Human BPH Research Articles

In vitro modulation of steroid 5α-reductase activity by phospholipases in epithelium and stroma of human benign prostatic hyperplasia

Membrane components, such as phospholipids, play an important role in the regulation of prostatic 5α-reductase activity. To describe in more detail the impact of such regulation on 5α-reductase activity, epithelial and stromal cell homogenates of human BPH were treated with phospholipases to specifically alter the structure of cellular phospholipid components. Phospholipase A 2 (PLA 2) was used to alter the structure of the nonpolar, hydrophobic region of the membrane bilayer. Various types of phospholipase C (PLC) affect the polar, hydrophilic region of phospholipids. In epithelium and stroma, 5α-reductase activity was dose-dependently inhibited by PLA 2 and PLC type III. In epithelium and stroma, the mean IC 50 values of PLA 2 were 9.4 ± 1.1 and 13.9 ± 2.6 [U/mg protein ± SEM], respectively. The mean IC 50 values of PLC type III in epithelium and stroma were 4.5 ± 1.2 and 1.7 ± 0.2 [U/mg protein ± SEM], respectively. In epithelium as well as in stroma, 5α-reductase activity was more greatly inhibited by PLC type III than by PLA 2. Both in epithelium and stroma, PLA 2 significantly decreased the V max of 5α-reductase whereas its K m remained unaffected. A similar decrease in V max was found with PLC type III in epithelium and stroma. Furthermore, the K m of epithelial 5α-reductase increased significantly following the addition of PLC type III. The two phospholipases, with their specific substrate affinities and sites of hydrolysis, exhibited significantly different effects on 5α-reductase, indicating that 5α-reductase activity is not unspecifically affected by modification of the hydrophilic milieu. Rather, 5α-reductase activity is specifically modulated by various phospholipids and/or phospholipolysis mediated degradation products. These findings suggest that the structural composition of the lipid environment plays a fundamental role in the post-translational regulation of 5α-reductase activity in the epithelium and stroma of human BPH. Thus, changes in membrane phospholipid content seem to be instrumental in the expression of DHT-dependent processes.

Fatty acid composition of phospholipids in epithelium and stroma of human benign prostatic hyperplasia.

Although it is well known that prostatic 5alpha-reductase is active only in its membrane-bound form, rather limited information is available concerning the composition of cellular lipids in human BPH. Therefore, in the present study, the phospholipid fatty acid composition and content in epithelium and stroma of human BPH have been investigated for the first time. Phospholipids separated on TLC plates were methylated and fatty acid methyl esters were analyzed by capillary gas chromatography. The fatty acid composition of total phospholipids was significantly different between epithelium and stroma. In particular, the percentage of oleic acid was significantly higher in epithelium as compared with stroma, whereas that of arachidonic acid was significantly lower in epithelium than in stroma. In addition, significant differences between epithelium and stroma were found in regard to the fatty acid composition of the main phospholipid subclasses. Another remarkable finding were the age-dependent changes of the fatty acid composition in human BPH. This study shows that the fatty acid composition of phospholipids is significantly different between epithelium and stroma of human BPH. Furthermore, age-dependent alterations of the fatty acid composition were found. Further studies are needed to determine whether the endogenous hormonal milieu in the prostate modulates the fatty acid composition of the prostatic cells, as well as what impact such modulation could have on the properties of membrane proteins, i.e., enzymes like the 5alpha-reductase and receptors, which are thought to be affected by alterations in membrane fluidity or composition, or both.

5α-Reductase inhibition by finasteride (proscar ®) in epithelium and stroma of human benign prostatic hyperplasia

