Christian Huidobro

The expression of syndecan-1 and -2 is associated with Gleason score and epithelial-mesenchymal transition markers, E-cadherin and β-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.

Expression of Multidrug Resistance Proteins in Prostate Cancer Is Related With Cell Sensitivity to Chemotherapeutic Drugs

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.

Gadolinium-enhanced MRI in the evaluation of minimally invasive treatments of the prostate: Correlation with histopathologic findings

OBJECTIVES To explore the use of magnetic resonance imaging (MRI) with gadolinium enhancement as a noninvasive method to image the extent of ablation after minimally invasive treatment. Minimally invasive methods for ablating prostatic tissue have emerged as a viable option in the treatment of prostate disease. As these devices enter the mainstream of patient care, imaging methods that verify the exact location, extent, and pattern of the ablation are needed. METHODS Nineteen patients with prostate cancer were evaluated. All received some type of minimally invasive treatment, post-treatment gadolinium-enhanced MRI sequences, and radical retropubic prostatectomy for histopathologic evaluation. Visual comparisons of gadolinium defects and areas of coagulation necrosis as seen on histopathologic evaluation were made by us. Volumetric and two-dimensional area measurements of the ablation lesions were also compared for correlation between the MRI and histopathologic results. RESULTS Gadolinium-enhanced MRI could be matched to histopathologic findings by visual comparison in 17 of the 19 cases. Surgically distorted histopathologic specimens and a small periurethral lesion caused 2 patients to have MRI and histopathologic results that could not be matched. Complete volumetric measurements were available for 16 of the 19 patients and correlated strongly (r = 0.924). The two-dimensional area data for all patients also showed significant correlation (r = 0.886). CONCLUSIONS Correlation with histopathologic findings showed gadolinium-enhanced MRI to be useful for determining the location, pattern, and extent of necrosis caused within the prostate by minimally invasive techniques. Gadolinium-enhanced MRI gives the urologist a useful tool to evaluate the effectiveness of new minimally invasive therapies.

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 thermal therapy in patients with localized prostate cancer: histologic analysis

OBJECTIVES To examine, by way of histologic examination, the destruction of excised prostate glands treated with thermal ablation. Thermal ablation treatment with permanently implanted temperature self-regulating rods is being used in the treatment of localized prostate cancer. METHODS Four patients with biopsy-proven prostate cancer, who had been scheduled for routine radical prostatectomy with a gland size of less than 70 g, Gleason sum of 7 or less, and prostate-specific antigen values less than 10.0 ng/mL, were implanted with 70 degrees C rods under ultrasound and fluoroscopic control. The patients were then given multiple thermal treatments. Glands were removed and histologically analyzed to access the thermal destruction. RESULTS Histologic examination revealed confluent thermal destruction within the rod array when the rods were placed end-to-end and no farther than 1 cm apart. Little necrosis was seen outside the array. To ensure the necessary destruction, the rods must be placed at the capsule, including posteriorly near the rectum. The results indicated that energy levels greater than 40 W-min/g of tissue should be used. This can be achieved by implanting 1.5 rods/g of prostate and treating the patient for 60 minutes. In 3 of the 4 patients, no residual cancer was found in the gland after thermal treatment. CONCLUSIONS Histologic examination has aided in determining the implant density and treatment time and, therefore, the necessary energy, for adequate necrosis. The technique demonstrates the ability to destroy the prostate adequately, including tissue at the capsule. This new procedure appears promising in the treatment of localized prostate cancer.

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.

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

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.

Proapoptotic effect of endocannabinoids in prostate cancer cells

In the early stages, prostate cancer is androgen- dependent; therefore, medical castration has shown significant results during the initial stages of this pathology. Despite this early effect, advanced prostate cancer is resilient to such treatment. Recent evidence shows that derivatives of Cannabis sativa and its analogs may exert a protective effect against different types of oncologic pathologies. The purpose of the present study was to detect the presence of cannabinoid receptors (CB1 and CB2) on cancer cells with a prostatic origin and to evaluate the effect of the in vitro use of synthetic analogs. In order to do this, we used a commercial cell line and primary cultures derived from prostate cancer and benign prostatic hyperplasia. The presence of the CB1 and CB2 receptors was determined by immunohistochemistry where we showed a higher expression of these receptors in later stages of the disease (samples with a high Gleason score). Later, treatments were conducted using anandamide, 2-arachidonoyl glycerol and a synthetic analog of anandamide, methanandamide. Using the MTT assay, we proved that the treatments produced a cell growth inhibitory effect on all the different prostate cancer cultures. This effect was demonstrated to be dose-dependent. The use of a specific CB1 receptor blocker (SR141716) confirmed that this effect was produced primarily from the activation of the CB1 receptor. In order to understand the MTT assay results, we determined cell cycle distribution by flow cytometry, which showed no variation at the different cell cycle stages in all the cultures after treatment. Treatment with endocannabinoids resulted in an increase in the percentage of apoptotic cells as determined by Annexin V assays and caused an increase in the levels of activated caspase-3 and a reduction in the levels of Bcl-2 confirming that the reduction in cell viability noted in the MTT assay was caused by the activation of the apoptotic pathway. Finally, we observed that endocannabinoid treatment activated the Erk pathway and at the same time, produced a decrease in the activation levels of the Akt pathway. Based on these results, we suggest that endocannabinoids may be a beneficial option for the treatment of prostate cancer that has become nonresponsive to common therapies.

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.

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.