Fouad K. Habib

Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer

Earlier studies have demonstrated an unexplained depletion of the epidermal growth factor receptor (EGFR) protein expression in prostatic cancer. We now attribute this phenomenon to the presence of a variant EGFR (EGFRvIII) that is highly expressed in malignant prostatic neoplasms. In a retrospective study, normal, benign hyperplastic and malignant prostatic tissues were examined at the mRNA and protein levels for the presence of this mutant receptor. The results demonstrated that whilst EGFRvIII was not present in normal prostatic glands, the level of expression of this variant protein increased progressively with the gradual transformation of the tissues to the malignant phenotype. The selective association of high EGFRvIII levels with the cancer phenotype underlines the role that this mutant receptor may maintain in the initiation and progression of malignant prostatic growth, and opens the way for new approaches in the management of this disease including gene therapy.

Serenoa repens (Permixon®) : A 5α-reductase types I and II inhibitor-New evidence in a coculture model of BPH

The aim of this study was to determine the effect of the phytotherapeutic agent, Permixon®, on a novel coculture model of benign prostatic hyperplasia (BPH) in an effort to better understand the mode of action of the drug in vivo.

Estrogen and androgen signaling in the pathogenesis of BPH

Estrogens and androgens have both been implicated as causes of benign prostatic hyperplasia (BPH). Although epidemiological data on an association between serum androgen concentrations and BPH are inconsistent, it is generally accepted that androgens play a permissive role in BPH pathogenesis. In clinical practice, inhibitors of 5α-reductase (which converts testosterone to the more potent androgen dihydrotestosterone) have proven effective in the management of BPH, confirming an essential role for androgens in BPH pathophysiology. To date, multiple lines of evidence support a role for estrogens in BPH pathogenesis. Studies of the two estrogen receptor (ER) subtypes have shed light on their differential functions in the human prostate; ERα and ERβ have proliferative and antiproliferative effects on prostate cells, respectively. Effects of estrogens on the prostate are associated with multiple mechanisms including apoptosis, aromatase expression and paracrine regulation via prostaglandin E2. Selective estrogen receptor modulators or other agents that can influence intraprostatic estrogen levels might conceivably be potential therapeutic targets for the treatment of BPH.

The relevance of a hypoxic tumour microenvironment in prostate cancer

Research into the hypoxic tumour microenvironment is accelerating and the reversal of hypoxia is increasingly being suggested as a mechanism for improving cancer treatment. Recent studies have suggested that hypoxia is also a feature in prostate cancer and is associated with a poor prognosis. Hypoxia has been shown to cause radio‐resistance and hence hamper one of the major treatments for prostate cancer. However, unlike other solid tumours, such as cervical and head‐and‐neck cancer, there are inconsistencies and unanswered questions about the relevance of hypoxia in prostate cancer. This review outlines the role of low‐oxygen conditions in prostate cancer and the areas where further studies are required.

Epidermal growth factor receptors in human prostate cancer: correlation with histological differentiation of the tumour

The presence of specific and high affinity epidermal growth factor receptors (EGF-R) has been demonstrated in human prostate cancer (CaP). Scatchard analysis of the binding data revealed a linear plot consistent with a single class of binding sites with a mean dissociation constant (Kd) +/- s.d. = 1.6 +/- 0.4 nmol 1-1. Additionally the binding was specific for EGF since no other competitor than EGF was able to displace the binding of the labelled ligand from its receptor. Comparison of the concentrations of EGF-R in tissues from 19 patients with CaP with those measured in a group of 18 patients with benign prostatic hyperplasia (BPH) reveal that the expression of EGF-R was significantly higher in BPH (mean +/- s.d. = 125 +/- 7 fmol mg protein-1) than in CaP (52 +/- 11 fmol mg protein-1; P less than 0.01). Furthermore, in CaP the expression of EGF-R varied according to the histological grade of the cancer: well differentiated tumours demonstrated more receptors (84 +/- 13 fmol mg protein-1) than poorly differentiated tumours (22 +/- 5 fmol mg protein-1; P less than 0.01). Clearly the depletion in the expression of EGF receptors in CaP is a function of the histological grade of the cancer and as such EGF receptors could be used as a biochemical marker for tumour differentiation.

Serenoa repens (Permixon®) inhibits the 5α‐reductase activity of human prostate cancer cell lines without interfering with PSA expression

The phytotherapeutic agent Serenoa repens is an effective dual inhibitor of 5α‐reductase isoenzyme activity in the prostate. Unlike other 5α‐reductase inhibitors, Serenoa repens induces its effects without interfering with the cellular capacity to secrete PSA. Here, we focussed on the possible pathways that might differentiate the action of Permixon from that of synthetic 5α‐reductase inhibitors. We demonstrate that Serenoa repens, unlike other 5α‐reductase inhibitors, does not inhibit binding between activated AR and the steroid receptor‐binding consensus in the promoter region of the PSA gene. This was shown by a combination of techniques: assessment of the effect of Permixon on androgen action in the LNCaP prostate cancer cell line revealed no suppression of AR and maintenance of PSA protein expression at control levels. This was consistent with reporter gene experiments showing that Permixon failed to interfere with AR‐mediated transcriptional activation of PSA and that both testosterone and DHT were equally effective at maintaining this activity. Our results demonstrate that despite Serenoa repens effective inhibition of 5α‐reductase activity in the prostate, it did not suppress PSA secretion. Therefore, we confirm the therapeutic advantage of Serenoa repens over other 5α‐reductase inhibitors as treatment with the phytotherapeutic agent will permit the continuous use of PSA measurements as a useful biomarker for prostate cancer screening and for evaluating tumour progression.

