Silencing of the transcriptional factor ZEB1 alters the steroidogenic pathway, and increases the concentration of testosterone and DHT in DU145 cells.

Prostate cancer (PCa) is the second most common type of male malignancy worldwide. The transcription factor zinc finger E­box binding homeobox 1 (ZEB1) is associated with epithelial­mesenchymal transition and is also involved in regulation of androgen receptor (AR) expression, the main ligands of which are testosterone and dihydrotestosterone (DHT). These androgens are synthesized through the steroidogenic pathway within the prostate, and their synthesis is altered in PCa. The present study aimed to determine the ZEB1­induced alterations in androgen synthesis and AR expression in the DU145 PCa cell line. Reverse transcription­quantitative polymerase chain reaction, western blotting and immunocytochemistry were used to determine the mRNA and protein expression levels, and cellular localization of steroidogenic pathway enzymes in the DU145 cell line in response to ZEB1 silencing. Furthermore, the concentrations of testosterone and DHT were detected in cell culture medium using ELISA. ZEB1­silenced cells exhibited an increase in testosterone and DHT production, an increase in AR expression and an alteration in the steroidogenic pathway. In particular, steroidogenic acute regulatory protein and 5α­reductase 2 expression levels were decreased, whereas cytochrome P450 family 17 subfamily A member 1, 5α­reductase 1, aldo­keto reductase family 1 member D1 and aldo­keto reductase family 1 member C2 expression levels were increased. In conclusion, the present study provided novel information regarding the regulation of intratumoral androgen production in PCa, which is relevant for the progression of the disease to a castration­resistant form.

Proapoptotic effect of endocannabinoids in prostate cancer cells.

In the early stages, prostate cancer is androgen­ dependent; therefore, medical castration has shown significant results during the initial stages of this pathology. Despite this early effect, advanced prostate cancer is resilient to such treatment. Recent evidence shows that derivatives of Cannabis sativa and its analogs may exert a protective effect against different types of oncologic pathologies. The purpose of the present study was to detect the presence of cannabinoid receptors (CB1 and CB2) on cancer cells with a prostatic origin and to evaluate the effect of the in vitro use of synthetic analogs. In order to do this, we used a commercial cell line and primary cultures derived from prostate cancer and benign prostatic hyperplasia. The presence of the CB1 and CB2 receptors was determined by immunohistochemistry where we showed a higher expression of these receptors in later stages of the disease (samples with a high Gleason score). Later, treatments were conducted using anandamide, 2-arachidonoyl glycerol and a synthetic analog of anandamide, methanandamide. Using the MTT assay, we proved that the treatments produced a cell growth inhibitory effect on all the different prostate cancer cultures. This effect was demonstrated to be dose-dependent. The use of a specific CB1 receptor blocker (SR141716) confirmed that this effect was produced primarily from the activation of the CB1 receptor. In order to understand the MTT assay results, we determined cell cycle distribution by flow cytometry, which showed no variation at the different cell cycle stages in all the cultures after treatment. Treatment with endocannabinoids resulted in an increase in the percentage of apoptotic cells as determined by Annexin V assays and caused an increase in the levels of activated caspase-3 and a reduction in the levels of Bcl-2 confirming that the reduction in cell viability noted in the MTT assay was caused by the activation of the apoptotic pathway. Finally, we observed that endocannabinoid treatment activated the Erk pathway and at the same time, produced a decrease in the activation levels of the Akt pathway. Based on these results, we suggest that endocannabinoids may be a beneficial option for the treatment of prostate cancer that has become nonresponsive to common therapies.

Influence of embryonic conditioned media upon the endometrial beta-adrenergic receptor.

