Analysis of prostate cancer association with four single-nucleotide polymorphisms from genome-wide studies and serum phyto-estrogen concentrations.

BACKGROUND: Both genetics and the environment are implicated as risk factors for prostate cancer (PCa). This population-based case-control study evaluated four single-nucleotide polymorphisms (SNPs) previously identified by genome-wide association studies to be associated with increased PCa susceptibility. Potential relationships between serum concentrations of phyto-estrogens and SNPs were also investigated. METHODS: Four SNPs (rs10993994, rs2660753, rs1016343 and rs6983267) were genotyped in 247 PCa patients, 125 BPH patients and 274 control men recruited in Scotland. Serum concentrations of the phyto-estrogens enterolactone, equol, genistein and daidzein were measured by isotope dilution gas chromatography-mass spectrometry. RESULTS: Increased PCa risk was associated with TT genotype of rs10993994 compared with CC and CT genotypes combined (odds ratio (OR)=1.87; 95% confidence interval (CI), 1.26-2.77). TT homozygotes who had low serum enterolactone concentrations (below median) were more likely to have PCa (OR=2.90; 95% CI, 1.28-6.57) than individuals with CC/CT genotype and high serum enterolactone concentrations (above median). PCa was not associated with the other three SNPs tested. CONCLUSIONS: PCa susceptibility was associated with TT genotype of SNP rs10993994 in this cohort of Scottish men and the increased risk of PCa was modified by serum enterolactone concentrations.

FGFR4 Gly388Arg polymorphism and prostate cancer risk in Scottish men.

Fibroblast growth factor receptor 4 (FGFR4), a member of the fibroblast growth receptor family, was recently reported to be more abundantly expressed in malignant than benign prostate cells. A single nucleotide polymorphism at position 388 of the FGFR4 amino-acid sequence results in the substitution of glycine (Gly) with arginine (Arg) and higher frequency of the ArgArg genotype was previously found in prostate cancer patients. DNA was extracted from the blood drawn from 399 prostate cancer patients, 150 BPH patients and 294 healthy community controls. Polymerase chain reaction was carried out and single nucleotide polymorphisms of FGFR4 were identified by restriction enzyme digestion. No overall association is detectable between the Arg allele and increased prostate cancer risk. Subgroup analysis shows a higher incidence of the heterozygous ArgGly genotype in cancer cases than in the combined group of BPH and controls (P<0.05); this difference is statistically significant between cancer and BPH patients but not between cancer cases and community controls. The single nucleotide polymorphism Gly(388)Arg in FGFR4 is not associated with increased risk of prostate cancer in Scottish men. This observation is in contrast with results from two previous studies conducted in the USA and Japan.

Variation in dermcidin expression in a range of primary human tumours and in hypoxic/oxidatively stressed human cell lines.

Dermcidin acts as a survival factor in a variety of cancer cell lines under hypoxia or oxidative stress. The aim of this study was to evaluate dermcidin expression in cell lines following simulation of tumour microenvironmental conditions and in a range of primary tumours. Tumour tissues were collected from patients with oesophageal (28 samples), gastric (20), pancreatic (five), bile duct (one) and prostatic (52) carcinomas as well as 30 benign tissue samples, for assessment of dermcidin mRNA levels using real-time PCR. Dermcidin expression was assessed in prostatic and pancreatic cancer cell lines, with and without induction of hypoxia or oxidative stress. Dermcidin mRNA expression was very low or absent in both unstressed and stressed prostate cell lines. None of the primary prostate tissue, benign or malignant, expressed dermcidin mRNA. Only two (4%) of the gastro-oesophageal cancer samples expressed moderate quantities of dermcidin mRNA. However, three (60%) of the pancreatic cancer samples and the single cholangiocarcinoma specimen had moderate/high levels of dermcidin expression. Of the two pancreatic cancer cell lines, one expressed dermcidin moderately but neither showed a response to hypoxia or oxidative stress. Expression of dermcidin in human primary tumours appears highly variable and is not induced substantially by hypoxia/oxidative stress in cell line model systems. The relationship of these findings to dermcidin protein levels and cell survival remains to be determined.

Micro-engineered remote palpation device for assessing tissue compliance.

