Pharmacoperone IN3 enhances the apoptotic effect of leuprolide in prostate cancer cells by increasing the gonadotropin-releasing hormone receptor in the cell membrane.

Gonadotropin-releasing hormone (GnRH) agonists are widely used for the treatment of advanced prostate cancer (PCa). Agonists activate the GnRH receptor (GnRH-R), triggering apoptosis in PCa cells. In gonadotropes, the amount of GnRH-R in the plasma membrane is regulated by protein folding and endoplasmic reticulum retention, mechanisms that can be overcome by the pharmacoperone IN3. Our aim was to describe the intracellular distribution of GnRH-R in PCa cells and its relation to response to GnRH analog treatments. The expressions of GnRH-R in PCa biopsies were evaluated by immunohistochemistry and the intracellular distribution was determined by immunofluorescence in primary cell cultures from human PCa samples. Cultured cells were pretreated with IN3 and then with leuprolide. Cell survival was evaluated by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) thiazolyl blue formazan and cell cycle and apoptosis by flow cytometry. We observed that the expression of GnRH-R decreased according to malignant progression. Most GnRH-R are located inside the cell, colocalizing with endoplasmic reticulum markers. The treatment with IN3 decreased cellular GnRH-R retention, increasing plasma membrane expression in approximately 60%. Pretreatment with IN3 decreased PCa cell survival compared with leuprolide-alone treatment, primarily because of an increase in apoptosis. We conclude that the response of PCa cells to leuprolide is related to the amount of GnRH-R in the plasma membrane. Therefore, pretreatment evaluation of the amount of these receptors may be a predictor of the outcome of leuprolide treatment in PCa patients. Assessment of systemic IN3 effect would be necessary to determine its utility as an adjuvant treatment in hormone-resistant tumors.

Molecular signature of cancer stem cells isolated from prostate carcinoma and expression of stem markers in different Gleason grades and metastasis.

Prostate cancer (PCa) is the most frequently diagnosed malignancy in men worldwide. Chemotherapy response is very poor and resistance to hormone-based treatments is frequent in advances stages. Recently, tumor-initiating cells or cancer stem cells (CSCs) have been identified in several cancers, including PCa. These cells are thought to be responsible for therapy resistance, relapse and metastasis. In the present work, enriched populations of CSCs were obtained using a mixed procedure that included differential clone-forming ability, sphere growing induction (prostatospheres) and magnetic-associated cell sorting (MACS). Also, stem marker expression was determined in PCa biopsies of different histological grades and metastasis samples. The signature for stem markers of the isolated CSCs was CD133+/CD44+/ABCG2+/ CD24-. Expression of stem markers (CD133, CD44, and ABCG2) was higher in medium Gleason biopsies than in lower and higher grades, and lymph-node and bone metastasis samples. These results suggest that the CSCs in PCa reach an important number in medium Gleason grades, when the tumor is still confined into the gland. At this stage, the surgical treatment is usually with curative intention. However, an important percentage of patients relapse after treatment. Number and signature of CSCs may be a prognosis factor for PCa recurrence.

Chemotherapy sensitivity recovery of prostate cancer cells by functional inhibition and knock down of multidrug resistance proteins.

BACKGROUND: In several cancer types, expression of multidrug resistance (MDR) proteins has been associated with lack of chemotherapy response. In advanced prostate cancer (PCa) the use of chemotherapy is mainly palliative due to its high resistance. Previously, we described that MDR phenotype in PCa could be related with high basal and drug-induced expression of MDR proteins P-Glycoprotein (P-Gp), MRP1, and LRP. METHODS: Using primary cell cultures from PCa patients, we evaluated the effect of function and expression inhibition of P-Gp, MRP1, and LRP, on cell survival after chemotherapy exposure. Cells were treated with specific MDR protein substrates (docetaxel and mitoxantrone for P-Gp, methotrexate for MRP1 and cisplatin for LRP) and pharmacological inhibitors (cyclosporine A, genistein and 3-aminobenzamide), and cell survival was evaluated trough 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell cycle analysis. MRP1 activity was evaluated by FACS using the specific inhibitor MK571. Cells were transfected with MDR proteins siRNAs and treated with the corresponding substrates. RESULTS: PCa cell resistance to MDR protein substrates was partially reversed, decreasing cell survival in around 20%, by treating primary cell cultures with specific pharmacological inhibitors. PCa cells transfected with siRNAs against MDR proteins decreased cell survival when treated with the corresponding drugs. Docetaxel was the most effective chemotherapeutic drug to induce cell death and decrease survival. CONCLUSION: Low chemotherapy response in PCa could be explained, in part, by over-expression of functional MDR proteins. Expression and function of these proteins should be evaluated to enhance efficacy of docetaxel-based therapies of patients with hormone-resistant PCa.

