Jean-Pierre Raynaud

Differences in steroid 5α-reductase iso-enzymes expression between normal and pathological human prostate tissue

We studied the expression level and cell-specific expression patterns of 5alpha-reductase (5alpha-R) types 1 and 2 iso-enzymes in human hyperplastic and malignant prostate tissue by semi-quantitative RT-PCR and in situ hybridisation analyses. In situ hybridisation established that 5alpha-R1 mRNA is preferentially expressed by epithelial cells and little expressed by stromal cells whereas 5alpha-R2 mRNA is expressed by both epithelium and stroma. Semi-quantitative RT-PCR has been performed on total RNA from different zones of normal prostate, BPH tissues and liver. We found that 5alpha-R1 and 5alpha-R2 mRNAs expression was near the same in all zones of normal prostate. In BPH tissue, 5alpha-R1 and 5alpha-R2 mRNAs expression was slightly but significantly increased, when it was compared to the levels recorded for normal prostate. In cancer samples, 5alpha-R1 mRNA expression was higher than in normal and hyperplastic prostate but the level of 5alpha-R2 mRNA was not statistically different from that observed in the different zones of normal prostate. In liver, 5alpha-R2 mRNA level was similar to that measured in BPH but 5alpha-R1 mRNA expression was ten times higher. The increase observed in 5alpha-R isoenzymes expression in BPH tissue could play an important role in the pathogenesis and/or maintenance of the disease.

Human prostatic steroid 5α-reductase isoforms—A comparative study of selective inhibitors

The present study describes the independent expression of the type 1 and 2 isoforms of human 5 alpha-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6-8) pH optimum and the type 2 isoform an acidic (5-6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (Km = 0.5 and 2.9 microM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (Ki (type 2) = 7.3 and 21.7 nM compared to Ki (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (Ki (type 1) = 8.4 nM and 7.2 micrograms/ml, respectively; Ki (type 2) = 7.4 nM and 4.9 micrograms/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5 alpha-reductase. Partially purified recombinant 5 alpha-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5 alpha-reductase.

Effect of the Lipidosterolic Extract of Serenoa repens (Permixon®) and Its Major Components on Basic Fibroblast Growth Factor-Induced Proliferation of Cultures of Human Prostate Biopsies

Objective: To assess the effect of the lipidosterolic extract of Serenoa repens (LSESr) on in vitro cell proliferation in biopsies of human prostate Material and Methods: Cell proliferation was assessed by incorporation of [3H]thymidine followed by historadiography. Results: Basic fibroblast growth factor (b-FGF) induced a considerable increase in human prostate cell proliferation (from +100 to +250%); the glandular epithelium was mainly affected, minimal labeling being recorded in the other regions of the prostate. Similar results were observed with epidermal growth factor (EGF), although the increase in cell proliferation was not recorded in some cases. Lovastatin, an inhibitor of hydroxymethylglutaryl coenzyme A, antagonized both the basal proliferation and the growth factor-stimulated proliferation of human prostate epithelium (EGF, mean inhibition ≈80–95%; b-FGF, mean inhibition ≈40–90%). Geraniol, a precursor of both farnesyl pyrophosphate and geranylgeranyl pyrophosphate, and farnesol, the precursor of farnesyl pyrophosphate, increased cell proliferation only in some prostate specimens, this effect being antagonized by lovastatin. LSESr did not affect basal prostate cell proliferation, with the exception of two prostate specimens in which a significant inhibition of basal proliferation was observed with the highest concentration of LSESr (30 µg/ml). In contrast, LSESr inhibited b-FGF-induced proliferation of human prostate cell cultures; this effect was significant for the highest concentration of LSESr (30 µg/ml). In some prostate samples, a similar inhibition was also noted with lower concentrations. Unsaturated fatty acids (UFA), in the range 1–30 ng/ml, did not affect the basal prostate cell proliferation, only a slight increase in cell proliferation was noted in 1 prostate specimen. UFA (1, 10 or 30µg/ml) markedly inhibited the b-FGF-induced cell proliferation down to the basal value. Lupenone, hexacosanol and the unsaponified fraction of LSESr markedly inhibited the b-FGF-induced cell proliferation, whereas a minimal effect on basal cell proliferation was noted. Conclusions: Despite the large variability in the response of the prostate samples to b-FGF, these results indicate that LSESr and its components affect the proliferative response of prostate cells to b-FGF more than their basal proliferation.