This paper concerns the operation of the actuator for a prototype micro-engineered mechanical palpation device for deployment via a cystoscope to measure the dynamic mechanical properties of the prostate gland in vivo. The subassembly consists of a 400x200 microm silicon (Si) piston manufactured using deep reactive ion etching (DRIE) housed within an anodically bonded glass-Si-glass sandwiched housing. The micro-channel on the Si layer was formed by powder blasting and contains the micro-piston with one end pointing to the side of the housing and the other facing a via hole leading to a capillary tube. The opening on the side of the housing was sealed by a 5 microm thick silicone membrane which acts to retain the micro-piston and act as a return spring. A 320 microm diameter capillary forms the connection between the micro-channel and a micro-syringe which is operated by a programmable syringe pump to produce a reciprocating action. A pressure sensor is connected along the capillary tube to measure the dynamic pressure within the system. The micro-piston has already been used, separately actuated to measure the dynamic mechanical properties of known viscoelastic materials and prostate tissue. The purpose of the present work is to assess the functionality of the actuator assembly.

A variant epidermal growth factor receptor protein is similarly expressed in benign hyperplastic and carcinomatous prostatic tissues in black and white men.

BACKGROUND AND OBJECTIVE: Anti-epidermal growth factor receptor strategies are now established in cancer treatment We have recently described the presence of EGFRvIII (a variant EGFR) in prostatic tumours from UK white men and this is now a target for anti-prostate cancer treatments. However, there has been no report on the expression of this abnormal protein in black men. MATERIALS AND METHODS: We determined EGFRvIII expression in sections of normal, benign hyperplastic (BPH) and carcinomatous (CaP) prostatic archival tissues from Nigerian men and UK white men using streptavidin immunohistochemical techniques. The EGFRvIII immunoreactivity was scored visually using a semi-quantitative method and the results compared statistically. RESULTS: EGFRvIII expression increased with increasing malignancy in both study populations (CaP > BPH > Normal p, <0.0001). Furthermore, EGFRvIII expression was similar in both BPH and CaP tissues in black and white men (p, 0.86 and 0.31 respectively). CONCLUSION: These results demonstrate that EGFRvIII immunoreactivity in prostatic tumours in black men is similar to that in white men. Anti-cancer treatments directed at the EGFRvIII should be equally effective in men from both subpopulations.

cAMP-responsive element-binding protein regulates vascular endothelial growth factor expression: implication in human prostate cancer bone metastasis.

Aberrant expression of vascular endothelial growth factor (VEGF) is associated with human prostate cancer (PCa) metastasis and poor clinical outcome. We found that both phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and VEGF levels were significantly elevated in patient bone metastatic PCa specimens. A PCa ARCaP progression model demonstrating epithelial-to-mesenchymal transition exhibited increased CREB phosphorylation and VEGF expression as ARCaP cells became progressively more mesenchymal and bone-metastatic. Activation of CREB induced, whereas inhibition of CREB blocked, VEGF expression in ARCaP cells. CREB may regulate VEGF transcription via a hypoxia-inducible factor-dependent mechanism in normoxic conditions. Activation of CREB signaling is involved in the coordinated regulation of VEGF and may pre-dispose to PCa bone metastasis.

In-vitro dynamic micro-probing and the mechanical properties of human prostate tissues.

In vitro macro- and micro-indentation test systems have been designed to measure the dynamic micro-mechanical properties of human prostate tissues at actuation frequencies between 5 Hz and 30 Hz, and 0.5 Hz and 20 Hz, respectively. The development of in vitro test systems was aimed at assessing the capacity of such an in vivo medical probe to provide information useful for the diagnosis of various prostate diseases. The macro-indentation test system is an established one, which we have used to determine structure-property relationships in human and canine prostate tissues and here we use it to validate a newly-developed micro-indentation test system using a tissue phantom. Mechanical testing was also carried out on sections of prostate tissue harvested from cystectomy and radical prostatectomy, diagnosed with bladder cancer and benign prostatic hyperplasia. Dynamic probing under displacement control was carried at pre-strains between 5% and 8% for macro-probing and at 5% pre-strain for micro-probing, and the general effect of pre-strain on the dynamic mechanical properties (described by the amplitude ratio between stress and strain, and the phase lag between strain and stress) of phantom and prostate tissues is presented. Specific point probing on epithelial and stromal histological components was also carried out showing a significant difference between the amplitude ratios of epithelial and stromal components for actuation frequencies exceeding 5 Hz. However, no significant difference was found between phase lags for epithelial and stromal tissues.

Measurement of tissue mechanical characteristics to distinguish between benign and malignant prostatic disease.