Membrane translocation and activation of GnRH receptor sensitize prostate cancer cells to radiation.

BACKGROUND: GnRH analogs are widely used as neoadjuvant agents for radiotherapy in prostate cancer (PCa) patients, with well-documented effects in reducing tumor bulk and increasing progression-free survival. GnRH analogs act locally in the prostate by triggering apoptosis of PCa cells via activation of the GnRH receptor (GnRHR). During PCa progression, the distribution of GnRHR within the cell is altered, with reduced expression in the cell membrane and remaining sequestered in the endoplasmic reticulum. Pharmacoperone IN3 is able to relocalize GnRHR to the cell membrane. The aim of this study was to evaluate the effect of radiation on PCa cells pretreated with leuprolide, alone or in combination with IN3, as radiosensitizers. MATERIAL AND METHODS: PC3 and human PCa primary cell cultures were treated with IN3 for 24 h, followed by different doses of leuprolide for 48 h and, finally, single doses of radiation (3, 6, and 9 Gy). After radiation, cell survival, apoptosis, cell cycle distribution, and colony growth were evaluated. RESULTS: Radiation reduced cell survival and increased apoptosis in a dose-dependent manner. This effect was also directly related to leuprolide concentration. Pretreatment with IN3 enhanced apoptosis and decreased cell survival, also observing a higher proportion of cells arrested in G2. CONCLUSION: Neoadjuvant leuprolide increases radiation-mediated apoptosis of PCa cells. This effect was enhanced by pretreatment with pharmacoperone IN3. Clinical use of IN3 as a radiosensitizer combined with androgen deprivation therapy to improve survival of patients with PCa remains to be evaluated.

Gonadotropin releasing hormone analogs induce apoptosis by extrinsic pathway involving p53 phosphorylation in primary cell cultures of human prostatic adenocarcinomas.

BACKGROUND: Gonadotropin-releasing-hormone (GnRH) analogs are widely used to block hypothalamic-pituitary-gonadal axis and inhibit blood androgen levels in patients with prostate cancer (PCa). In addition, GnRH analogs induce proliferation arrest and apoptosis through GnRH receptors expressed on the membrane of PCa cells. Possible molecular mechanisms involved in GnRH-mediated apoptosis on prostate cancer cells were studied. METHODS: Primary cultures from PCa and benign prostatic hyperplasia (BPH) (non-malignant control) were derived from samples provided by our Institutional Hospital. Cell cultures were incubated for 24 hr with 20 ng/ml of GnRH agonist Leuprolide (Lp) or antagonist Cetrorelix (Cx). Apoptosis was evaluated by studying the expression of Bax and Bcl-2 and the activation of caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and caspase-3. Also, mRNA level, protein expression and phosphorylation of p53 were studied. RESULTS: Cleaved caspase-8 and -3, but not -9, increased in presence of Lp and Cx in PCa cell cultures. Bax and Bcl-2 mRNA levels showed no changes after GnRH-analog treatments. Only Bax protein showed an increase after Cx treatment in PCa cell cultures. p53 mRNA level was higher in PCa than in BPH cell cultures. Lp and Cx increased p53 expression and phosphorylation in PCa cell cultures. CONCLUSIONS: Apoptosis induced by GnRH analogs seems to be mediated by extrinsic pathway involving p53 phosphorylation. Phosphorylated-p53 might be associated with the increase in apoptotic NGF receptor, p75, previously reported by our laboratory. These findings reinforce the concept of clinical use of GnRH analogs for PCa suggesting that intraprostatic treatment may be more effective.

