Overexpression of REST Represses the Epithelial-Mesenchymal Transition Process and Decreases the Aggressiveness of Prostate Cancer Cells.

The RE-1 silencing transcription factor (REST) is a repressor factor related to neuroendocrine prostate cancer (PCa) (NEPC), a poor prognostic stage mainly associated with castration-resistant PCa (CRPC). NEPC is associated with cell transdifferentiation and the epithelial-mesenchymal transition (EMT) in cells undergoing androgen deprivation therapy (ADT) and enzalutamide (ENZ). The effect of REST overexpression in the 22rv1 cell line (xenograft-derived prostate cancer) on EMT, migration, invasion, and the viability for ENZ was evaluated. EMT genes, Twist and Zeb1, and the androgen receptor (AR) were evaluated through an RT-qPCR and Western blot in nuclear and cytosolic fractions of REST-overexpressing 22rv1 cells (22rv1-REST). The migratory and invasive capacities of 22rv1-REST cells were evaluated via Transwell® assays with and without Matrigel, respectively, and their viability for enzalutamide via MTT assays. The 22rv1-REST cells showed decreased nuclear levels of Twist, Zeb1, and AR, and a decreased migration and invasion and a lower viability for ENZ compared to the control. Results were expressed as the mean + SD of three independent experiments (Mann-Whitney U test, Kruskal-Wallis, Tukey test). REST behaves like a tumor suppressor, decreasing the aggressiveness of 22rv1 cells, probably through the repression of EMT and the neuroendocrine phenotype. Furthermore, REST could represent a response marker to ENZ in PCa patients.

The Transcription Factors Zeb1 and Snail Induce Cell Malignancy and Cancer Stem Cell Phenotype in Prostate Cells, Increasing Androgen Synthesis Capacity and Therapy Resistance.

Prostate cancer (PCa) incidence has increased during the last decades, becoming one of the leading causes of death by cancer in men worldwide. During an extended period of prostate cancer, malignant cells are androgen-sensitive being testosterone the main responsible for tumor growth. Accordingly, treatments blocking production and action of testosterone are mostly used. However, during disease progression, PCa cells become androgen insensitive producing a castration-resistant stage with a worse prognosis. Overcoming castration-resistant prostate cancer (CRPC) has become a great challenge in the management of this disease. In the search for molecular pathways leading to therapy resistance, the epithelial-mesenchymal transition (EMT), and particularly the transcription factors zinc finger E-box-binding homeobox 1 (Zeb1) and zinc finger protein SNAI1 (Snail), master genes of the EMT, have shown to have pivotal roles. Also, the discovery that cancer stem cells (CSCs) can be generated de novo from their non-CSCs counterpart has led to the question whereas these EMT transcription factors could be implicated in this dynamic conversion between non-CSC and CSC. In this review, we analyze evidence supporting the idea that Zeb1 and Snail induce cell malignancy and cancer stem cell phenotype in prostate cells, increasing androgen synthesis capacity and therapy resistance.

Cancer Stemness/Epithelial-Mesenchymal Transition Axis Influences Metastasis and Castration Resistance in Prostate Cancer: Potential Therapeutic Target.

Prostate cancer (PCa) is a leading cause of cancer death in men, worldwide. Mortality is highly related to metastasis and hormone resistance, but the molecular underlying mechanisms are poorly understood. We have studied the presence and role of cancer stem cells (CSCs) and the Epithelial-Mesenchymal transition (EMT) in PCa, using both in vitro and in vivo models, thereby providing evidence that the stemness-mesenchymal axis seems to be a critical process related to relapse, metastasis and resistance. These are complex and related processes that involve a cooperative action of different cancer cell subpopulations, in which CSCs and mesenchymal cancer cells (MCCs) would be responsible for invading, colonizing pre-metastatic niches, initiating metastasis and an evading treatments response. Manipulating the stemness-EMT axis genes on the androgen receptor (AR) may shed some light on the effect of this axis on metastasis and castration resistance in PCa. It is suggested that the EMT gene SNAI2/Slug up regulates the stemness gene Sox2, and vice versa, inducing AR expression, promoting metastasis and castration resistance. This approach will provide new sight about the role of the stemness-mesenchymal axis in the metastasis and resistance mechanisms in PCa and their potential control, contributing to develop new therapeutic strategies for patients with metastatic and castration-resistant PCa.

SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvß3 and the Transcription Factor ZEB1 in Prostate Cancer Cells.

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a matricellular protein that modulates interactions between cells and their microenvironment. SPARC is expressed during extracellular matrix remodeling and is abundant in bone marrow and high-grade prostate cancer (PCa). In PCa, SPARC induces changes associated with epithelial-mesenchymal transition (EMT), enhancing migration and invasion and increasing the expression of EMT transcriptional factor Zinc finger E-box-binding homeobox 1 (ZEB1), but not Zinc finger protein SNAI1 (Snail) or Zinc finger protein SNAI2 (Slug). It is unknown whether the SPARC-induced downregulation of E-cadherin in PCa cells depends on ZEB1. Several integrins are mediators of SPARC effects in cancer cells. Because integrin signaling can induce EMT programs, we hypothesize that SPARC induces E-cadherin repression through the activation of integrins and ZEB1. Through stable knockdown and the overexpression of SPARC in PCa cells, we demonstrate that SPARC downregulates E-cadherin and increases vimentin, ZEB1, and integrin ß3 expression. Knocking down SPARC in PCa cells decreases the tyrosine-925 phosphorylation of FAK and impairs focal adhesion formation. Blocking integrin αvß3 and silencing ZEB1 revert both the SPARC-induced downregulation of E-cadherin and cell migration enhancement. We conclude that SPARC induces E-cadherin repression and enhances PCa cell migration through the integrin αvß3/ZEB1 signaling pathway.

Epidemiological and clinical description of patients with oropharyngeal cancer treated in a public oncology referral hospital in Chile.

Introduction: The incidence of squamous carcinoma of the oropharynx (OPSCC) has presented an increase worldwide, a fact that occurs along with a phenomenon of epidemiological transition, whose pathogenesis is linked to human papilloma virus (HPV) in a significant part of the cases. Published evidence at the Latin American level is scarce. The present study aims to evaluate the epidemiological and clinical characteristics of patients with oropharyngeal cancer treated in a public oncology reference centre in Chile. Methodology: A cross-sectional study was carried out. Patients with histological confirmation of OPSCC aged 18 years or older, referred to the National Cancer Institute of Chile between 2012 and 2023 were included. The association with HPV was determined by immunohistochemistry for p16. Results: 178 patients were analysed, most of them in locoregionally advanced stages involving the palatine tonsil. Seventy-seven percent were male, with a median age of 60 years. Sixty-seven percent of patients were positive for p16, with a progressive increase to 85% in the last 2 years of the study. The p16(+) patients were younger and had fewer classical risk factors. Primary treatment was radiotherapy in 94% of patients. Conclusion: The epidemiological profile of patients with OPSCC treated in a Chilean public oncology referral centre reflects the epidemiological transition observed in developed countries. This change justifies the need to adapt health policies and conduct research that considers the characteristics of this new epidemiological profile.

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.

Knockdown of ZEB1 reverses cancer stem cell properties in prostate cancer cells.

