Activation of estrogen receptor induces differential proteomic responses mainly involving migration, invasion, and tumor development pathways in human testicular embryonal carcinoma NT2/D1 cells.

The aims of the present study were to investigate the global changes on proteome of human testicular embryonal carcinoma NT2/D1 cells treated with 17ß-estradiol (E2), and the effects of this hormone on migration, invasion, and colony formation of these cells. A quantitative proteomic analysis identified the presence of 1230 proteins in both E2-treated and control cells. The analysis revealed 75 differentially abundant proteins (DAPs), out of which 43 proteins displayed a higher abundance and, 30 proteins showed a lower abundance in E2-treated NT2/D1 cancer cells. Functional analysis using IPA highlighted some activation processes such as migration, invasion, metastasis, and tumor growth. Interestingly, the treatment with E2 and ERß-selective agonist DPN increased the migration of NT2/D1 cells. On the other hand, ERα-selective agonist PPT did not modify cell migration, indicating that ERß is the upstream receptor involved in this process. The activation of ERß increased the invasion and anchorage­independent growth of NT2/D1 cells more intensely than ERα. ERα and ERß may play overlapping roles on invasion and colony formation of these cells. Further studies are required to clarify the mechanism underlying these effects. The molecular mechanisms revealed by proteomic and functional studies might also guide the development of potential targets for a better understanding of the biology of these cells and novel treatments for non-seminoma in the future.

Molecular regulation of prostate cancer by Galectin-3 and estrogen receptor.

Prostate cancer remains the most prevalent cancer among men worldwide. This cancer is hormone-dependent; therefore, androgen, estrogen, and their receptors play an important role in development and progression of this disease, and in emergence of the castration-resistant prostate cancer (CRPC). Galectins are a family of ß-galactoside-binding proteins which are frequently altered (upregulated or downregulated) in a wide range of tumors, participating in different stages of tumor development and progression, but the molecular mechanisms which regulate its expression are still poorly understood. This review provides an overview of the current and emerging knowledge on Galectin-3 in cancer biology with focus on prostate cancer and the interplay with estrogen receptor (ER) signaling pathways, present in androgen-independent prostate cancer cells. We suggest a molecular mechanism where ER, Galectin-3 and ß-catenin can modulate nuclear transcriptional events, such as, proliferation, migration, invasion, and anchorage-independent growth of androgen-independent prostate cancer cells. Despite a number of achievements in targeted therapy for prostate cancer, CRPC may eventually develop, therefore new effective drug targets need urgently to be found. Further understanding of the role of Galectin-3 and ER in prostate cancer will enhance our understanding of the molecular mechanisms of prostate cancer development and the future treatment of this disease.

Estrogen receptors regulate galectin­3 in androgen­independent DU­145 prostate cancer cells.

The aim of the present study was to investigate the role of estrogen receptor (ER)α and ERß, and galectin­3 (GAL­3) in migration and invasion of androgen­independent DU­145 prostate cancer cells, and to examine the regulation of the expression of GAL­3 by the activation of these receptors. Wound healing and cell invasion assays were performed using the control (basal level of cellular function) and treated DU­145 cells. At 24 h of treatment, 17ß­estradiol (E2), the ERα­selective agonist, 4,4',4"­(4­propyl­(1H)­pyrazole­1,3,5­triyl)trisphenol (PPT), or the ERß­selective agonist, 2,3­bis(4­hydroxyphenyl)­propionitrile (diarylprepionitrile; DPN), increased the migration and invasion of the DU­145 cells. Pre­treatment with the ERα­ and ERß­selective antagonists blocked these effects, indicating that ERα and ERß are upstream receptors regulating these processes. Western blot analysis and immunofluorescence staining for the detection of the GAL­3 were performed using the control and treated DU­145 cells. Treatment of the DU­145 cells with E2, PPT or DPN for 24 h increased the expression of the GAL­3 compared to the control. Furthermore, a specific inhibitor of GAL­3 (VA03) inhibited the migration and invasion of DU­145 cells, indicating the involvement of the complex ERα/GAL­3 and ERß/GAL­3 in the regulation of these processes. On the whole, the present study demonstrates that the activation of both ERs increases the expression and signaling of GAL­3, and promotes the migration and invasion of DU­145 cells. The findings of the present study provide novel insight into the signatures and molecular mechanisms of ERα and ERß in DU­145 cells.

Activation of estrogen receptor ESR1 and ESR2 induces proliferation of the human testicular embryonal carcinoma NT2/D1 cells.