Expression of multidrug resistance proteins in prostate cancer is related with cell sensitivity to chemotherapeutic drugs.

BACKGROUND: Multidrug resistance (MDR) proteins have been associated with the lack of chemotherapy response. Expression of these proteins has been described in the prostate, but there is no information about their role in the chemotherapy response of prostate cancer (PC). We studied the gene and protein expression of MDR proteins in primary cell cultures from PC tumors and PC cell lines, their relationship with chemotherapy and their effects on cell survival. METHODS: Primary cell cultures from PC were obtained from samples provided by our Institutional Hospital. Cell lines LNCaP, PC3, and DU145 were also examined. Cells were treated during 72 hr with several chemotherapeutic drugs. Protein and mRNA expressions of P-glycoprotein (P-Gp), MRP1 and LRP, before and after drug treatment, were evaluated by RT-PCR and Western blot analyses. The effect on cell survival was evaluated by proliferation assays (MTT), and cell cycle and apoptosis by flow cytometry. RESULTS: Primary PC cultures exhibited higher MDR protein expression and lower drug sensitivity than cell lines, in which P-Gp was not detected. Docetaxel and mitoxantrone displayed the highest apoptotic effect. Exposure to chemotherapeutic drugs increased apoptosis, cell cycle arrest, and MDR expression. Long-term treatment with doxorubicin diminished apoptosis elicited by all drugs examined in this study, suggesting a cross-resistance phenomenon. CONCLUSIONS: Low chemotherapy response observed in PC primary cultures could be explained, in part, by the high levels of MDR proteins (intrinsic MDR phenotype), and also, by their over-expression induced after long-term exposure to drugs (acquired MDR phenotype), which increase treatment resistance. Prostate 69: 1448-1459, 2009. (c) 2009 Wiley-Liss, Inc.

Evaluation of MENT on primary cell cultures from benign prostatic hyperplasia and prostate carcinoma.

7-alpha-Methyl-19-Nortestosterone (MENT) is a synthetic androgen more potent than testosterone (T) and cannot be reduced at 5-alpha position. No important effects of MENT on prostate growth have been reported. However, little is known about the effect of MENT on benign prostatic hyperplasia (BPH) or prostate carcinoma (CaP). We evaluate the effect of MENT, T and dihydrotestosterone (DHT) on secretion, proliferation and gene expression of primary cell cultures from human BPH and CaP. Moreover, the effect of these androgens was examined in the presence of finasteride to determine the influence of the 5-alpha reductase (5-AR) activity on the androgenic potency. BPH and CaP primary cultures were treated with 0, 1, 10 and 100 nM of T, MENT or DHT during 24 and 48 h. Prostate-specific antigen (PSA) was measured by micro particles immunoassay and proliferation rate by spectrophotometric assay (MTT) and by the immunochemical detection of the proliferation marker Ki-67. Gene expression of FGF8b (androgen sensitive gene) was evaluated by semi-quantitative RT-PCR. Results showed that MENT treatments increased PSA secretion and proliferation rate with a potency ranged between T and DHT. Similar effects of MENT were observed in both BPH and CaP cultures. The studies with finasteride showed that in BPH and CaP cells, the conversion of T into DHT significantly contributes to its effect on the proliferation and PSA secretion, and corroborated the resistance of MENT to the 5-AR. The effect of MENT on the gene expression of FGF8b in CaP cells was similar to T and lower than DHT. It is concluded that MENT increases proliferative and secretory activities and gene expression on pathological prostate cells although in less extent than the active metabolite DHT. Furthermore, the fall of endogenous concentration of T during MENT treatment anticipates that this androgen will be of low impact for the prostate.

