Differential effects of estrogen receptors in the rostral ventrolateral medulla in Goldblatt hypertension.

Previous studies have shown that 17ß-estradiol plays a cardioprotective role in the central nervous system (CNS) of male rats. The aim of the present study was to determine the influence of 17ß-estradiol on sympathetic vasomotor activity and blood pressure in a renovascular hypertensive Goldblatt two-kidney one-clip (2K-1C) male rat model. We also determined the influence of angiotensin II AT1 receptor on the expression of estrogen receptors (ERα, ERß, and G protein-coupled ER (GPER)) in the rostral ventrolateral medulla (RVLM) of Goldblatt rats. Experiments were performed in Goldblatt and age-matched control rats six weeks after clipping of renal artery to induce hypertension. Microinjection of 17ß-estradiol into the RVLM led to a greater reduction in mean arterial pressure and renal sympathetic nerve activity in controls than in 2K-1C rats. Microinjection of the GPER agonist G-1 into the RVLM led to a significantly greater increase in mean arterial pressure and renal sympathetic nerve activity in 2K-1C rats. Expression levels of estrogen receptors GPER and ERα, but not ERß, were significantly higher in the RVLM of 2K-1C rats than in that of the control rats. Chronic treatment with losartan significantly reduced the expression levels of estrogen receptors in the RVLM of 2K-1C rats. Taken altogether, the data suggest that the imbalance of actions between ERα and GPER, particularly with the predominance of GPER in the RVLM, contributes to sympathetic overactivation in male rats with Goldblatt hypertension. AT1-Angiotensin II receptor in the RVLM upregulated estrogen receptor expression in male Goldblatt rats.

Simultaneous Application of Mixotrophic Culture and Magnetic Fields as a Strategy to Improve Spirulina sp. LEB 18 Phycocyanin Synthesis.

Spirulina is a filamentous microalga which is considered a promising alternative source of essential nutrients and active biomolecules. High production cost and the space required to install a photobioreactor are two of the greatest challenges in the industrial application of microalga-based products. Thus, this study aimed to improve Spirulina sp. LEB 18 biomass and phycocyanin content by combining the application of mixotrophic culture and magnetic fields (MF). Zarrouk medium was modified with 1 and 3 g/L liquid molasses and the application of 30 mT for 1·h/d was investigated. Mixotrophic culture with 1 g/L molasses showed the highest biomass concentration (1.62 g/L), carbohydrate content (25.6%), and lipid contents (8.7%) after 15 days. Although the combination of 30 mT and 1 g/L liquid molasses decreased biomass production (1.44 g/L), there was increase in protein yield (76.9%) and protein productivity (73.8 mg/L·d). The proposed method increased phycocyanin production by 145% and its purity from 0.584 in the control culture to 0.627. Data described by this study show that the combination of mixotrophic culture and MF application is a promising alternative to increase microalga protein and phycocyanin production.

Antitumoral activity of liraglutide, a new DNMT inhibitor in breast cancer cells in vitro and in vivo.

Breast cancer (BC) is the most frequently diagnosed female cancer and second leading cause of death. Despite the discovery of many antineoplastic drugs for BC, the current therapy is not totally efficient. In this study, we investigated the potential of repurposing the well-known diabetes type II drug liraglutide to modulate epigenetic modifications in BC cells lines in vitro and in vivo via Ehrlich mice tumors models. The in vitro results revealed a significant reduction on cell viability, migration, DNMT activity and displayed lower levels of global DNA methylation in BC cell lines after liraglutide treatment. The interaction between liraglutide and the DNMT enzymes resulted in a decrease profile of DNA methylation for the CDH1, ESR1 and ADAM33 gene promoter regions and, consequently, increased their gene and protein expression levels. To elucidate the possible interaction between liraglutide and the DNMT1 protein, we performed an in silico study that indicates liraglutide binding in the catalytic cleft via hydrogen bonds and salt bridges with the interdomain contacts and disturbs the overall enzyme conformation. The in vivo study was also able to reveal that liraglutide and the combined treatment of liraglutide and paclitaxel or methotrexate were effective in reducing tumor growth. Moreover, the modulation of CDH1 and ADAM33 mouse gene expression by DNA demethylation suggests a role for liraglutide in DNMT activity in vivo. Altogether, these results indicate that liraglutide may be further analysed as a new adjuvant treatment for BC.