The aims of the present study were to investigate the expression of the classic estrogen receptors ESR1 and ESR2, the splicing variant ESR1-36 and GPER in human testicular embryonal carcinoma NT2/D1 cells, and the effects of the activation of the ESR1 and ESR2 on cell proliferation. Immunostaining of ESR1, ESR2, and GPER were predominantly found in the nuclei, and less abundant in the cytoplasm. ESR1-36 isoform was predominantly expressed in the perinuclear region and cytoplasm, and some weakly immunostained in the nuclei. In nonstimulated NT2/D1 cells (control), proteins of the cell cycle CCND1, CCND2, CCNE1 and CDKN1B are present. Activation of ESR1 and ESR2 increases, respectively, CCND2 and CCNE1 expression, but not CCND1. Activation of ESR2 also mediates upregulation of the cell cycle inhibitor CDKN1B. This protein co-immunoprecipitated with CCND2. Also, E2 induces an increase in the number and viability of the NT2/D1 cells. These effects are blocked by simultaneous pretreatment with ESR1-and ESR2-selective antagonists, confirming that both estrogen receptors regulate NT2/D1 cell proliferation. In addition, E2 increases SRC phosphorylation, and SRC mediates cell proliferation. Our study provides novel insights into the signatures and molecular mechanisms of estrogen receptor in NT2/D1 cells.

The role of estrogen receptors in rat Sertoli cells at different stages of development.

The aim of the study was to investigate the effects of estrogen receptors (ESR1 and ESR2) on the expression of the proteins involved with proliferation (CCND1) and differentiation (CDKN1B and CTNNB) of Sertoli cells from rat in different stages of development. ESR1-selective agonist PPT, but not ESR2-selective agonist DPN, increased CCND1 expression in Sertoli cells from 5- and 15-day old rats. PPT did not have any effect on CCND1 expression in Sertoli cells from 20- and 30-day-old rats. DPN, but not PPT, increased CDKN1B expression in Sertoli cells from 15-, 20-, 30-day-old rats. DPN did not have any effect on Sertoli cells from 5-day-old rats. 17ß-estradiol (E2) and PPT enhanced the [Methyl-3H] thymidine incorporation in Sertoli cells from 15-day-old rats, whereas the treatment did not have any effect in 20-day-old rats. E2 and DPN, but not PPT, increased non-phosphorylated CTNNB expression in Sertoli cells from 20-day-old rats. This upregulation was blocked by ESR2-selective antagonist PHTPP. The activation of ESR1 and ESR2, respectively, plays a role in the proliferation and differentiation of Sertoli cells in a critical period of testicular development. Furthermore, in Sertoli cells from 20-day-old rats, upregulation of non-phosphorylated CTNNB by E2/ESR2, via c-SRC/ERK1/2 and PI3K/AKT, may play a role in the interaction between Sertoli cells and/or in cell-germ cell adhesion and/or in the stabilization and accumulation of CTNNB in the cytosol. CTNNB could be translocated to the nucleus and modulate the transcriptional activity of specific target genes. The present study reinforces the important role of estrogen in normal testis development.

Crosstalk between FSH and relaxin at the end of the proliferative stage of rat Sertoli cells.

Follicle-stimulating hormone (FSH) stimulates the proliferation of immature Sertoli cells through the activation of PI3K/AKT/mTORC1 and MEK/ERK1/2 pathways. Mature Sertoli cells stop proliferating and respond to FSH by stimulating cAMP production. To gain insight into possible mechanisms involved in this switch as well as the impact of paracrine factors that stimulate cell proliferation, we analyzed the effects of FSH and relaxin on intracellular signaling pathways involved with proliferation and differentiation in Sertoli cells from 15-day-old rats, which are close to the transition between the two stages. FSH stimulated 3H-thymidine incorporation and cyclin D1 expression, changes associated with proliferation. In contrast, FSH inhibited AKT and ERK1/2 phosphorylation, activated cAMP production and induced changes in several cell cycle genes that were compatible with differentiation. Relaxin also stimulated 3H-thymidine incorporation but increased phosphorylation of ERK1/2 and AKT. When both hormones were added simultaneously, relaxin attenuated FSH-mediated inhibition of ERK1/2 and AKT phosphorylation and FSH-mediated activation of cAMP production. FSH but not relaxin increased CREB phosphorylation, and relaxin but not FSH shifted NF-κB expression from the cytoplasm to the nucleus. Relaxin did not inhibit the effects of FSH on inhibin α and Bcl2 expression. We propose that at this time of Sertoli cell development, FSH starts to direct cells to differentiation through activation of cAMP/CREB and inhibition of ERK1/2 and AKT pathways. Relaxin counteracts FSH signaling through the inhibition of cAMP and activation of ERK1/2, AKT and NF-κB, but does not block the differentiation process triggered by FSH.