Increased risk of breast cancer is a critical side effect associated with the use of a menopausal hormone therapy (MHT). Estetrol (E4) is a natural estrogen produced by the human fetal liver and is a promising compound for clinical use in MHT. However, its impact on breast cancer is controversial and poorly defined. In this preclinical study, we show that E4 acts as a weak estrogen by stimulating the growth of hormone-dependent breast cancer only at concentrations exceeding menopausal therapeutic needs. E4 presents also an antitumor activity by decreasing the strong proliferative effect of estradiol (E2). While estrogen receptor alpha (ERα) is the predominant receptor mediating its effects, the dual weak-estrogenic/anti-estrogenic feature of E4 results from differential signaling pathways activation. Both nuclear and rapid extra-nuclear signaling pathway are necessary for a complete estrogenic effect of E4. However, the antitumor action of E4 is not due to a capacity to antagonize E2-induced nuclear activity. Altogether, our results highlight that E4 has a limited impact on breast cancer and may offer a safe therapeutic window for the treatment of menopausal symptoms.

Estetrol (E 4 ) is a natural estrogen produced exclusively by the human fetal liver during pregnancy. Its physiological activity remains unknown. In contrast to ethinyl estradiol and estradiol (E 2 ), E 4 has a minimal impact on liver cell activity and could provide a better safety profile in contraception or hormone therapy. The aim of this study was to delineate if E 4 exhibits an activity profile distinct from that of E 2 on mammary gland. Compared with E 2 , E 4 acted as a low-affinity estrogen in both human in vitro and murine in vivo models. E 4 was 100 times less potent than E 2 to stimulate the proliferation of human breast epithelial (HBE) cells and murine mammary gland in vitro and in vivo respectively. This effect was prevented by fulvestrant and tamoxifen, supporting the notion that ERa (ESR1) is the main mediator of the estrogenic effect of E 4 on the breast. Interestingly, when E 4 was administered along with E 2 , it significantly antagonized the strong stimulatory effect of E 2 on HBE cell proliferation and on the growth of mammary ducts. This study characterizes for the first time the impact of E 4 on mammary gland. Our results highlight that E 4 is less potent than E 2 and exhibits antagonistic properties toward the proliferative effect of E 2 on breast epithelial cells. These data support E 4 as a potential new estrogen for clinical use with a reduced impact on breast proliferation.

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Estetrol (E4), estradiol (E2) and progesterone (P4) have important antioxidative and neuroprotective effects in neuronal system. We aimed to study the consequence of combined steroid therapy in neonatal hypoxic-ischemic encephalopathy (HIE). In vitro the effect of E4 combined with other steroids on oxidative stress and the cell viability in primary hippocampal cultures was evaluated by lactate dehydrogenase and cell survival assays. In vivo neuroprotective and therapeutic efficacy of E4 combined with other steroids was studied in HIE model of immature rats. The rat pups rectal temperature, body and brain weights were evaluated. The hippocampus and the cortex were investigated by histo/immunohistochemistry: intact cell number counting, expressions of markers for early gray matter lose, neuro- and angiogenesis were studied. Glial fibrillary acidic protein was evaluated by ELISA in blood samples. In vitro E4 and combinations of high doses of E4 with P4 and/or E2 significantly diminished the LDH activity and upregulated the cell survival.In vivopretreatment or treatment by different combinations of E4 with other steroids had unalike effects on body and brain weight, neuro- and angiogenesis, and GFAP expression in blood. The combined use of E4 with other steroids has no benefit over the single use of E4.

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Oxidative stress with elevated intracellular Ca(2+) concentration as well as endothelial dysfunction is a component of pre-eclampsia. Our aim was to investigate the oxidative stress-dependent expression of Endoglin and Ca(2+)-binding S100B protein from villous and amniotic tissue cultures, and to assess sEng expression from S100B protein-stimulated endothelial cells. We initially examined Endoglin and Hydroxy-nonenal-(HNE)-modified proteins in the placentas and amnion obtained from women with pre-eclampsia (n = 8), and healthy controls (n = 8) by immunohistochemistry. To examine oxidative stress and the S100B protein effect on sEng expression from endothelial cells, normal villous and amniotic tissue cultures were stimulated by 4-HNE, sodium fluoride and xanthine/xanthine oxidase, whereas human umbilical vein endothelial cell cultures were treated with S100B protein in a dose- and time-dependent manner at 37 degrees C in an environment of 95% air and 5% of CO(2). Culture supernatants were assessed using ELISA. Cell viability was determined using MTS assay. The concentrations of sEng and S100B protein were significantly increased in the villous and amniotic tissue culture supernatants under oxidative stress. S100B protein-stimulated endothelial cells released sEng into conditioned media with a significantly higher expression levels at a concentration of 200 pM-20 nM S100B by 2 h, whereas treated with 200 nM of S100B endothelial cells significantly expressed sEng by 12 h and stimulated the cell proliferation by the same period of time. Our findings show that oxidative stress affects sEng and S100B protein expression from villous and amniotic tissues, and picomolar and low nanomolar concentrations of S100B protein significantly up-regulate sEng release from endothelial cells leading to endothelial dysfunction.