The main objective of this study was to evaluate the influence of the products secreted by the human embryo upon the three subtypes of beta-AR (beta1, beta2, beta3). Cell cultures were developed using endometrial biopsies, taken on day 7 after ovulation, from four healthy women <35 years of age, with regular cycles and infertility due only to male factors. Embryos from women with a normal uterine cavity and endometrial lining were incubated until they reached the 4-cell stage, before being transferred to their mother's uterus. Culture media for embryo incubation were derived from two groups: (i) embryos that achieved pregnancy, (ii) embryos which failed to implant. Control and experimental endometrial cell culture plates were treated with the two embryo culture media, with or without 10(-9) mol/l oestradiol and 10(-7) mol/l progesterone for 48 h. Expression of the three subtypes of beta-AR was assessed by RT-PCR. Beta1-AR was expressed in both control and experimental plates; beta2-AR was expressed only in plates incubated with embryonic culture media of embryos which achieved pregnancy, in both hormonal conditions, with or without oestradiol and progesterone. Beta3-AR was not expressed in any condition. Thus secretory products of human embryos may influence gene expression of beta2-AR concentrations in the human endometrium, and this subtype of beta-AR may be involved in implantation.

Glutathione-related enzymes in cell cultures from different regions of human epididymis.

Protection of maturing sperm from potential endogenous or exogenous harmful substances during their transit throughout the epididymis is a critical event. The authors studied the activity of gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase (GST), and glutathione (GSH) levels in epithelial cell cultures from human caput, corpus, and cauda epididymides. Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Enzymatic activity was measured in conditioned media and cellular fractions. Androgen influence was also evaluated. Both enzymatic activities were found in cellular homogenates and conditioned media from cultures of all epididymal regions. GGT activity was highest in cultures from cauda epididymis, both in conditioned media and cell fractions, while GST activity did not show regional differences in conditioned media, but exhibited higher activity in cell homogenates from cauda cultures than those obtained from corpus and caput epididymis. GSH level showed no regional difference in cell homogenates and it could not be detected in conditioned media by the method used. Presence of different concentrations of dihydrotestosterone (DHT) had no influence neither on the enzymatic activities nor GSH concentration. The results indicate that GGT and GST are present along the human epididymis and a fraction or isoform of these enzymes might be secreted to the luminal fluid to play a detoxificative role in sperm maturation.

Secretion of glycosidases in human epididymal cell cultures.

The dynamics of glycosidase secretion was evaluated in human epididymal cell culture. Epithelial cells from caput, corpus, and cauda epididymis were isolated from tissue obtained from patients undergoing therapeutic orchidectomy due to prostatic carcinoma. The activities of alpha-glucosidase, N-acetylglucosaminidase, beta-glucuronidase, and alpha-mannosidase were analyzed in conditioned culture media. Glycosidase activity was significantly higher in corpus and/or cauda than in caput epididymis. There was a time-dependent increase in enzyme activities that was maximal between 10 and 14 days of culture in all epididymal regions. Epididymal glycosidases are secreted by cultured epithelial cell from human epididymis with an increase toward the distal regions of this organ, which may be related to the dynamics of sperm maturation. Cultures from different epididymal regions may represent a valuable tool to study of human epididymal function.

Expression of GnRH receptor in mouse and rat testicular germ cells.

The expression of gonadotrophin-releasing hormone receptor (GnRH-R) in germinal cells of mouse testis, whole testes, pituitary glands, and mouse ovaries was determined by means of Northern hybridization using a mouse GnRH-R [(32)P]-labelled cDNA probe. Also, the expression of GnRH-R in rat germinal cells, testis and pituitary gland was determined by Northern blot analysis using the same mouse-specific probe. Three receptor transcripts were detected in all cases. In mouse pituitary, ovary and testis, we found two GnRH-R transcripts in the proximity of 4.6-4.7 and 3.4 kb, as well as a 1.6 kb transcript in the pituitary and a 2.0-2.1 kb transcript in both the ovary and testis. Mouse germ cells also exhibited three GnRH-R transcripts of 4.7, 3.5 and 2.2 kb. Two distinct GnRH-R transcripts were also detected in the rat pituitary (4.6 and 2.1 kb), testis (4. 7 and 3.5 kb) and germ cells (4.5 and 3.5 kb). In addition, a third transcript was detected in rat pituitary (1.9 kb) and in rat testis and germinal cells (2.1 kb). The present study demonstrates that GnRH-R mRNA is expressed in rat and mouse testicular germ cells. We suggest that GnRH-R present in these cells may interact with GnRH or GnRH-like peptides produced in the testis and may be part of a paracrine system. The presence of multiple GnRH-R encoding transcripts is also of interest and warrants further studies to evaluate their regulation and function.