OBJECTIVES: To investigate the relationship between the morphology and mechanical properties of benign and malignant prostatic tissues measured in vitro. METHODS: Fresh tissue specimens were collected from patients undergoing transurethral resection of the prostate (TURP) for benign or malignant prostatic enlargement. Individual TURP chippings underwent immediate mechanical testing by applying a dynamic compressive strain to the samples. The amplitude ratio (E*) and phase difference (tan delta), measures of tissue elastic and viscous components respectively, were derived. Individual sections from the processed specimens underwent immunohistochemical staining and computerized image analysis was used to measure the morphologic characteristics of each TURP chipping. Linear regression analysis was used to assess correlations between morphologic and mechanical measurements, and the unpaired t test, assuming equal variances, was used to compare the mechanical and morphologic characteristics of benign and malignant prostates. RESULTS: Significant differences were noted between the morphology of the benign and malignant prostates. Tan delta was significantly smaller within the malignant prostates (P = 001). No difference was found between the benign and malignant prostates with respect to E*. Within the malignant prostates, a strong negative correlation was found between the epithelial tissue content and tan delta (R2 = 0.50, P = 0.031). CONCLUSIONS: The results of this study showed that measurable differences exist between the mechanical characteristics of benign and malignant prostatic tissue and provide further evidence that significant correlations exist between prostatic tissue morphology and mechanical characteristics. We believe that the ability to quantify prostatic tissue mechanical characteristics in vivo may be of clinical benefit in the future assessment of prostatic diseases, both benign and malignant.

Measurement of the mechanical characteristics of benign prostatic tissue: a novel method for assessing benign prostatic disease.

OBJECTIVES: To investigate the relationship between the morphology and the mechanical properties of benign prostatic tissues measured in vitro. METHODS: Fresh tissue specimens were collected from 17 patients undergoing transurethral resection of the prostate for benign prostatic obstruction. Individual tissue specimens underwent immediate mechanical testing, by applying a dynamic compressive strain to the samples. The amplitude ratio (E*) and phase difference (tan delta), measures of tissue elastic and viscous components, were derived. Individual sections from the processed specimens underwent immunohistochemical staining and computerized image analysis to measure the morphologic characteristics of each transurethral resection of the prostate chipping. Correlations between the morphologic and mechanical measurements were assessed. RESULTS: A strong positive correlation was found between prostatic smooth muscle content and (E*) (R2 = 0.58, P = 0.009). CONCLUSIONS: The results of this study have demonstrated that strong correlations exist between prostatic tissue morphology and mechanical characteristics. We believe that the ability to quantify prostatic tissue mechanical characteristics in vivo may be of clinical benefit in the future assessment and treatment of benign prostatic disease.

Not all brands are created equal: a comparison of selected components of different brands of Serenoa repens extract.

Recommendations regarding the use of plant-derived medications for the treatment of lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) state that every brand should be fully evaluated and considered separately. Disparity between a number of brands in terms of their stated and actual doses has been recently highlighted. The aim of this study was to fully quantify the variation in Serenoa repens extracts (SrE) commercially available for the treatment of BPH-associated LUTS. To this end, 14 brands of SrE were compared. Concentrations of free fatty acids (FFAs), methyl and ethyl esters, long-chain esters and glycerides were assessed using liquid and gas chromatography. Many of the brands showed a significantly different proportional content which may have an impact on their clinical efficacy and safety. The high concentrations of FFAs in particular, which previous research has suggested as comprising the active agent of SrE for the treatment of LUTS, may influence the clinical benefit derived from each product. Our findings lend further weight to recommendations by the 5th International Consultation on BPH that plant-derived treatments should be analysed and considered as independent entities despite their common origin. Only extracts with demonstrated pharmacological activities and proven clinical efficacy should be considered for the treatment of patients with BPH.

Alterations in the expression of androgen receptor, wild type-epidermal growth factor receptor and a mutant epidermal growth factor receptor in human prostate cancer.