Finasteride is a specific 5α-reductase inhibitor that has been shown to reduce the size of human benign prostatic hyperplasia (BHP) by inhibiting the intraprostatic conversion of testosterone to 5α-dihydrotestosterone. The aim of the present in vitro study was to describe in more detail the inhibitory effect of finasteride on 5α-reductase in epithelium and stroma of human BPH. 5α-Reductase activity in epithelium and stroma was inhibited dose-dependently by finasteride. The mean IC 50 (50% inhibitory concentration) values, determined in the presence of various testosterone concentrations, were generally 2- to 4-fold lower in epithelium than in stroma. With finasteride concentrations greater the 5 nM, competitive inhibition of 5α-reductase occurred both in epithelium and stroma. The mean inhibition constant K i [ nM ± SEM] was 7 ± 3 and 31 ± 3 in epithelium and stroma, respectively. In the presence of finasteride concentrations ≤5 nM, the epithelial 5α-reductase seems to be inhibited in an umcompetitive manner, whereas such low finasteride concentrations cause either no inhibition (1–2 nM) or competitive inhibition (5 nM) in stroma. Our present study provides evidenmce that the inhibitory effect of finasteride on 5α-reductase is much stronger in epithelium than in stroma. Therefore, it is conceivable that the global size-reduction of BPH under finasteride treatment due to the regression of BPH epithelium.

Transplantation of human benign hyperplastic prostate tissue into nude mice: first results of systemic therapy.

Xenografts of human benign hyperplastic prostate tissue (BPH) have been established as a model for the investigation of the etiology of BPH. In this paper it is our aim to answer the question of whether this model is useful for the established therapy of the disease. Additionally we try to evaluate the value of a commonly used plant extract for the therapy of BPH. Is there any influence of the extract from Sabal serrulatum on the BPH tissue in our model? Human BPH tissue from 2 patients was transplanted into athymic nude mice and treated with three different regimens. Animals of group I did not receive any treatment and served as control, in groups II and III the tissue was stimulated by application of silicone tubes containing crystalline 5 alpha-dihydrotestosterone and estradiol. Animals of group II additionally were treated orally with the lipophilic extract of S. serrulatum (contained in Prostagutt N), which is commonly used for the treatment of BPH clinically. Significant inhibition of tissue growth was observed in group III when compared to group II. In group I (control) atrophy of the graft was observed as expected. However, histologically no differences were visible between groups II and III. Our experiment shows a significant growth-inhibiting effect of the plant extract for human BPH tissue in our model (p less than 0.05). We conclude that the nude mouse model can be useful for the evaluation of systemic therapy modalities of human BPH and that the plant extract may have a certain value for clinical treatment, which is not only due to subjective criteria.

Endocrine treatment of benign prostatichypertrophy: Current concepts

To summarize the endocrine approach for the treatment of BPH: much clinical data have accumulated over the past forty years. Until recently, scientists and physicians mainly concentrated on the reduction of androgens as a possible solution. We have come a long way from surgical castration, through the administration of hormones such as estrogen and progesterone, gonadotropin-releasing hormone agonists to the inhibition of an enzymatic reaction reducing testosterone to DHT--the now recognized active intracellular androgen metabolite. Recently, the role of estrogens has been emphasized with the finding that stromal hyperplasia is the main change occurring in BPH. Lately, research has been initiated to examine the clinical effect aromatase inhibitors would have in the treatment of human BPH. Since there is enough evidence that both the epithelial and stromal components of the human prostate undergo hyperplasia in BPH, and individuals vary with respect to their relative epithelial/stromal components, both structures would have to be reduced for therapy to be successful. Therefore, the combination of an antiandrogenic and antiestrogenic effect is theoretically promising. Indeed, prostates of beagles shrunken after treatment with an aromatase inhibitor, further decreased in weight after additional treatment with cyproterone acetate, an antiandrogen. We are now approaching the stage where these "antihormones" are both enzyme inhibitors with actually no side effects that preclude the use of the earlier generation's "antihormonal" hormonal drugs. Furthermore, it has recently been reported that the aromatase inhibitor, 4-hydroxy-androstenedione also inhibits human prostatic 5-alpha reductase, at least in vitro. The in vivo relevance of this finding awaits further classification. Thus, a good hormonal treatment that will be both scientifically sound, and clinically safe and effective, seems feasible in the near future. Two main factors have encouraged our interest and research into methods of inhibiting prostatic growth or reducing its obstructive symptomatology: the enormous cost of prostatic operations for outlet obstruction secondary to BPH, and the natural aging process of the population accompanied by deteriorated health precluding anesthesia and prostatic surgery. Medical treatment of BPH has to result in symptomatic improvement, elimination of residual urine, and improvement of flow to be considered successful. These are usually accomplished by surgery and results at least as good as those obtained by operation should be aimed at, if medical treatment is to replace surgery. Although indications for surgery and outcome of operations are well-defined, this is not the case when alternatives to prostatectomy are chosen.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical and Experimental Studies of Benign Prostatic Hyperplasia