Androgen regulation of glycosidase secretion in epithelial cell cultures from human epididymis.

The human epididymis and its secretions actively promote sperm fertilizing capacity and provide protection for spermatozoa against harmful influences. Among epididymal secretions, glycosidases have been recently studied and associated with molecular changes on the sperm surface. In the present work, we studied the influence of different concentrations of testosterone, dihydrotestosterone and cyproterone acetate on the secretion of alpha-glucosidase, N-acetyl-glucosaminidase, beta-glucuronidase and alpha-mannosidase by isolated and cultured epithelial cells from human caput, corpus and cauda epididymides. Cell cultures were obtained from aggregates of isolated tubule fragments plated on extracellular matrix-covered multi-well plates. Activities of the glycosidases were measured in conditioned culture media and were higher in the distal regions of the epididymis. Testosterone and dihydrotestosterone significantly increase the enzyme secretion in a concentration-dependent manner. This increase was higher in corpus and/or cauda than in caput epididymis. Cyproterone acetate caused a dose-dependent decrease in glycosidase secretion in cultures from all epididymal regions. It is concluded that the secretion of epididymal glycosidases is regulated by androgen, being stimulated by dihydrotestosterone and testosterone and inhibited by the androgen antagonist cyproterone acetate.

Influence of age, hormones and germ cells on glutathione S-transferase activity in cultured Sertoli cells.

Glutathione S-transferase (GSH-S-T) activity was measured, using 1-Cl-2,4-dinitrobenzene as substrate, in Sertoli cell cultures obtained from rats aged 10, 18, and 26 days. The GSH-S-T activity showed a significant increase with age of the Sertoli cell donor. When cultures were treated with hypotonic solution, in order to eliminate residual contaminating germ cells, the age dependent increase in enzyme activity was less pronounced. FSH, but not testosterone, increased enzyme activity in all cultures. Addition of freshly isolated germ cells (mainly pachytene spermatocytes) to hypotonic-treated Sertoli cell monolayers enhanced GSH-S-T activity at all ages. It is concluded that GSH-S-T activity can be measured in cultured Sertoli cells during the period of onset of spermatogenesis (10-26 days). This enzyme activity is dependent on age of the Sertoli cell donor and is influenced by FSH and germ cells. Since GSH-S-Ts are actively engaged in cell detoxificative functions through conjugation of xenobiotics with glutathione, the present findings suggest that this enzyme may have a relevant protective role during the critical period when spermatogenesis is being established.

Glutathione and gamma-glutamyl cycle enzymes in rat testis during sexual maturation.

The main enzymes of the gamma-glutamyl cycle in the testis were studied during the onset of spermatogenesis. The activities of gamma-glutamyl transpeptidase, 5-oxoprolinase, and gamma-glutamyl cysteine synthetase, and levels of glutathione were measured in testis homogenates and Sertoli cell preparations obtained from 10-, 18-, and 26-day-old rats. A significant increase of all enzyme activities with the animal age was observed. Level of glutathione also increased in an age-dependent manner. Since the gamma-glutamyl cycle is involved in the cellular incorporation of amino acids, the present findings suggest that this uptake mechanism may be relevant during spermatogenic onset in which synthesis and secretion of specific proteins are essential for germ cell development.