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Are oxytocin preprotein and the oxytocin receptor expressed in human spermatozoa and is their mRNA expression different between normal semen samples and samples with at least one abnormal parameter?An in-vitro prospective study of 175 semen samples from Greek men, according to World Health Organization criteria, 2010. mRNA expression levels were compared between different categories of semen samples, classified according to their concentration, total number, motility and morphology. Immunohistochemistry was used to detect oxytocin preprotein and its receptor on spermatozoa smears.Compared with normal samples (normal motility and normal concentration), samples with at least one abnormal sperm parameter had statistically significantly lower oxytocin preprotein mRNA expression (P = 0.019) and higher oxytocin receptor mRNA expression levels (P0.001). Oligozoospermic samples had statistically significantly higher oxytocin preprotein mRNA expression levels (P = 0.002) and lower oxytocin receptor mRNA expression levels (P = 0.047). Asthenozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P0.001). Teratozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P = 0.049) and higher oxytocin receptor mRNA expression levels (P0.001). Oxytocin preprotein mRNA expression was positively associated with total progressive motility (P0.001) and negatively associated with the percentage of immotile spermatozoa (P = 0.001). Oxytocin receptor mRNA expression was negatively associated with the percentage of normal forms (P0.001).Oxytocin preprotein and oxytocin receptor mRNA expression in spermatozoa could be used as a novel and unbiased diagnostic tool for male infertility.

Accumulating evidence during the last decades revealed that androgens exert membrane-initiated actions leading to the modulation of significant cellular processes, important for cancer cell growth and metastasis (including prostate and breast), that involve signaling via specific kinases. Collectively, many nonclassical, cell surface-initiated androgen actions are mediated by novel membrane androgen receptors (mARs), unrelated to nuclear androgen receptors. Recently, our group identified the G protein coupled oxo-eicosanoid receptor 1 (OXER1) (a receptor of the arachidonic acid metabolite, 5-oxoeicosatetraenoic acid, 5-oxoETE) as a novel mAR involved in the rapid effects of androgens. However, two other membrane proteins, G protein-coupled receptor family C group 6 member A (GPRC6A) and zinc transporter member 9 (ZIP9) have also been portrayed as mARs, related to the extranuclear action of androgens. In the present work, we present a comparative study of in silico pharmacology, gene expression and immunocytochemical data of the three receptors in various prostate and breast cancer cell lines. Furthermore, we analyzed the immunohistochemical expression of these receptors in human tumor and non-tumoral specimens and provide a pattern of expression and intracellular distribution.

The expression profile of estrogen receptors (ER) in Non-Small Cell Lung Carcinoma (NSCLC) remains contradictory. Here we investigated protein and transcriptome expression of ERα wild type and variants. Tissue Micro-Arrays of 200 cases of NSCLC (paired tumor/non-tumor) were assayed by immunohistochemistry using a panel of ERα antibodies targeting different epitopes (HC20, 6F11, 1D5, ERα36 and ERα17p). ERβ epitopes were also examined for comparison. In parallel we conducted a probe-set mapping (Affymetrix HGU133 plus 2 chip) meta-analysis of 12 NSCLC tumor public transcriptomic studies (1418 cases) and 39 NSCLC cell lines. Finally, we have investigated early transcriptional effects of 17β-estradiol, 17β-estradiol-BSA, tamoxifen and their combination in two NSCLC cell lines (A549, H520). ERα transcript and protein detection in NSCLC specimens and cell lines suggests that extranuclear ERα variants, like ERα36, prevail, while wild-type ERα66 is minimally expressed. In non-tumor lung, the wild-type ERα66 is quasi-absent. The combined evaluation of ERα isoform staining intensity and subcellular localization with sex, can discriminate NSCLC subtypes and normal lung. Overall ERα transcription decreases in NSCLC. ERα expression is sex-related in non-tumor tissue, but in NSCLC it is exclusively correlating with tumor histologic subtype. ERα isoform protein expression is higher than ERβ. ERα isoforms are functional and display specific early transcriptional effects following steroid treatment. In conclusion, our data show a wide extranuclear ERα-variant expression in normal lung and NSCLC that is not reported by routine pathology ER evaluation criteria, limited in the nuclear wild type receptor.

In prostate cancer, calcium homeostasis plays a significant role in the disease's development and progression. Intracellular calcium changes are an important secondary signal, triggered by a variety of extracellular stimuli, that controls many cellular functions. One of the main events affecting calcium is androgen signaling. Indeed, via calcium changes, androgens regulate cell processes like cell growth, differentiation and motility. In the present work we explored the nature of the receptor involved in calcium response induced by membrane-acting testosterone in prostate cancer cells. We report that testosterone, independently of the presence of the classical androgen receptor, can rapidly increase intracellular calcium from calcium stores, through the oxoeicosanoid receptor 1 (OXER1) and a specific signaling cascade that triggers calcium release from the endoplasmic reticulum. These findings reveal for the first time the receptor involved in the rapid calcium changes induced by androgens. Moreover, they further support the notion that androgens, even in the absence of AR, can still exert specific effects that regulate cancer cell fate.