The canine prostate appears to be a suitable model for investigating many aspects of abnormal prostate growth related to BPH. This model has indicated that BPH is a complex pathologic process that varies with age and produces a gland that is composed of a mixture of glandular, cystic, and stromal hyperplasia juxtapositioned with foci of atrophy. Canine BPH first occurs very early in life as a glandular hyperplasia before the onset of more complex BPH. As the prostate increases in size with age, the tissue dihydrotestosterone content falls, as does the secretory capacity of the gland. The fall in glandular dihydrotestosterone content is counterbalanced by a concomitant increase in the metabolism of androgens toward dihydrotestosterone and an increase in nuclear androgen receptors. As the prostate ages, it thus becomes more sensitive to androgens, which are required for maintaining the abnormal size of the gland. Established BPH can be maintained in the absence of the testes in the presence of normal testosterone levels, and it appears that the abnormal growth is primarily attributable to an inherent change that has occurred in the aged prostate. In the dog and man, both the normal prostate and BPH are rich in growth factors and dihydrotestosterone receptor in the nucleus, yet mitotic cells are rare. Established BPH is not growing rapidly; the abnormal size of the aged prostate is maintained, not by an increase in the rate of cell replication, but rather by an apparent decrease in the rate of cell death. It appears that a combination of estrogens and 5 alpha-reduced androgens can cause this decrease in the rate of cell death and replication and that this imbalance can produce abnormal cell accumulation and an experimental prostatic hyperplasia. Besides affecting cell death and replication, estradiol has a marked effect in stimulating the stroma and collagen synthesis. Current investigations are focusing on determining the distribution of stem cells for both the epithelial and the stromal elements of the prostate, how these stem cells are recruited to form transiently proliferating cells, and how they mature and subsequently are programmed for cell death. It is believed that steroid imprinting in the neonatal and prepubertal period may be of great importance in determining the ultimate growth of the prostate. Preliminary attempts to control abnormal canine prostate growth by forcing maturation with retinoic acid, or by interfering with growth factors through the use of suramin, have not proved effective; only androgen ablation is. Important leads in the search for the etiology and control of BPH can be studied in the canine model. These concepts must ultimately be tested and confirmed for human BPH in well-controlled clinical investigations.

Study of the effect of an anti-androgen (Oxendolone) on experimentally induced canine prostatic hyperplasia

To investigate the effect of anti-androgens on BPH, Oxendolone (OXD), a pure anti-androgen, was tested in experimentally induced BPH in 17 beagle dogs, alone or in combination with medroxyprogesterone acetate (MPA) which displays both anti-androgenic and anti-estrogenic activity. The relatively early stage of canine BPH was induced by administration of 3 alpha-androstanediol (3 alpha-A) plus estradiol (E2) for 6 months and followed by testosterone propionate (TP) plus E2 for another 6 months during the anti-androgenic treatment. By the manipulation with T, a decrease in volume of glandular component associated with a relative increase in stromal tissue was achieved, which mimics human BPH histology. The prostate substituted with T and E2, however, gradually decreased in size. Therefore the effect of OXD or OXD + MPA was not significant against the untreated controls (T-E control). The weight of the prostate in these OXD +/- MPA groups was however significantly reduced as compared to that of BPH controls which received 3 alpha-A and E2 throughout the experimental period. On histological examination, atrophic changes were observed in the hormone-treated groups compared to the T-E control. The finding was the most striking in the OXD + MPA group with small non-involuted acini scattered in the abundant stomal tissue. This was almost identical to the appearances of castrated control groups. Atrophy may be due not only to the anti-androgenic but also to the anti-estrogenic property of MPA. A report on the hormonal background of this experiment will appear in the second article.