Characterizing Prostiva RF treatments of the prostate for BPH with gadolinium-enhanced MRI.

Transurethral needle ablation (TUNA) is an accepted and effective therapy for the treatment of lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH). Prostiva (Medtronic, Shoreview, MN) is the newest-generation device, which includes a new needle design and radio frequency (RF) generator. This device creates temperatures of 120 degrees C and necrotic lesions in less than 2.5 min. Using previously described techniques, we analyzed dynamic, gadolinium-enhanced MRIs to characterize the ablative properties of the new Prostiva RF device. Ten men with LUTS due to BPH were treated with the standard Prostiva manufacturer-recommended protocol. The bladder neck and lateral lobes received treatment based on prostate volume and prostatic urethral length. Gadolinium-enhanced MRI sequences were obtained prior to and 1 week post-treatment. Analyze software (Mayo Clinic Biomedical Imaging Resource, Rochester, MN) was used to evaluate MRIs. New gadolinium defects were seen in all patients following Prostiva treatments. All lesions coalesced within the prostate. No defects were seen beyond the prostate, and the urethra was spared in all patients. The mean volume of necrosis was 7.56 cc, representing a mean of 11.28% of total prostate volume. Dynamic, gadolinium-enhanced MRIs demonstrate new vascular defects representing necrosis caused by Prostiva RF therapy of the prostate. The standard Prostiva RF protocol produces lesions that coalesce to create larger lesions in the bladder neck and lateral lobes. Compared to the TUNA Precision Plus device, the ablative lesions appear comparable while produced with a shorter burn time.

Characterizing TUNA ablative treatments of the prostate for benign hyperplasia with gadolinium-enhanced magnetic resonance imaging.

BACKGROUND AND PURPOSE: Transurethral Needle Ablation of the prostate TUNA has been accepted as an office-based treatment for benign prostatic hyperplasia (BPH) for many years. Clinical outcomes have been reported, but the amount and location of the necrosis produced have yet to be characterized. The necrosis caused by TUNA was evaluated by gadolinium-enhanced magnetic resonance imaging (MRI) of the pelvis. PATIENTS AND METHODS: Twelve patients with BPH/lower urinary-tract symptoms underwent standard TUNA, and MRI scans with gadolinium enhancement were performed before and 1 week after treatment. The images were studied using Analyze software to quantify the amount of necrosis compared with the prostatic volume. Transverse, coronal, and sagittal images were obtained to identify the location of the necrosis. RESULTS: New gadolinium defects were seen in all patients after TUNA. The lesions coalesced into continuous areas of necrosis and correlated with the site of needle placement. The mean volume of necrosis was 6.84 cc and equated to 8.6% of the prostate volume. No lesions were found near the apex, urethra, or rectum; and none extended beyond the prostate capsule. CONCLUSIONS: Gadolinium-enhanced MRI demonstrates new vascular defects representing necrosis caused by TUNA of the prostate. This therapy for BPH produces necrotic lesions that can be placed strategically by the surgeon. The standard protocol produces lesions that coalesce to create larger lesions. This MRI study has characterized, for the first time, the heating pattern and intraprostatic necrosis of a complete TUNA procedure.

Joint effect among p53, CYP1A1, GSTM1 polymorphism combinations and smoking on prostate cancer risk: an exploratory genotype-environment interaction study.

AIM: To assess the role of several genetic factors in combination with an environmental factor as modulators of prostate cancer risk. We focus on allele variants of low-penetrance genes associated with cell control, the detoxification processes and smoking. METHODS: In a case-control study we compared people carrying p53cd72 Pro allele, CYP1A1 M1 allele and GSTM1 null genotypes with their prostate cancer risk. RESULTS: The joint risk for smokers carrying Pro* and M1*, Pro* and GSTM1null or GSTM1 null and CYP1A1 M1* variants was significantly higher (odds ratio [OR]: 13.13, 95% confidence interval [CI]: 2.41-71.36; OR: 3.97, 95% CI: 1.13-13.95 and OR: 6.87, 95% CI: 1.68-27.97, respectively) compared with that for the reference group, and for non-smokers was not significant. OR for combinations among p53cd72, GSTM1 and CYP1A1 M1 in smokers were positively and significantly associated with prostate cancer risk compared with non-smokers and compared with the putative lowest risk group (OR: 8.87, 95% CI: 1.25-62.71). CONCLUSION: Our results suggest that a combination of p53cd72, CYP1A1, GSTM1 alleles and smoking plays a significant role in modified prostate cancer risk on the study population, which means that smokers carrying susceptible genotypes might have a significantly higher risk than those carrying non-susceptible genotypes.