Prostate cancer (PCa) is the second most diagnosed type of cancer in men worldwide. Advanced PCa is resistant to conventional therapies and high recurrence has been associated with high rates of metastasis. Cancer stem cells (CSCs) have been proposed to be responsible for this, due to their ability of self­renewal and differentiation into other cell types. Zinc finger E­box­binding homeobox 1 (ZEB1), a transcription factor involved in the regulation of epithelial­mesenchymal transition (EMT), has been associated with the activation of several mechanisms that lead to resistance to treatment. As recent evidence has shown that CSCs may originate from non­CSCs during EMT, it was hypothesized that knocking down ZEB1 expression in PCa cell lines could revert some properties associated with CSCs. Using lentiviraltransduction, ZEB1 expression was silenced in the PCa DU145 and LNCaP cell lines. The mRNA and protein expression levels of key canonical CSC markers (Krüppel­like factor 4, SOX2, CD44 and CD133) were determined using reverse transcription­-quantitative PCR and western blot analysis, respectively. In addition, the colony forming ability of the ZEB1­knockdown cells was evaluated, and the type of colonies formed (holoclones, paraclones and meroclones) was also characterized. Finally, the ability to form prostatospheres was evaluated in vitro. It was found that in ZEB1­knockdown DU145 cells, the expression levels of CSC phenotype markers (CD44, CD133 and SOX2) were decreased compared with those in the control group. Furthermore, ZEB1­knockdown cells exhibited a lower ability to form prostatospheres and to generate colonies. In conclusion, stable silencing of ZEB1 reversed CSC properties in PCa cell lines. Since ZEB1 is associated with malignancy, therapy resistance and a CSC phenotype in PCa cell lines, targeting ZEB1 may be a key factor to eradicate CSCs and improve the prognosis of patients with advanced PCa.

Endothelin-1 induces changes in the expression levels of steroidogenic enzymes and increases androgen receptor and testosterone production in the PC3 prostate cancer cell line.

Endothelin­1 (ET­1) is involved in the regulation of steroidogenesis. Additionally, patients with castration­resistant prostate cancer (PCa) have a higher ET­1 plasma concentration than those with localized PCa and healthy individuals. The aim of the present study was to evaluate the effect of ET­1 on steroidogenesis enzymes, androgen receptor (AR) and testosterone (T) production in PCa cells. The expression levels of endothelin receptors in prostate tissue from patients with localized PCa by immunohistochemistry, and those in LNCaP and PC3 cells were determined reverse transcription­quantitative PCR (RT­qPCR) and western blotting. Furthermore, the expression levels of ET­1 were determined in LNCaP and PC3 cells by RT­qPCR and western blotting. The ET­1 receptor activation was evaluated by intracellular calcium measurement, the expression levels of AR and enzymes participating in steroidogenesis [cytochrome P450 family 11 subfamily A member 1 (CyP11A1), cytochrome P450 family 17 subfamily A member 1, aldo­keto reductase family member C2 and 3ß­hydroxysteroid dehydrogenase/isomerase 2 (3ß HSD2)] were determined by western blotting and T concentration was determined by ELISA using PC3 cells. The present results revealed higher expression levels of endothelin A receptor (ETAR) in tissues obtained from samples of patients with PCa with a low Gleason Score. No changes were identified for endothelin B receptor (ETBR). PC3 cells expressed higher levels of ET­1 and ETAR, while LNCaP cells exhibited higher expression levels of ETBR. Blocking of ETAR and endothelin B receptor decreased the expression levels of CyP11A1 and 3ß HSD2 enzymes and AR in PC3 cells, as well as T secretion. These findings suggested that ET­1 has a potential role in modulating the intratumoral steroidogenesis pathway and might have relevance as a possible therapeutic target.

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.

Cancer stem cell and mesenchymal cell cooperative actions in metastasis progression and hormone resistance in prostate cancer: Potential role of androgen and gonadotropin­releasing hormone receptors (Review).