The complexity of estrogen actions mainly relies to the presence of different identified receptors (ERα, ERβ, their isoforms, and GPR30/GPER) and their discrete cellular distribution. Depending on the localization of the receptor that mediates estrogen effects, nuclear and extra-nuclear actions have been described. The latter can trigger a number of signaling events leading also to transcriptional modifications. In an attempt to clarify the nature of the receptor(s) involved in the membrane initiated effect of estrogens on gene expression, we performed a whole transcriptome analysis of breast cancer cell lines with different receptor profiles (T47D, MCF7, MDA-MB-231, SK-BR-3). A pharmacological approach was conducted with the use of estradiol (E(2)) or membrane-impermeable E(2)-BSA in the absence or presence of a specific ERα-β or GPR30/GPER antagonist. Our results clearly show that in addition to the ERα isoforms and/or GPR30/GPER that mainly mediate the transcriptional effect of E(2)-BSA, there is a specific transcriptional signature (found in T47D and MCF-7 cells) suggesting the presence of an unidentified membrane ER element (ERx). Analysis of its signature and phenotypic verification revealed that important cell function such as apoptosis, transcriptional regulation, and growth factor signaling are associated with ERx.

Triple negative breast cancer is deprived of estrogen receptor alpha (ERα), progesterone receptor (PR) and HER-2 protein. It constitutes the most heterogeneous and aggressive group of breast carcinomas, for which identification of novel characteristics and characterization of putative targets becomes very demanding. In the present work we have assayed the expression of ERα36, a recently identified ERα variant of 36 kDa, in a series of triple negative breast cancers, in relation to the clinical behavior and other clinico-pathological features of the tumors. While widely expressed within the cytoplasm in almost all tumors, we found that exclusively the membrane/submembrane expression of the receptor exhibits a correlation with patient’s survival. Moreover, membrane ERα36 correlates in an inverse manner with the expression of miRNA210, a pro-angiogenic miR, with high prognostic relevance in triple negative carcinomas. A thorough transcriptomic, pharmacological-based approach in breast cancer cell lines, revealed an early (direct) transcriptional signature of the receptor activation, related to immune system processes and T-cell differentiation, RNA biosynthesis, regulation of metabolism, VEGF signaling and regulation of the cell cycle, with a down-regulation of CREB, NFκB and STATs transcription factors. Finally, ERα36 expression is not limited within breast cancer epithelial linen, but is equally identified in tumor vasculature, peritumoral fat tissue, lymphocytic infiltrate and stromal fibroblasts. In light of the above, ERα36 could represent a major counterpart in triple negative breast cancer.

Estrogens exert their effect through ERα and ERβ intracellular transcription factors and rapid, usually membrane-initiated receptors, influencing cytosolic signaling and transcription. The nature of extranuclear estrogen elements has not been elucidated so far; classical or alternatively transcribed ER isoforms (ERα36, ERα46) anchored to the plasma membrane and GPR30 (GPER1) have been reported to exert early estrogen actions. Here, we used E2-BSA, an impermeable estradiol analog for a transcriptome analysis in four GREP1 positive breast cancer cell lines with different estrogen receptor profiles (T47D, MCF-7, MDA-MB-231 and SKBR3) in order to evaluate GPER1 transcriptional effects. Early effects of E2-BSA were assayed after 3h of incubation, in the absence/presence of ICI182,780 (ER-inhibitor) or G15 (GREP1-specific inhibitor). E2-BSA specifically modified 277-549 transcripts in the different cell lines. Two different clusters of transcripts could be identified: (1) the majority of transcripts were inhibited by both ICI182,780 and G15, suggesting an interaction of E2-BSA with a common ER-related element, or a direct ER-GPER1 interaction; (2) a small number of G15-only modified transcripts, in two cell lines (T47D and SKBR3 cells), indicative of specific GPER1-related effects. The latter transcripts were significantly related to pathways including FOXA2/FOXA3 transcription factor networks, RNA-Polymerases Transcription Regulation and lipid metabolism, while ICI/G15 inhibited transcripts affected pathways related to apoptosis, erythropoietin signaling, metabolic effects through the citric acid cycle, IL-4 and IL-5 mediated events and homologous DNA recombination. Finally, we review the current literature of GPER1 actions, in view of our results of ER-dependent and independent GPER1-modified pathways.

Quercetin is a flavonol modifying a number of cell processes in different cell lines. Here, we present evidence that nonconjugated quercetin enters cells possibly via organic anion transporter polypeptides and quickly accumulates in the nucleus where it concentrates at distinct foci. Furthermore, it induces major transcriptional events with a high number of transcripts being modified over time and about 2200 transcripts being continuously influenced by the agent. The latter transcripts are related to cell cycle and adhesion, xenobiotic metabolism, immune-related factors and transcription. In addition, quercetin up-regulates the expression of estrogen receptors α and β. The overall outcome on cell fate is reflected by an inhibition of cell proliferation, cell cycle arrest in the G1 phase and reduction of the cells' migratory potential due to actin cytoskeleton disorganization. Finally, we report that the flavonol modifies the transcription and/or activity of numerous transcription factors. In conclusion, our data support the idea that quercetin may actively accumulate in discrete cell structures and exert more than just antioxidant actions on epithelial cells by regulating mechanisms related to gene transcription.