Prostatic carcinogenesis has been associated with alterations in the expression of the androgen receptor (AR) and the epidermal growth factor receptor (WT-EGFR), and over-expression of the constitutively active variant epidermal growth factor receptor (EGFRvIII). Changes in the expression of AR, WT-EGFR and EGFRvIII were evaluated in serial sections from 26 normal and 26 benign hyperplastic and 50 prostate cancer tissues using specific immunostaining techniques. The loss of AR expression in peri-epithelial stroma as prostatic tissues de-differentiated correlated strongly with the depletion of WT-EGFR and with increasing expression of the EGFRvIII in the adjacent epithelium. In contrast, changes in epithelial AR immunopositivity in these tissues correlated weakly with the changes in normal and variant EGFR levels. This is the first report correlating the changes in the expression of these three proteins in archival material from the different human prostatic tissue histotypes. The loss of expression of proteins that contribute to the regulation of prostatic homeostasis (AR and WT-EGFR) correlates strongly with the expression of a constitutively active variant EGF receptor (EGFRvIII) in human prostate cancer. These changes occur at an early stage of neoplastic transformation and may contribute to the progression of the disease to hormone independence.

MUC1 expression, splice variant and short form transcription (MUC1/Z, MUC1/Y) in prostate cell lines and tissue.

OBJECTIVE: To detect the expression and transcription pattern of MUC1 in benign and malignant disease, and in two widely studied cell lines, and to investigate the glycosylation of MUC-1 in bone metastasis of prostate cancer, as mucins have been implicated in the progression and behaviour of several cancers. MATERIALS AND METHODS: RNA extracted from cell lines (DU145 and PC3), five samples of BPH and five samples of prostate cancer was reverse transcribed before amplification of MUC1-specific sequences by polymerase chain reaction. Paraffin-embedded sections were stained for glycosylated MUC1 and MUC1 core epitopes by HMFG1 and B27.29 antibodies, respectively. Steroid-treated cell lines were analysed by fluorescence-activated cell sorting, using the same antibodies. RESULTS: MUC1, in an under-glycosylated form, was widely expressed in the prostate and in metastatic lesions. MUC1/Z and MUC1/Y RNA were differentially expressed in BPH and prostate cancer, with no detectable expression of splice variant mRNA. This is in contrast to prostate cancer cell line cells (PC3 and DU145), which express splice variant mRNA. CONCLUSIONS: BPH, prostate cancer and metastatic prostate cancer all express high levels of under-glycosylated MUC1. This may explain the inability of previous studies to detect MUC1 in prostate tissue, as the antibody used was specific for a carbohydrate epitope which is not expressed on the under-glycosylated MUC1.

Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)-integrin expression.

A major impediment to our understanding of the biology of stem cells is the inability to distinguish them from their differentiating progeny. We made use of the known association of stem cells with basement membranes to isolate prostate epithelial stem cells. We show that, in vivo, putative stem cells express higher levels of the alpha(2)-integrin subunit than other cells within the basal layer. Approximately 1% of basal cells examined by confocal microscopy were integrin "bright", and these cells can be selected directly from the tissue on the basis of rapid adhesion to type I collagen. This selected population has a basal phenotype, as determined by expression of CK5 and CK14 and lack of expression of the differentiation-specific markers prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), and has a fourfold greater ability to form colonies in vitro than the total basal population. These putative stem cells are distinguished from other basal cells by their ability to generate prostate-like glands in vivo with morphologic and immuno-histochemical evidence of prostate-specific differentiation. These properties are consistent with a stem cell origin. Furthermore, the presence of surface integrins on prostate stem cells suggests that these cells share common pathways with stem cells in other tissues.

Further characterization of storage-related alterations in immunoreactivity of archival tissue sections and its implications for collaborative multicenter immunohistochemical studies.

Storage of unstained paraffin slides may lead to the deterioration of specimens and failure to detect cellular proteins immunohistochemically. Although the implication of age-induced alterations on multicenter immunohistochemical studies would be considerable, they have not been investigated previously. The current study was undertaken to examine the effect of this factor further and to explore new ways of overcoming the resultant shortcomings. The authors now report on the immunodetection of a host of antigens in similarly preserved unstained serial paraffin slides obtained from three centers using a panel of eight antibodies. Staining of recently prepared sections from the authors' centers resulted in similar strong patterns in seven of eight antibodies, with one antibody demonstrating variable immunoreactivity. However, storage of unstained paraffin sections at room temperature resulted in a variable but progressive decrease in expression of several tissue antigens. Although the loss in antigenicity was proportional to the length of storage, the effect was reversible if super antibody concentrations were used. The authors conclude that recently prepared paraffin sections from centers with similar fixation protocols have similar immunoreactivity and are suitable for use in comparative multicenter studies. However, in view of the delays that may attend tissue transportation during these projects, the authors suggest that test systems should be checked for age-induced antigen degradation by incubating sections with higher antibody concentrations.