Inhibition of type 1 and type 2 5α-reductase activity by free fatty acids, active ingredients of Permixon®

In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%. To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector. The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml). The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.

Induction of apoptosis and inhibition of cell proliferation by the lipido-sterolic extract of Serenoa repens (LSESr, Permixon in benign prostatic hyperplasia.

To determine the mechanism by which prostate volume increases during the development of BPH and to evaluate the effect of LSESr (Permixon®), a phytotherapeutic agent, we investigated apoptosis and cell proliferation in the stroma and epithelium of normal prostate and of BPH tissues from patients treated with or without LSESr.

Testosterone metabolism in primary cultures of human prostate epithelial cells and fibroblasts

We compare testosterone (T) metabolism in primary cultures of epithelial cells and fibroblasts separated from benign prostate hypertrophy (BPH) and prostate cancer tissues. In all cultures, androstenedione (delta 4) formed by oxidation of T by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) represented 80% of the metabolites recovered. The amounts of 5 alpha-dihydrotestosterone (DHT), formed by reduction of T by 5 alpha-reductase (5 alpha-R), were small: 5 and 2% (BPH) and 8 and 15% (adenocarcinoma) for epithelial cells and fibroblasts, respectively. Northern blot analysis of total RNA from epithelial cells (BPH or adenocarcinoma) attributed the reductive activity to the 5 alpha-reductase type 1 isozyme and oxidative activity to the 17 beta-HSD type 2. In cancer fibroblasts, only little 17 beta-HSD type 2 mRNA was detected. The 5 alpha-reductase inhibitors, 4-MA (17 beta-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) and finasteride, inhibited DHT formation with a preferential action of 4-MA on epithelial cells (BPH or adenocarcinoma) and of finasteride on fibroblasts from adenocarcinoma. Neither inhibitor acted on delta 4 formation. On the other hand, the lipido-sterol extract of Serenoa repens (LSESr, Permixon) inhibited the formation of all the T metabolites studied [IC50 S = 40 and 200 micrograms/ml (BPH) and 90 and 70 micrograms/ml (adenocarcinoma) in epithelial cells and fibroblasts, respectively]. These results have important therapeutic implications when selecting appropriate treatment options for BPH.

Pharmacological effects of the lipidosterolic extract of Serenoa repens (Permixon®) on rat prostate hyperplasia induced by hyperprolactinemia : Comparison with finasteride

The growth of the prostate gland is mainly dependent on androgens. Other hormones, like prolactin (PRL), also influence prostate development. Our purpose was to analyze and compare the effects of two drugs (5α‐reductase inhibitor) used in the therapy of benign prostatic hyperplasia: lipidosterolic extract of Serenoa repens (LSESR), and finasteride in an in vivo model of rat prostate hyperplasia induced by hyperprolactinemia.

High Incidence of Predominant Gleason Pattern 4 Localized Prostate Cancer is Associated With Low Serum Testosterone