The mode of action of steroid hormones has been extended in recent years. In addition to their classical nuclear action (acting as transcription factors), they can also regulate cell-signaling phosphorylation cascades and exert actions that are initiated at the membrane and which, in most cases, are rapid. Even though research in this field was intensified during the last decade the nature of the up-stream receptor targets that mediates these rapid non-genomic actions remains to be better established. However, it became obvious that steroid signaling is not uniform, with a variety of modes of rapid action being described. There are several studies speculating a classical steroid receptor involvement in the rapid effects of steroids, localized at the cytoplasmic membrane and mediating effects directly or indirectly, via interactions with specific membrane structures (estrogen receptor (ER) isoforms have been shown to localize in caveolae) and/or other membrane receptors (like growth factor receptor). In addition, there are reports that suggest the existence of a distinct receptor, associated to the plasma membrane, being different from the classical, intracellular one. Non-genomic/extranuclear actions of steroids have been described in a number of different normal or cancer tissues independently of the presence of classical nuclear steroid receptors. In the present work, we review briefly the identification and signaling events of membrane-initiated steroid (androgen and estrogen) action in breast and prostate cancer cell lines and clinical specimens. Furthermore, we discuss the interaction of cytokine/growth factor receptors with membrane-acting steroids and their potential clinical implications.

The stilbene resveratrol exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines, through diverse mechanisms, including antioxidant effects, enzyme, growth factor and hormone receptor binding, and nucleic acid direct or indirect interactions. Although resveratrol accumulates in the liver, its effect on hepatocellular carcinoma has not been extensively studied. We have used the human hepatocyte-derived cancer cell line HepG2 to address the possible action of resveratrol on cell growth and to examine some possible mechanisms of action. Our results indicate that the stilbene inhibits potently cell proliferation, reduces the production of reactive oxygen species and induces apoptosis, through cell cycle arrest in G1 and G2/M phases. Furthermore it modulates the NO/NOS system, by increasing iNOS and eNOS expression, NOS activity and NO production. Inhibition of NOS enzymes attenuates its antiproliferative effect. These data could be of value in possible prevention or adjuvant treatment of hepatocellular carcinoma, through an increased consumption of resveratrol-rich foods and beverages.

Rapid, non-genomic, steroid actions have been identified for more than 20 years. In the last decade however, a great expansion of research was observed. In the present review we report the identification and the subsequent signaling cascades involved in these rapid steroid effects. In the current state of knowledge, with the exception of progesterone for which a seven-loop G protein-coupled receptor has been identified, two major lines of evidence exist for membrane-related steroid actions: (1) a binding to the intracellular receptor, coupled to the plasma membrane, or interacting with other growth factor receptors, and (2) the existence of specific membrane steroid receptors. In addition, major intracellular signaling cascades involved in cell survival and/or apoptosis are activated by non-genomic steroid actions. Finally, it appears that cancer cells and tumors express membrane steroid sites, related to cancer aggressiveness. These lines of evidence may implicate, in the forthcoming years, membrane steroid receptors in cancer control as major or adjuvant chemotherapeutic agents, providing new possible targets for cancer chemotherapy.

Neuropeptides influence cancer cell replication and growth. Opioid peptides, and opiergic neurons are found in the prostate gland, and they are proposed to exert a role in tumor regulation, influencing cancer cell growth, as opioid agonists inhibit cell growth in several systems, including the human prostate cancer cell line LNCaP. In the same cell line, the existence of membrane testosterone receptors was recently reported, which increase, in a non-genomic manner, the secretion of PSA, and modify actin cytoskeleton dynamics, through the signaling cascade FAK→PI-3 kinase→Cdc42/Rac1. In the present work, we present data supporting that the general opioid agonist Ethylketocyclazocine (EKC) decreases testosterone-BSA (a non-internalizable testosterone analog) induced PSA secretion. Furthermore, we report that this opioid affects this non-genomic testosterone action, by modifying the distribution of the actin cytoskeleton in the cells, disrupting the above signaling cascade. In addition, after long (>24 h) incubation, opioids decrease the number of membrane testosterone receptors, and reverse their effect on the signaling molecules. In conclusion, our results provide some new insights of a possible action of opioids in prostate cancer control by interfering with the action and the expression of membrane testosterone receptors and signaling.

The present invention relates to the use of membrane steroid receptor agonists as potentiators of the action of erythropoietin (EPO). The present invention also relates to the combined use of membrane steroid receptor agonists and erythropoietin to control apoptosis, proliferation, differentiation, migration and regeneration of cells, in different organs and tissues. Compositions comprising (i) a membrane steroid receptor agonist and (ii) erythropoietin are also provided, as are kits comprising (i) a membrane steroid receptor agonist and (ii) erythropoietin.