAI-1 is augmented only with higher doses. Although DHT exerts these actions through androgen receptors (AR), T acts in part through aromatase-dependent conversion to 17β-estradiol. Ovariectomy is associated with significant changes in eNOS, t-PA and PAI-1 expression in the aorta of Wistar rats and T and DHT result in modifications on eNOS, PAI-1 and t-PA that are in line with the in vitro experiments. In conclusion, T and DHT act on endothelial cells through AR or via conversion to estradiol. Physiological, but not higher amounts are associated with enhanced NO synthesis and an increased t-PA/PAI-1 ratio. These findings are useful to understand the impact of androgens in ageing individuals.

While progesterone plays multiple roles in the process of breast development and differentiation, its role in breast cancer is less understood. We have shown previously that progestins stimulate breast cancer cell migration and invasion because of the activation of rapid signaling cascades leading to modifications in the actin cytoskeleton and cell membrane that are required for cell movement. In this study, we have investigated the effects of progesterone on the formation of focal adhesion (FA) complexes, which provide anchoring sites for cell attachment to the extracellular matrix during cell movement and invasion. In T47-D breast cancer cells, progesterone rapidly enhances FA kinase (FAK) phosphorylation at Tyr 397 in a time-and concentration-dependent manner. As a result, exposure to progesterone leads to increased formation of FA complexes within specialized cell membrane protrusions. The cascade of events required for this phenomenon involves progesterone receptor interaction with the tyrosine kinase c-Src, which activates the phosphatidylinositol-3-kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase complex. In the presence of progesterone, T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices, which is reversed by small interfering RNAs abrogating FAK. In conclusion, progesterone promotes breast cancer cell movement and invasion by facilitating the formation of FA complexes via the rapid regulation of FAK. These results provide novel mechanistic views on the effects of progesterone on breast cancer progression, and may in the future be helpful to develop new strategies for the treatment of endocrine-sensitive breast cancers.

Estrogen and selective estrogen receptor modulators (SERMs) differentially impact endometrial cell function, however, the biological basis of these differences is not established. Deregulated cell adhesion to the extracellular matrix, cell movement and invasion are related to endometrial disorders, such as endometriosis or endometrial cancer. Remodeling of the actin cytoskeleton is required to achieve cell adhesion and movement. Estrogen receptor (ER) regulates actin and cell membrane remodeling through extra-nuclear signaling cascades. In this article, we show that administration of 17beta-estradiol (E2) and tamoxifen (TAM) to immortalized Ishikawa endometrial cells or to human endometrial stromal cells (ESC) results in remodeling of actin fibers and cell membrane. This is linked to rapid phosphorylation on Thr(558) of the actin-binding protein moesin and enhanced migration and invasion of normal and Ishikawa cells. Raloxifene (RAL) does not result in moesin activation or actin remodeling. When endometrial cells are exposed to E2 in the presence of TAM or RAL, both SERMs interfere with the recruitment of moesin, with the remodeling of the cytoskeleton, and with cell movement and migration induced by E2. The differential actions of E2, TAM and RAL are linked to a distinct modulation of the extra-nuclear signaling of ER to G proteins and to the Rho-associated kinase. These findings increase our understanding of the actions of estrogen and SERMs in endometrial cells and highlight potential molecular targets to interfere with the estrogen-related altered cell adhesion encountered in endometrial disorders.

IntroductionHormone replacement therapy (HRT) Abstract Raloxifene (RAL) is a selective oestrogen receptor modulator (SERM) approved for the prevention and treatment of osteoporosis and for the prevention of breast cancer in postmenopausal women. However, little is known on the effects of this SERM on breast cancer cell metastasis, which is the main cause of morbidity and death. Cell movement is critical for local progression and distant metastasis of cancer cells. These processes rely on the dynamic control of the actin cytoskeleton and of cell membrane morphology. The aim of the present study was to characterize the effects of RAL or of 17␤-estradiol (E2) plus RAL on oestrogen receptor (ER) positive T47-D breast cancer cell cytoskeletal remodelling, migration and invasion. Our findings show that, when given alone, RAL induces a weak actin cytoskeleton remodelling in breast cancer cells, with the formation of specialized cell membrane structures implicated in cell motility

Postmenopausal hormone therapy is associated with increased incidence of breast cancer. For this reason alternative therapeutic options to treat menopausal symptoms have been developed. Red clover extracts (RCE) are rich in isoflavones, particularly genistein and daidzein and they have been proved to be effective in reducing vasomotor symptoms in a number of studies. Due to their partial selectivity of action on estrogen receptors (ERs) these compounds have been claimed to be safer on the breast. In this article, we explored the action of RCE on motility and invasion of ER positive breast cancer cells and we partially characterized the signaling mechanisms. The principal isoflavones contained in RCE acted as weak estrogenic compounds when administered alone. However, when provided in association with physiological amounts of estradiol, RCE acted as estrogen antagonist on remodeling of actin cytoskeleton that are requested to enact cell movement and with related modifications of the activity of actin-binding proteins, such as moesin. These results offer novel information on the molecular actions of isoflavones contained in red clover on breast cancer cells, supporting a possible action of these molecules as natural selective estrogen receptor modulators in the presence of physiological amounts of estrogens.