PURPOSE We characterized the aggressiveness of prostate cancer by Gleason score and predominant Gleason pattern in relation to preoperative serum testosterone. MATERIALS AND METHODS In a prospective study serum total testosterone was measured preoperatively in patients referred to our department from January 2007 to January 2011 for radical prostatectomy. Gleason score and predominant Gleason pattern were determined in prostate biopsy and prostate tissue specimens. RESULTS A total of 431 patients were enrolled in the study. In biopsies a predominant Gleason pattern 4 was observed in 72 patients (17%). In prostate specimens the predominant Gleason pattern 4 increased to 132 patients (31%). In the 132 patients total testosterone was lower than in the 299 with predominant Gleason pattern 3 (4.00 vs 4.50 ng/ml, p = 0.001), prostate specific antigen was higher (8.4 vs 6.6 ng/ml, p <0.00001), and extraprostatic extension and positive margins were noted more often (49% vs 20% and 23% vs 14%, p <0.000001 and 0.02, respectively). The 62 patients with total testosterone less than 3.0 ng/ml were larger (mean 7 kg, p = 0.0001) with a higher body mass index (mean 0.5 kg/m(2), p <0.000001). They had a higher percent of Gleason score with predominant Gleason pattern 4 (47% vs 28%, p = 0.002). CONCLUSIONS Low total testosterone is associated with a higher percent of predominant Gleason pattern 4, a signature of prostate cancer aggressiveness. Tumor aggressiveness cannot be accurately estimated by biopsy Gleason score and predominant Gleason pattern. Preoperative total testosterone should be added to prostate specific antigen determination to improve management for prostate cancer.

Estrone sulfate (E1S), a prognosis marker for tumor aggressiveness in prostate cancer (PCa).

Seeking insight into the possible role of estrogens in prostate cancer (PCa) evolution, we assayed serum E2, estrone (E1), and estrone sulfate (E1S) in 349 PCa and 100 benign prostatic hyperplasia (BPH) patients, and in 208 control subjects in the same age range (50-74 years). E1 (pmol/L+/-S.D.) and E1S (nmol/L+/-S.D.) in the PCa and BPH patients (respectively 126.1+/-66.1 and 2.82+/-1.78, and 127.8+/-56.4 and 2.78+/-2.12) were significantly higher than in the controls (113.8+/-47.6 and 2.11+/-0.96). E2 was not significantly different among the PCa, BPH, and control groups. These assays were also carried out in PCa patients after partition by prognosis (PSA, Gleason score (GS), histological stage, and surgical margins (SM)). Significantly higher E1S levels were found in PCa with: PSA>10 ng/L (3.05+/-1.92) versus PSA<or=10 ng/mL (2.60+/-1.55), stage pT3-T4 (2.99+/-1.80) versus pT2 (2.58+/-1.58), and positive (3.26+/-1.95) versus negative margins (2.52+/-1.48). E1 was higher in poor- than in better-prognosis PCa. E2 was significantly higher in PCa with GS>or=4+3 (109.5+/-43.8) versus GS<or=3+4 (100.6+/-36.5) and increased significantly when GS increased from 3+3 to 4+4. Estrogens, especially E1S appeared to be possible markers of PCa progression. Attempting to identify potential sources of E2 in PCa according to prognosis, as well as in BPH, we found a significant correlation coefficient between E1S and E2 (0.266-0.347) in poor-prognosis PCa and no correlation in BPH (0.026) and better-prognosis PCa (0.013-0.104). It is as though during progression of PCa from good to poor prognosis there were a shift in the E1 to E2 metabolic pathway from predominantly oxidative to predominantly reductive.

Prostate-specific antigen (PSA) concentrations in hypogonadal men during 6 years of transdermal testosterone treatment

Hypogonadism affects an estimated 2–4 million men in the USA, but only 5% receive treatment. Testosterone replacement therapy reduces the effects of testosterone deficiency on sexual function, mood and energy in hypogonadal patients. Long‐term hypogonadism management requires testosterone treatment to restore serum concentrations of testosterone and its active metabolites, within physiological ranges; a testosterone preparation that achieves physiological plasma concentrations without supra‐physiological escape is a preferred option. A previous 1‐year study European clinical study showed the efficacy and safety of a transdermal testosterone patch (Testopatch®). The present study shows the long‐term (6‐year) safety and efficacy of Testopatch in patients with primary or secondary hypogonadism. We show that, over the long‐term, Testopatch was associated with no relevant changes in PSA concentration and PSA velocity, or any significant prostate risks (there were no cases of prostate cancer).