Intraprostatic injection of alcohol gel for the treatment of benign prostatic hyperplasia: preliminary clinical results.

Benign prostatic hyperplasia (BPH) is one of the most common diseases ailing older men. Office-based procedures offer the advantage of being more effective than medications, while limiting the adverse effects, cost, and recovery of surgery. This study presents preliminary data on a new procedure that utilizes intraprostatic alcohol gel injection to ablate prostatic tissue. The purpose of this study is to evaluate the feasibility of using this gel as a treatment for BPH. A total of 65 patients with lower urinary tract symptoms (LUTS) due to BPH were treated with intraprostatic injections of alcohol gel. The gel is composed of 97% denatured alcohol and a patented polymer to cause viscosity. Three different methods of injection were utilized: transrectal (TR) injections (8), transurethral (TU) injections (36), and transperineal (TP) injections guided by biplaned ultrasound (21). Each method provided easy access to the center of the prostate, where a volume of gel, approximately 20-30% of the prostatic volume, was injected. Follow-up was based on changes in peak urinary flow (Qmax), IPSS scores, quality of life scores (QoL), adverse effects, and failures. Data are available at 3 and 12 months. The procedure was well tolerated with only local or no anesthesia in the TR and TP groups; the TU group received spinal anesthesia. All groups showed statistically significant (p < 0.0001) improvements in Qmax, IPSS, and QoL. The mean amount of gel injected was 8.05 ml, representing 21.56% of the prostatic volume. Qmax increased from a baseline mean of 8.50 to 12.01 ml/s at 3 months, and to 11.29 ml/s at 12 months. IPSS scores improved from a baseline mean of 21.12 to 10.00 at 3 months, and to 11.84 at 12 months. QoL scores were only available for 55 patients. QoL scores improved from a baseline of 3.93 to 1.98 at 3 months, and to 2.18 at 12 months. No extraprostatic injury or adverse effects were reported due to treatment. This preliminary study presents significant results showing that intraprostatic injection of alcohol gel could be an option for the treatment of BPH and LUTS. The viscosity of the gel allows for accurate imaging under ultrasound, no run back along the needle allowing for multiple methods of delivery, and the gel does not spread to extraprostatic tissue. This new technique could provide a simple and possibly less expensive clinic procedure for treating BPH, and warrants further study.

Ablation of stage T1/T2 prostate cancer with permanent interstitial temperature self-regulating rods.

PURPOSE: To determine if stage T(1)/T(2) prostate cancer can be treated safely and effectively with interstitial thermal ablation. PATIENTS AND METHODS: Twenty patients with biopsy-confirmed prostate cancer were enrolled in the protocol. The average age was 71.0 years, and the pretreatment prostate specific antigen (PSA) concentration ranged from 2.5 to 10.7 ng/mL and the Gleason sum from 3 to 7. An array of small biocompatible magnetic alloy rods was placed in the patients percutaneously in a procedure analogous to the placement of brachytherapy seeds. Rods were placed end-to-end and no further than 1 cm apart; rods extended to the capsule and were placed at the capsule in the rectal grove. The rods are temperature self-regulating and heat to 70 degrees C when placed in an alternating magnetic field. Each patient was treated in a coil system that supplies a uniform magnetic field throughout the patient's pelvis for a single 60-minute session. Urethral cooling and rectal temperature monitoring was performed. Serial PSA was followed, and biopsy was performed 1 year post-treatment. RESULTS: Immediately after treatment, most PSA values increased dramatically but then fell to <1.0 ng/mL within 8 weeks. After 1 year, five patients had positive biopsies; these patients had significantly lower rodimplant densities. Eight patients reported erectile dysfunction, but none reported incontinence. Other complications were minor. CONCLUSION: The data suggest that this technique is well tolerated and safe and may be useful in certain patients with T(1)/T(2) prostate cancer.