Development of a new in vitro model for the study of benign prostatic hyperplasia.

BACKGROUND: The search for novel agents for the treatment of the lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) is dependent on an increased understanding of the pathophysiology of the disease. Unfortunately, in vitro and animal models have been of limited value. METHODS: This article describes a novel model system in which the interactions of the stromal and epithelial components of the prostate gland can be determined. RESULTS: The coculture system provides a simple model of the cellular interactions occurring in the adult human prostate. CONCLUSIONS: This coculture system could potentially be used to determine the precise molecular site of action of agents such as terazosin on prostatic apoptosis.

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.

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 5alpha-reductase types I and II inhibitor-new evidence in a coculture model of BPH.

BACKGROUND: 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. METHODS: The effect of Permixon, at the calculated therapeutic concentration, on the activity of 5alpha-reductase isoenzymes was evaluated utilizing a pH-specific assay. Prostate-specific antigen (PSA) secretions into the medium were measured in the presence and absence of Permixon and quantified by an ELISA assay. The morphological patterns before and following Permixon treatment were also examined by electron microscopy. All results were compared to controls. RESULTS: Permixon at a concentration of 10 micrograms/ml (calculated plasma concentration in patient receiving recommended therapeutic dosage) was shown to be an effective inhibitor of both 5alpha-reductase types I and II isoenzymes without influencing the secretion of PSA by the epithelial cells, even after stimulation with testosterone. The morphology of Permixon-treated cells was found to be markedly different from that of untreated controls. Cells which had been treated with the drug demonstrated extensive accumulation of lipids in the cytoplasm and widespread damage of intracellular membranes, including mitochondrial and nuclear membranes. CONCLUSIONS: Permixon is an effective dual inhibitor of 5alpha-reductase isoenzyme activities in the prostate. Unlike other 5alpha-reductase inhibitors, Permixon induces this effect without interfering with the cells' capacity to secrete PSA, thus permitting the continued use of PSA measurements for prostate cancer screening.

Factors controlling the expression of 5alpha-reductase in human prostate: A possible new approach for the treatment of prostate cancer.

The functional activity of the 5alpha-reductase isoenzyme in the human prostate is gradually depleted as the gland undergoes transformation from the normal to the metastatic state. There are two isoforms of this enzyme expressed in the prostate and recent studies have demonstrated that the type II isoenzyme is the one most affected by the onset of neoplasia. To elucidate the mechanism(s) responsible for the down-regulation of the isoenzyme activity, we re-examined the nature of the interaction between stroma and epithelial cells in the human prostate. We noted that the control of the isoenzyme expression in the epithelial cells was regulated by a paracrine factor specific to the prostate fibroblast. In the absence of this factor the human prostate loses its capacity to express the 5alpha-reductase type II isoenzyme - a characteristic manifested simultaneously with the loss of differentiation in the tissues. This finding presents a totally new concept to the control of 5alpha-reductase in the human prostate and offers a possibility for an interesting and alternative approach to the management of prostate cancer.

Malignant transformation of human prostatic epithelium is associated with the loss of androgen receptor immunoreactivity in the surrounding stroma.

The cellular pathways involved in the pathogenesis of hormone resistance remain unclear. Studies evaluating the role of changes in human androgen receptor (hAR) expression in the progression of prostatic tumors have been inconclusive. Androgenic influence over prostatic growth is mediated via the regulation of interactions between stromal and epithelial cells. We hypothesized that neoplastic transformation of the prostate would be associated with alterations in hAR expression in the adjacent stroma. Using immunohistochemical techniques, we determined hAR positivity in the epithelium and adjacent stroma of sections from 17 benign and 39 malignant prostatic glands. We found that whereas the expression of the receptor decreased in both cellular compartments as the tissues dedifferentiated, the depletion was more pronounced in the stromal nuclei (P<0.0001). However, in sections from both untreated and hormone-resistant prostate cancer tissues, although heterogeneity of hAR expression in malignant epithelia was increased, there appeared to be a unique field effect around the cancerous prostate glands that resulted in a decreased expression of the receptor in the adjacent benign glands and its total loss in the surrounding stroma. The loss of hAR in the stroma adjacent to malignant prostatic epithelium may play an important role in prostate cancer progression. Furthermore, the similarity of the lack of stromal hAR expression in newly diagnosed and hormone-resistant prostate cancer (P = 0.85) may be an indication that the mechanisms responsible for the acquisition of hormone independence are established early in the malignant transformation process.