Evaluation of estradiol binding in human benign prostatic hyperplasia

AbstractWe have systematically evaluated the effect of tris/phosphate buffers, ethylenediamine tetraacetate (EDTA), dithiothreitol (DTT), molybdate, and phenylmethylsulfonyl fluoride (PMSF) on the binding of 17β‐estradiol to human BPH cytosol. Competition binding assays demonstrated that the estrogen receptor was highly specific for 17β‐estradiol. There was statistically no difference in binding observed with tris or phosphate buffers. Two classes of binding (high affinity—type I, low affinity—type II) were observed. DTT (1 mM) showed maximum inhibition of type II binding. There was no significant difference in binding observed in the presence of 1.0 mM EDTA. However, 10 mM EDTA stimulated estradiol binding in both saturation analysis and glycerol gradients. Stimulation of estradiol binding by 10 mM EDTA was observed even in the presence of 1 mM DTT. Molybdate (20 mM), a reagent used to stabilize steroid receptors, significantly inhibited estradiol binding. PMSF inhibited estradiol binding at all concentrations studied and the nature of the inhibition appeared to be uncompetitive. A Bmax (maximum number of binding sites) of 84.4 ± 68.5 fmoles/mg protein with a KD (equilibrium dissociation constant) of 2.5 ± 1.7 mM was observed for type I estrogen receptor binding. Our Bmax values are higher than those reported in other studies even after allowing for possible overestimation. In view of the potential importance of estrogen receptors in human prostate, careful reevaluation of estrogen receptors must be undertaken using proper assay conditions.

IN VITRO EFFECTS OF AN AROMATASE INHIBITOR ON 5α-REDUCTASE ACTIVITY IN HUMAN HYPERTROPHIC PROSTATIC TISSUE

To determine the effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the in vitro conversion of testosterone (T) to 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone, DHT), 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstan-3 beta, 17 beta-diol (diols), human benign hypertrophic prostatic (BPH) tissue was incubated with 4-14C-T as substrate, in the presence of 4-OH-A (10(-8) to 10(-6) M); the amounts of the 5 alpha-reduced metabolites formed were quantitated. The effects of 4-OH-A were compared with those of 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA), a known inhibitor of the 5 alpha-reductase. In the absence of 4-OH-A and 4-MA, human BPH tissue converted T to DHT and the diols readily. Both 4-OH-A and 4-MA induced significant and dose-related decreases in the formation of both DHT and the diols. The degree of inhibition induced by the different concentrations of 4-OH-A and 4-MA were 31, 41, 72% and 57, 87, 97%, respectively. The decreased formation of the diols was a consequence of the decreased availability of DHT (the immediate precursor of the diols) and was not due to direct effects of the inhibitors on the 3-hydroxysteroid dehydrogenases; both 4-OH-A and 4-MA were totally unable to modify the conversion of DHT to the diols, when 4-14C-DHT was used as substrate. Thus, 4-OH-A inhibits the process of 5 alpha-reduction of T in BPH tissue. This molecule might represent a potential new agent for the prevention and/or treatment of human BPH.

Effect of dextran-coated charcoal treatment on 17β-estradiol receptor in human benign prostatic hyperplasia

Using glycerol density gradient centrifugation technique, single saturation dose assay, and Scatchard plot, the effect of dextran-coated charcoal (DCC) treatment of homogenate or crude cytosol on estradiol binding protein in human benign prostatic hyperplasia was investigated. Receptor binding is increased after thirty minutes DCC treatment of homogenate or cytosol. Increase in estradiol binding is accompanied by loss in cytosolic protein. A 75 per cent increase in binding of estradiol to its receptor was observed after two hours incubation of DCC with homogenate. The concomitant increase in estradiol binding and decrease in protein concentration in both homogenate or cytosol after DCC treatment indicate the possible removal of some protein(s) which inactivate(s) the estradiol receptor. Removal of cofactors required for activation of proteases and removal of endogenous steroids which could be occupying the estradiol sites also are possible. This simple experimental procedure has improved significantly the methodology for the measurement andcharacterization of estrogen receptor in human BPH.

A Histochemical Comparison of Acid Phosphatase Activity in Human BPH and Rat Ventral Prostate

Acid phosphatase activity and its sensitivity to formalin inhibition were compared histo-chemically in cryostat sections of human BPH and rat ventral prostate. While the glandular epithelium in both human and young adult rat ventral prostate exhibits positive acid phosphatase activity, the intensity of the reaction is greater in human tissue. Moreover, human prostatic tissue remains acid phosphatase positive following 24 hours immersion in formalin, whereas the enzyme reaction in young adult rat ventral prostate disappears after 5 hours of formalin treatment. Unlike both human and young adult rat ventral prostate, sites of acid phosphatase activity in aged rat ventral prostate are not homogeneously distributed throughout the glandular epithelium but, rather, appear as large clumps, which persist after 5 hours of formalin treatment. Procedures used for the histochemical demonstration of formalin-resistant human prostatic acid phosphatase are not, therefore, directly applicable to rat ventral prostate.