In vivo temperature mapping of prostate during treatment with TherMatrx TMx-2000 device: heat field and MRI determinations of necrotic lesions.

BACKGROUND AND PURPOSE: The effectiveness of any thermotherapy device is determined by the temperatures created and how long they are applied. Understanding the heating characteristics of a thermotherapy device is vital to its correct implementation. Interstitial temperature mapping was used to determine the heat field created within the prostate by the TherMatrx TMx-2000 transurethral microwave thermotherapy (TUMT) device. Gadolinium-enhanced MRI was used to determine the extent, type, and pattern of coagulation necrosis caused by TUMT. PATIENTS AND METHODS: Interstitial temperature mapping was performed during treatment in five patients with benign prostatic hyperplasia using 24 temperature sensors inserted through the perineum and arrayed throughout the prostate under ultrasound and X-ray guidance. Gadolinium-enhanced MRI scans were performed on all patients 1 week after treatment. RESULTS: Interstitial temperature mapping found the heat field created to peak at the urethral surface near the central part of the catheter antenna. Temperatures decreased at consistent rates of 1 to 1.4 degrees C/mm radially and 0.1 to 0.5 degrees C/mm longitudinally from the peak area. Gadolinium-enhanced MRI showed minimal dispersed necrosis and only in the periurethral area. CONCLUSIONS: The TMx-2000 can create temperatures >45 degrees C in areas 3 to 4 mm from the antenna. However, the heat pattern and protocol of the TMx-2000 produce necrosis-generating conditions only within a few millimeters of the urethra.

Glutathione peroxidase activity in cell cultures from different regions of human epididymis.

AIM: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. METHODS: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nmol/L testosterone. The effect of 1 micromol/L flutamide was also evaluated. RESULTS: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. CONCLUSION: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.

Functional characteristics of cancer stem cells and their role in drug resistance of prostate cancer.

Cancer stem cells (CSCs) have the ability to self-renew and differentiate to give rise to heterogeneous phenotype of the tumor cells. It is believed that these cells are involved in metastasis, recurrence and therapy resistance in various cancers. CSCs have been identified in prostate cancer (PCa), one of the most diagnosed malignancies in men over the world, for which chemotherapy resistance is a major problem in the treatment of castration-resistant advanced stages. Molecular signatures, gene expression and functional features have been reported for PCa CSCs. Most data come from cell lines which may not represent the actual tumor. In the present work, a CSCs enriched population obtained from PCa explants was functionally characterized and analyzed for drug resistance. Tumorsphere cultures positive for ABCG2 transporter, CD133, CD44, cytokeratins 5 and 18 (CK5 and CK18) and negatives for androgen receptor (AR) and prostate-specific antigen (PSA) showed higher clonogenic capacity, holoclone-forming ability, colony-forming capacity in soft agar and lower proliferative and apoptotic rate than control adherent cell cultures. Furthermore, exposing tumorsphere cultures to ABCG2 substrate drugs resulted in a high survival rate compared with control PCa cells. This high drug resistance was decreased using a selective inhibitor of ABCG2. According to these results, tumorspheres from PCa explants showed a functional stem phenotype and a marked drug resistance, probably mediated by high expression of the ABCG2 transporter, which might be considered as a suitable therapeutic target for CSCs.

The expression of syndecan-1 and -2 is associated with Gleason score and epithelial-mesenchymal transition markers, E-cadherin and beta-catenin, in prostate cancer.

