Chemical synthesis and biological activities of 16alpha-derivatives of 5alpha-androstane-3alpha,17beta-diol as antiandrogens

In our efforts to develop compounds with therapeutic potential as antiandrogens, we synthesized a series of 5alpha-androstane-3alpha,17beta-diol derivatives with a fixed side-chain length of 3-methylenes at C-16alpha, but bearing a diversity of functional groups at the end. Among these, the chloride induced the best antiproliferative activity on androgen-sensitive Shionogi cells. Substituting the OH at C-3 by a methoxy group showed the importance of the OH. Moreover, its transformation into a ketone increased the androgen receptor (AR) binding but decreased the antiproliferative activity and induced a proliferative effect on Shionogi cells. These results confirm the importance of keeping a 5alpha-androstane-3alpha,17beta-diol nucleus instead of a dihydrotestosterone nucleus. Variable side-chain lengths of 2-, 3-, 4-, and 6-methylenes at C-16alpha were investigated and the optimal length was found to be 3-methylenes. Although exhibiting a weak AR binding affinity, 16alpha-(3'-chloropropyl)-5alpha-androstane-3alpha,17beta-diol (15) provided an antiproliferative activity on Shionogi cells similar to that of pure non-steroidal antiandrogen hydroxy-flutamide (77% and 67%, respectively, at 0.1 microM). The new steroidal compound, 15, thus constitutes a good starting point for development of future antiandrogens with a therapeutic potential against prostate cancer.     

Reduced myotube diameter,atrophic signalling and elevated oxidative stress in cultured satellite cells from COPD patients

The mechanisms leading to skeletal limb muscle dysfunction in chronic obstructive pulmonary disease (COPD) have not been fully elucidated. Exhausted muscle regenerative capacity of satellite cells has been evocated, but the capacity of satellite cells to proliferate and differentiate properly remains unknown. Our objectives were to compare the characteristics of satellite cells derived from COPD patients and healthy individuals, in terms of proliferative and differentiation capacities, morphological phenotype and atrophy/hypertrophy signalling, and oxidative stress status. Therefore, we purified and cultivated satellite cells from progressively frozen vastus lateralis biopsies of eight COPD patients and eight healthy individuals. We examined proliferation parameters, differentiation capacities, myotube diameter, expression of atrophy/hypertrophy markers, oxidative stress damages, antioxidant enzyme expression and cell susceptibility to H₂O₂ in cultured myoblasts and/or myotubes. Proliferation characteristics and commitment to terminal differentiation were similar in COPD patients and healthy individuals, despite impaired fusion capacities of COPD myotubes. Myotube diameter was smaller in COPD patients (P = 0.015), and was associated with a higher expression of myostatin (myoblasts: P = 0.083; myotubes: P = 0.050) and atrogin‐1 (myoblasts: P = 0.050), and a decreased phospho‐AKT/AKT ratio (myoblasts: P = 0.022). Protein carbonylation (myoblasts: P = 0.028; myotubes: P = 0.002) and lipid peroxidation (myotubes: P = 0.065) were higher in COPD cells, and COPD myoblasts were significantly more susceptible to oxidative stress. Thus, cultured satellite cells from COPD patients display characteristics of morphology, atrophic signalling and oxidative stress similar to those described in in vivo COPD skeletal limb muscles. We have therefore demonstrated that muscle alteration in COPD can be studied by classical in vitro cellular models.     

Isolation and characterization of a cDNA encoding mouse 3alpha-hydroxysteroid dehydrogenase: an androgen-inactivating enzyme selectively expressed in female tissues

3alpha-Hydroxysteroid dehydrogenase catalyzes the transformation of 3-ketosteroids into 3alpha-hydroxysteroids, thus playing an important role in androgen and progesterone metabolism. So far, mouse cDNA and gene encoding 3alpha-HSD has not been reported. In this report, we describe the isolation of a mouse 3alpha-HSD cDNA and the characterization of its substrate specificity and tissue distribution. Sequence analysis indicates that m3alpha-HSD shares 87% amino acid identity with rat 3alpha-HSD. Cells stably transfected with this enzyme catalyze the transformation of dihydrotestosterone (DHT), 5alpha-androstanedione (5alpha-dione) and dihydroprogesterone (DHP) into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), androsterone (ADT) and 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone), respectively. Quantification of mRNA expression levels of this enzyme was determined in male and female mouse sex-specific tissues using quantitative Realtime PCR. We show that this enzyme is mainly expressed in female-specific tissues while being almost absent from male-specific tissues. In the liver, the same expression level is seen in both male and female, while there is 6-fold higher expression level in female pituitary than in male. These results strongly suggest that m3alpha-HSD could play an important role in the female mouse physiology similar to that of type 1 5alpha-reductase with which it works in tandem. This role could be related to the inactivation of excess of androgen and progesterone that are more severely regulated than in man.     

Androgen glucuronides, instead of testosterone, as the new markers of androgenic activity in women

Despite the long series of cohort studies performed during the last 20 years, the correlation between serum testosterone and any clinical situation believed to be under androgen control in women has remained elusive. This is likely related to the recent finding that the androgens made locally in large amounts in peripheral tissues from the precursor dehydroepiandrosterone (DHEA) act in the same cells where synthesis takes place and are not released in significant amounts in the circulation, thus making unreliable the measurement of serum testosterone as marker of total androgenic activity. The objective is to determine if serum androgen glucuronides can be replaced by testosterone or another steroid as measure of androgenic activity.