36 Steroid receptors (ER, PR, AR) in human BPH and prostate adenocarcinoma

36 Steroid receptors (ER, PR, AR) in human BPH and prostate adenocarcinoma

Testosterone Metabolism of Fibroblasts Grown From Prostatic Carcinoma, Benign Prostatic Hyperplasia and Skin Fibroblasts

The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3α, 17β-diol and androstane-3β, 17β-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5α-androstanes and 3α-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5α-androstane formation in BPH, whereas 5α-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC.

Acute therapy with megestrol acetate decreases nuclear and cytosol androgen receptors in human BPH tissue.

This study measures the effect of megestrol acetate (Megace), a progestational antiandrogen, on nuclear and cytosol receptor concentrations in human BPH prostates. Prostatic tissue was obtained at surgery from both untreated patients with BPH and patients pretreated for three to eleven days with 120 to 160 mg of Megace daily; tissues were homogenized and separated into cytosol and crude nuclear fractions. Cytosol and salt extractable nuclear fractions were subjected to saturation analysis with 3H R1881 over the range of 0.5 to 10 nmoles in exchange reactions for 20 hours at 15 degrees C. Cytosol receptor concentration decreased significantly from 32.7 to 8.7 femtomoles per mg protein (P less than 0.05); nuclear receptor also was significantly reduced from 317 to 43.8 femtomoles (fmole) per mg DNA (P less than .001). Plasma testosterone also decreased significantly from 3.65 to 1.01 ng/ml in Megace-treated patients. These effects of Megace on receptor concentration appear to be qualitatively quite different from those reported following castration in animals or following high-dose estrogen therapy in humans. The mechanism of action of Megace in decreasing both cytosol and nuclear androgen receptor concentrations is unknown. It is possible that decrease in receptor concentration is secondary to either the progestational or antiandrogenic effects of Megace. These possibilities are currently under study.

Phenotypic modulation of the canine prostate after long-term treatment with androgens and estrogens.

Fine structural alterations of the canine prostate induced by long-term treatment of castrated adult animals with estrogens and/or androgens and also in combination with antiandrogens and/or antiestrogens for six months have been studied with particular respect to their topographic location within the gland. Three major patterns of structural responses of the epithelium have been distinguished: squamous metaplasia, atrophy, and hypertrophy, while in stroma, regression, hypertrophy, or sclerosis were observed. In addition to cellular alterations of stromal fibrocytes and smooth muscle cells, characteristic changes in the arrangement, distribution, and pattern of the different stromal elements occurred. General squamous metaplasia of the epithelium and regressive alterations of stromal cells were most obvious in animals treated with estradiol plus androstanediol. Atrophy of the epithelium and stromal sclerosis were the salient features of antiandrogen-treated castrated animals, while hypertrophy or hyperplasia of both the epithelium and stroma was a major finding in androstanediol-substituted castrated animals. Combined treatment caused rather heterogeneous structural patterns seemingly dependent on the location within the gland. The results indicate that the prostatic epithelial cells dispose of a broad variety of structural reaction patterns that, in case of combined hormonal treatment, are expressed in a manner typical for their locations within the ductal system of the gland. However, with the exception of combined treatment with estradiol, tamoxifen, and androstanediol of castrated dogs, none of the experimental protocols used induced a morphologic response of the gland comparable to that seen in human benign prostatic hyperplasia. The canine prostate therefore is of rather limited value as a model for human BPH.

A method of in vivo maintenance of human prostatic tissue in immunosuppressed mice.

Summary A method for growing human prostatic tissue in immunosuppressed mice has been described. Many viable specimens were obtained from 2 weeks to 2 months after implantation. The implications of the method as a laboratory method for screening agents suitable for treating human BPH is discussed and the method is compared with other methods for the in vitro and in vitro study of human prostatic tissue.