The epithelial-mesenchymal transition (EMT) is considered a key step in tumor progression, where the invasive cancer cells change from epithelial to mesenchymal phenotype. During this process, a decrease or loss in adhesion molecules expression and an increase in migration molecules expression are observed. The aim of this work was to determine the expression and cellular distribution of syndecan-1 and -2 (migration molecules) and E-cadherin and beta-catenin (adhesion molecules) in different stages of prostate cancer progression. A quantitative immunohistochemical study of these molecules was carried out in tissue samples from benign prostatic hyperplasia and prostate carcinoma, with low and high Gleason score, obtained from biopsies archives of the Clinic Hospital of the University of Chile and Dipreca Hospital. Polyclonal specific antibodies and amplification system of estreptavidin-biotin peroxidase and diaminobenzidine were used. Syndecan-1 was uniformly expressed in basolateral membranes of normal epithelium, changing to a granular cytoplasmatic expression pattern in carcinomas. Syndecan-2 was observed mainly in a cytoplasmatic granular pattern, with high immunostaining intensity in areas of low Gleason score. E-cadherin was detected in basolateral membrane of normal epithelia showing decreased expression in high Gleason score samples. beta-Catenin was found in cell membranes of normal epithelia changing its distribution toward the nucleus and cytoplasm in carcinoma samples. We concluded that changes in expression and cell distribution of E-cadherin and beta-catenin correlated with the progression degree of prostate adenocarcinoma, suggesting a role of these molecules as markers of progression and prognosis. Furthermore, changes in the pattern expression of syndecan-1 and -2 indicate that both molecules may be involved in the EMT and tumor progression of prostate cancer.

Molecular signature of cancer stem cells isolated from prostate carcinoma and expression of stem markers in different Gleason grades and metastasis

Prostate cancer (PCa) is the most frequently diagnosed malignancy in men worldwide. Chemotherapy response is very poor and resistance to hormone-based treatments is frequent in advances stages. Recently, tumor-initiating cells or cancer stem cells (CSCs) have been identified in several cancers, including PCa. These cells are thought to be responsible for therapy resistance, relapse and metastasis. In the present work, enriched populations of CSCs were obtained using a mixed procedure that included differential clone-forming ability, sphere growing induction (prostatospheres) and magnetic-associated cell sorting (MACS). Also, stem marker expression was determined in PCa biopsies of different histological grades and metastasis samples. The signature for stem markers of the isolated CSCs was CD133+/CD44+/ABCG2+/ CD24-. Expression of stem markers (CD133, CD44, and ABCG2) was higher in medium Gleason biopsies than in lower and higher grades, and lymph-node and bone metastasis samples. These results suggest that the CSCs in PCa reach an important number in medium Gleason grades, when the tumor is still confined into the gland. At this stage, the surgical treatment is usually with curative intention. However, an important percentage of patients relapse after treatment. Number and signature of CSCs may be a prognosis factor for PCa recurrence.

Effect of leuprolide and cetrorelix on cell growth, apoptosis, and GnRH receptor expression in primary cell cultures from human prostate carcinoma.

Contradictory data have been reported regarding the effect of GnRH agonists and antagonists on cell growth and survival, using prostate cancer-derived cell lines expressing either endogenous or exogenous GnRH receptors. We addressed the issue studying the effect of leuprolide (agonist) and cetrorelix (antagonist) on cell growth, apoptosis and GnRH receptor expression using a primary cell coculture system. Also, binding characteristics of prostate GnRH receptor in this culture system are described. Epithelial and stromal cells were obtained from prostate adenocarcinoma samples and cocultured in a bicameral system. Expression of GnRH receptors was evaluated by semiquantitative RT-PCR (transcript level) and Western blot (protein level). Cell growth was estimated by MTT method and apoptosis by DNA fragmentation using COMET assay. Saturation and competition binding studies were carried out using 125I-GnRH as radioligand. GnRH receptors from cell cultures of prostate cancer exhibited a single class of binding sites with a Kd of 1.11 +/- 0.28 nM and a Bmax of 2.81 +/- 0.37 pmol/mg of membrane protein for GnRH. Leuprolide and cetrorelix showed no effect on GnRH receptor expression. Both analogues showed a significant reduction in cell growth rate and an increase in DNA-fragmented cell number. These effects were dependent on the analogue concentrations (from 5-20 ng/mL). Considering that the culture system used in this work represents more closely the in vivo conditions of tumor cells than metastatic derived cell lines, we conclude that GnRH analogues have a significant inhibitory effect on cell viability of cells expressing GnRH receptors. In addition, GnRH receptors expressed in tumor prostatic cells seem not discriminate between agonist and antagonist, both analogues activating these receptors. Also, leuprolide and cetrorelix treatments did not influence GnRH receptor expression in our culture system. These differences with pituitary receptors may be explained by differences in affinity, transduction mechanism and molecular context in prostatic tissue.