Since the glucuronide derivatives of androgens are the obligatory route of elimination of all androgens, these metabolites were measured by liquid chromatography tandem mass spectrometry under basal conditions in 377 healthy postmenopausal women aged 55–65 years as well as in 47 premenopausal women aged 30–35 years while testosterone was assayed by gas chromatography mass spectrometry. No correlation was found between the serum concentration of testosterone and that of androsterone glucuronide (ADT-G) or androstenediol glucuronide (3-diol-G), the androgen metabolites which account for the total pool of androgens.

The present data show that measurement of the total pool of androgens reflected by the serum levels of ADT-G and 3-diol-G cannot be replaced by serum testosterone or any other steroid, including DHEA or DHEA sulphate. These findings may have implications for women with androgen deficiency involving osteoporosis, obesity, type 2 diabetes, sexual dysfunction, loss of muscular strength and a series of other clinical situations affecting women's health. Measuring ADT-G and 3-diol-G might identify cases of true androgen deficiency and provide an opportunity to offer appropriate androgen therapy.     

Mixed analog/digital gonadotrope biosynthetic response to gonadotropin-releasing hormone

Mammalian reproduction requires gonadotropin-releasing hormone (GnRH)-mediated signaling from brain neurons to pituitary gonadotropes. Because the pulses of released GnRH vary greatly in amplitude, we studied the biosynthetic response of the gonadotrope to varying GnRH concentrations, focusing on extracellular-regulated kinase (ERK) phosphorylation and egr1 mRNA and protein production. The overall average level of ERK activation in populations of cells increased non-cooperatively with increasing GnRH and did not show evidence of either ultrasensitivity or bistability. However, automated image analysis of single-cell responses showed that whereas individual gonadotropes exhibited two response states, inactive and active, both the probability of activation and the average response in activated cells increased with increasing GnRH concentration. These data indicate a hybrid single-cell response having both digital (switch-like) and analog (graded) features. Mathematical modeling suggests that the hybrid response can be explained by indirect thresholding of ERK activation resulting from the distributed structure of the GnRH-modulated network. The hybrid response mechanism improves the reliability of noisy reproductive signal transmission from the brain to the pituitary.     

Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli

The morphological and functional integrity of the microcirculation is compromised in many cardiovascular diseases such as hypertension, diabetes, stroke, and sepsis. Angiotensin converting enzyme inhibitors (ACEi), which are known to favor bradykinin (BK) bioactivity by reducing its metabolism, may have a positive impact on preventing the microvascular structural rarefaction that occurs in these diseases. Our study was designed to test the hypothesis that BK, via B2 receptors (B2R), protects the viability of the microvascular endothelium exposed to the necrotic and apoptotic cell death inducers H₂O₂ and LPS independently of hemodynamics. Expression (RT‐PCR and radioligand binding) and functional (calcium mobilization with fura‐2AM, and p42/p44MAPK and Akt phosphorylation assays) experiments revealed the presence of functional B2R in pig cerebral microvascular endothelial cells (pCMVEC). In vitro results showed that the cytocidal effects of H₂O₂ and LPS on pCMVEC were significantly decreased by a BK pretreatment (MTT and crystal violet tests, annexin‐V staining/FACS analysis), which was countered by the B2R antagonist HOE 140. BK treatment coincided with enhanced expression of the cytoprotective proteins COX‐2, Bcl‐2, and ^Cu/ZnSOD. Ex vivo assays on rat brain explants showed that BK impeded (by ～40%) H₂O₂‐induced microvascular degeneration (lectin‐FITC staining). The present study proposes a novel role for BK in microvascular endothelial protection, which may be pertinent to the complex mechanism of action of ACEi explaining their long‐term beneficial effects in maintaining vascular integrity. J. Cell. Physiol. 222:168–176, 2010. © 2009 Wiley‐Liss, Inc.     

Morphological and functional recovery from diaphragm injury: an in vivo rat diaphragm injury model.

Our objective was to develop an in vivo model to study the timing and mechanisms underlying diaphragm injury and repair. Diaphragm injury was induced in anesthetized rats by the application of a 100 mM caffeine solution for a 10-min period to the right abdominal diaphragm surface. Diaphragms were removed 1, 4, 6, 12, 24, 48, 72, and 96 h and 10 days after the injury, with contractile function being assessed in strips in vitro by force-frequency curves. The extent of caffeine-induced membrane injury was indicated by the percentage of fibers with a fluorescent cytoplasm revealed by inward leakage of the procion orange dye. One hour after caffeine exposure, 32.9 +/- 3.1 (SE) % of fibers showed membrane injury that resulted in 70% loss of muscle force. Within 72-96 h, the percentage of fluorescent cells decreased to control values. Muscle force, however, was still reduced by 30%. Complete muscle strength recovery was observed 10 days after the injury. Whereas diaphragmatic fiber repair occurred within 4 days after injury induction, force recovery took up to 10 days. We suggest that the caffeine-damaged rat diaphragm is a useful model to study the timing and mechanisms of muscle injury and repair.