THE SELECTIVITY AND SPECIFICITY OF THE ACTIONS OF THE LIPIDO-STEROLIC EXTRACT OF SERENOA REPENS (PERMIXON®) ON THE PROSTATE

PURPOSE To investigate the effects of the phytotherapeutic agent, Permixon(R), on primary cultures of fibroblast and epithelial cells from the prostate, epididymis, testes, kidney, skin and breast and to determine the selectivity and specificity of the action of the drug. MATERIALS AND METHODS All primary cultures were examined by electron microscopy before and following treatment with Permixon(R) (10 microg./ml.). In addition the apoptotic index was assessed by flow cytometry employing propidium iodide as a fluorophore. The impact of the drug on 5alpha-reductase (5alphaR) isoenzymes was also tested utilizing a pH specific assay. RESULTS There were changes in the morphology of prostate cells after treatment including accumulation of lipid in the cytoplasm and damage to the nuclear and mitochondrial membranes; no similar changes were observed in other cells. Permixon(R) increased the apoptotic index for prostate epithelial cells by 35% and 12% in the prostate stromal/fibroblast. A lesser apoptotic effect was demonstrated in skin fibroblast (3%) whereas none of the other primary cultures showed any increase in apoptosis when compared with the controls. Permixon(R) was also an effective inhibitor of both 5alphaR type I and II isoenzymes in prostate cells, but other cells showed no inhibition of 5alphaR activity following treatment with the plant extract. CONCLUSIONS This investigation demonstrated the selectivity of the action of Permixon(R) for prostate cells. The morphological changes in the prostate are accompanied by an increase in the apoptotic index along with an inhibition in the activity of the nuclear membrane bound 5alphaR isoenzymes. No similar changes were observed in any of the other cells under investigation.

The effect of zinc on the 5α-reduction of testosterone by the hyperplastic human prostate gland

The present studies were performed to evaluate the role of zinc in the regulation of testosterone 5 alpha-reduction by the 800 g supernatants prepared from human benign prostate hyperplasia specimens. The results show that when zinc is added at low concentrations the 5 alpha-reduction of testosterone is increased but at higher cation concentrations the metabolism is significantly inhibited. This decrease was mediated by both a non-competitive inhibition of the binding of testosterone to the 5 alpha-reductase enzyme and by a reduction in the formation of the NADPH cofactor. We have also demonstrated that the decreased synthesis of NADPH was produced by a competitive inhibition of both G6P and NADP binding to the G6PD enzyme. The data also suggests that the increase in testosterone metabolism observed at low zinc concentrations does not produce any changes in the binding of testosterone to the 5 alpha-reductase enzyme. In spite of the above observations we were unable to establish any correlation between the endogenous zinc content of the tissue and the in vitro capacity of the BPH samples to 5 alpha-reduce testosterone. The present study suggests a possible physiological role for the regulation of testosterone metabolism by zinc in the human prostate gland.

Epidermal growth factor and transforming growth factor alpha concentrations in BPH and cancer of the prostate: their relationships with tissue androgen levels.

We measured immunoreactive EGF and TGF alpha in prostate tissue extracts obtained from 19 patients with benign prostatic hyperplasia (BPH) and 19 with cancer of the prostate (CaP). Whilst both BPH and CaP expressed EGF (BPH = 195.61 +/- 19.94 ng g-1 protein; CaP = 235.60 +/- 24.45 ng g-1 protein) and TGF alpha (BPH = 92.57 +/- 7.60 ng g-1 protein; CaP = 100.73 +/- 15.47 ng g-1 protein) in equal concentrations, the levels of EGF in any tissue extract were on average twice those of TGF alpha. Furthermore analysis of the individual growth factor data revealed a direct correlation between EGF and TGF alpha in both BPH (r = 0.72, P < 0.001) and CaP (r = 0.69, P < 0.001). When the tumours were classified according to their Gleason score, a slight but significant increase in growth factor concentrations was noted as the tumour became less differentiated. We also measured the concentrations of testosterone and dihydrotestosterone (DHT) in prostate extracts with a view of elucidating the relationship between androgen and growth factors in this gland. There was a small positive correlation only between testosterone and EGF (r = 0.62, P < 0.05) and testosterone and TGF alpha (r = 0.61, P < 0.05) in CaP. The absence of any similar correlation in BPH where DHT becomes the predominant hormone may suggest an indirect role for testosterone in the regulation of growth factor production.

Solubilization of human prostatic 5α-reductase

Abstract A sensitive assay for 5α-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5α-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5α-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 μM testosterone was found to stabilise 5α-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5α-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5α-reductase was studied. All detergents caused a concentration-dependent solubilization of 5α-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5α-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.