Expression and regulation of the estrogen receptors in PC-3 human prostate cancer cells.

The aim of this study was to identify the expression, cellular localization and regulation of classic estrogen receptors ERα and ERß, ER-α36 isoform and GPER in the androgen-independent prostate cancer cell line PC-3. In addition, we evaluated the relative contribution of these receptors to the activation of the ERK1/2 (extracellular signal-regulated protein kinases) signaling pathway. These four estrogen receptors were detected by Western blot assays and were shown by immunofluorescence assays to localize preferentially in extranuclear regions of PC-3 cells. In addition, treatment with 17ß-estradiol (E2) (1 µM) for 24 h led to down-regulation of the classic estrogen receptors, whereas E2 at physiological concentration (0.1 nM) for 24h tended to increase the levels of ERα and ERß. Furthermore, the ERα-selective agonist PPT selectively increased the expression of ERß and the ERß-selective agonist DPN increased ERα levels. None of these treatments affected expression of the ER-α36 isoform. The unusual cytoplasmic localization of the classic estrogen receptors in these cells differs from the nuclear localization in the majority of estrogen target cells and suggests that rapid signaling pathways may be preferentially activated. In fact, treatment with selective agonists of ERα, ERß and GPER induced ERK1/2 phosphorylation that was blocked by the respective antagonists. On the other hand, activation of ERK1/2 induced by E2 may involve additional mechanisms because it was not blocked by the three antagonists. Taken together, the results indicate that there is a crosstalk between ERα and ERß to regulate the expression of each other, and suggest the involvement of other receptors, such as ER-α36, in the rapid ERK1/2 activation by E2. The identification of new isoforms of ERs, regulation of the receptors and signaling pathways is important to develop new therapeutic strategies for the castration-resistant prostate cancer.

Estrogen receptor 1 agonist PPT stimulates Slc2a4 gene expression and improves insulin-induced glucose uptake in adipocytes.

Type 2 diabetes mellitus is characterized by disruption in glycemic homeostasis, involving impaired insulin-induced glucose disposal. For that, reduced glucose transporter GLUT4, encoded by Slc2a4 gene, plays a fundamental role. Conversely, increase in Slc2a4/GLUT4 expression improves glycemic homeostasis. Recent studies have proposed that estradiol is able to modulate Slc2a4 expression, according to distinct effects upon estrogen receptors ESR1/ESR2. We hypothesize that ESR1-agonist effect could stimulate Slc2a4 expression; thus, increasing cellular glucose disposal, which could be beneficial to glycemic control. Differentiated 3T3-L1 adipocytes were treated (24 hours) with selective ESR1- agonist PPT 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, selective ESR1-antagonist MPP 1,3-Bis(4- hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride, and selective ESR2 agonist DPN 2,3-bis(4-Hydroxyphenyl)-propionitrile, with/without 17ß-estradiol (E2). We analyzed Slc2a4 mRNA (real time PCR) and GLUT4 protein (Western blotting) expression, transcriptional activity of the Slc2a4 repressor Nuclear Factor- κB (NF-κB) (electrophoretic mobility shift assay), and cellular glucose disposal (2-deoxi-D-[(3)H]glucose uptake, 2-DG). ESR1-agonist PPT enhanced Slc2a4/GLUT4 expression (~30%) in the absence or presence of 0.1 and 10 nmol/L E2, and decreased the NF-κB binding activity (~50%). Conversely, ESR1-antagonist MPP, together with E2, decreased Slc2a4/GLUT4 expression (20-40%) and increased NF-κB binding activity (~30%). Furthermore, treatment with ESR2- agonist DPN decreased Slc2a4/GLUT4 expression (20-50%). 2-DG uptake was modulated in parallel to that observed in GLUT4 protein. The present results reveal that ESR1 activity enhances, whereas ESR2 activity represses, Slc2a4/GLUT4 expression. These effects are partially mediated by NF-κB, and allow parallel changes in adipocyte glucose disposal. Furthermore, the data provide evidences that ESR1-agonist PPT, as a Slc2a4/GLUT4 enhancer, can be a promising coadjuvant drug for diabetes mellitus therapy.