Interstitial temperature mapping during Prolieve transurethral microwave treatment: imaging reveals thermotherapy temperatures resulting in tissue necrosis and patent prostatic urethra.

OBJECTIVES: Temperature mapping of the prostate during transurethral microwave thermotherapy and imaging of the resultant zones of tissue necrosis have been previously performed using several commercial systems. This study was performed using the Prolieve Thermodilatation System, which simultaneously compresses the prostate with a 46F balloon circulating heated fluid and delivering microwave energy into the prostate. METHODS: Interstitial temperature mapping during Prolieve treatment was performed on 10 patients with benign prostatic hyperplasia using 24 temperature sensors arrayed throughout the prostate. Voiding cystourethrograms were performed on 3 additional patients treated without temperature mapping to document the patency of the prostatic urethra 1 hour after treatment. Gadolinium-enhanced magnetic resonance imaging studies were performed on all patients 1 week after treatment to determine the extent and pattern of tissue necrosis resulting from transurethral microwave thermotherapy. RESULTS: Interstitial temperature mapping found that the heating pattern generated by the Prolieve system created average peak temperatures of 51.8 degrees C an average of 7 mm away from the prostatic urethra. These temperatures were greater near the bladder neck and mid-gland than toward the prostatic apex. Subtherapeutic temperatures were seen adjacent to the urethra, consistent with the viable tissue seen on gadolinium-enhanced magnetic resonance imaging sequences. Magnetic resonance imaging also revealed necrotic zones that were consistent with sustained temperatures greater than 45 degrees C. Voiding cystourethrograms showed widely patent prostatic urethras 1 hour after treatment. CONCLUSIONS: Transurethral microwave thermotherapy with the Prolieve Thermodilatation System produced sustained therapeutic temperatures that resulted in tissue necrosis while maintaining viable tissue surrounding a temporarily dilated prostatic urethra.

Secretion of prostatic specific antigen, proliferative activity and androgen response in epithelial-stromal co-cultures from human prostate carcinoma.

We investigate the proliferative activity, prostatic specific antigen (PSA) secretion, morphology and androgen response of human prostate tumour epithelial cells co-cultured with stromal cells in a bicameral system. Stromal and epithelial cells were isolated from prostate adenocarcinoma by enzyme digestion and cultured in defined media. Immunocytochemistry for prostate carcinoma tumour antigen (PCTA-1) was performed for culture purity evaluation. Also, the morphology of the epithelial cells in co-culture was evaluated by electron microscopy. PSA was determined by microparticle enzyme immunoassay (MEIA) automatized protocol and the proliferation was evaluated by a commercial spectrophotometric kit, based on formazan salt formation. Both cell cultures showed more than 90% of purity. The epithelial cell co-cultures showed marked membrane processes and cell interdigitations. The proliferative activity of the epithelial cells was increased in presence of stromal cells. Also, PSA secretion was significantly increased and maintained for at least 14 days, whereas the androgen response for PSA secretion was evidenced only in co-culture condition. Primary co-cultures of epithelial and stromal cells from human prostate carcinoma are able to maintain, for a prolonged time, proliferative and secretory properties as well hormone response, and represent a valuable tool for cellular and molecular studies on prostate cancer.