Prostate cancer (PCa) is the leading cause of male cancer­associated mortality worldwide. Mortality is associated with metastasis and hormone resistance. Cellular, genetic and molecular mechanisms underlying metastatic progression and hormone resistance are poorly understood. Studies have investigated the local effects of gonadotropin­releasing hormone (GnRH) analogs (used for androgen deprivation treatments) and the presence of the GnRH receptor (GnRH­R) on PCa cells. Furthermore, cell subpopulations with stem­like properties, or cancer stem cells, have been isolated and characterized using a cell culture system derived from explants of human prostate tumors. In addition, the development of preclinical orthotopic models of human PCa in a nonobese diabetic/severe combined immunodeficiency mouse model of compromised immunity has enabled the establishment of a reproducible system of metastatic progression in vivo. There is increasing evidence that metastasis is a complex process involving the cooperative actions of different cancer cell subpopulations, in which cancer stem­like cells would be responsible for the final step of colonizing premetastatic niches. It has been hypothesized that PCa cells with stemness and mesenchymal signatures act cooperatively in metastatic progression and the inhibition of stemness genes, and that overexpression of androgen receptor (AR) and GnRH­R decreases the rate the metastasis and sensitizes tumors to hormone therapy. The aim of the present review is to analyze the evidence regarding this cooperative process and the possible influence of stem­like cell phenotypes, AR and GnRH­R in metastatic progression and hormone resistance. These aspects may represent an important contribution in the understanding of the mechanisms underlying metastasis and hormone resistance in PCa, and potential routes to blocking these processes, enabling the development of novel therapies that would be particularly relevant for patients with metastatic and castration­resistant PCa.

STAT3 inhibition with galiellalactone effectively targets the prostate cancer stem-like cell population.

Cancer stem cells (CSCs) are a small subpopulation of quiescent cells with the potential to differentiate into tumor cells. CSCs are involved in tumor initiation and progression and contribute to treatment failure through their intrinsic resistance to chemo- or radiotherapy, thus representing a substantial concern for cancer treatment. Prostate CSCs' activity has been shown to be regulated by the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Here we investigated the effect of galiellalactone (GL), a direct STAT3 inhibitor, on CSCs derived from prostate cancer patients, on docetaxel-resistant spheres with stem cell characteristics, on CSCs obtained from the DU145 cell line in vitro and on DU145 tumors in vivo. We found that GL significantly reduced the viability of docetaxel-resistant and patient-derived spheres. Moreover, CSCs isolated from DU145 cells were sensitive to low concentrations of GL, and the treatment with GL suppressed their viability and their ability to form colonies and spheres. STAT3 inhibition down regulated transcriptional targets of STAT3 in these cells, indicating STAT3 activity in CSCs. Our results indicate that GL can target the prostate stem cell niche in patient-derived cells, in docetaxel-resistant spheres and in an in vitro model. We conclude that GL represents a promising therapeutic approach for prostate cancer patients, as it reduces the viability of prostate cancer-therapy-resistant cells in both CSCs and non-CSC populations.

SNAIL expression correlates with the translocation of syndecan­1 intracellular domain into the nucleus in prostate cancer cell lines.

Zinc finger protein SNAI1 (SNAIL) and zinc finger protein SNAI2 (SLUG) transcription factors promote epithelial­mesenchymal transition, a process through which epithelial cells acquire a mesenchymal phenotype, increasing their migratory and invasive properties. In prostate cancer (PCa) progression, increased expression levels of SNAIL and SLUG have been described. In advanced PCa, a decrease in the cell surface proteoglycan syndecan­1 (SDC­1), which has a role in cell­to­extracellular matrix adhesion, has been observed. Notably, SDC­1 nuclear location has been observed in mesenchymal cancers. The present study aimed to determine if SNAIL and SLUG may be associated with the nuclear location of SDC­1 in PCa. To determine the location of SDC­1, antibodies against its intracellular domain (ID) or extracellular domain (ED) were used in benign prostatic hyperplasia (BPH) and PCa samples with high Gleason scores. Only ID­SDC­1 was located in the cell nuclei in advanced PCa samples, but not in the BPH samples. ED­SDC­1 was located in the cell membrane and cytoplasm, exhibiting decreased levels in PCa in comparison with those in BPH. Furthermore, LNCaP and PC3 PCa cell lines with ectopic SNAIL expression exhibited nuclear ID­SDC­1. No change was observed in the ED­SDC­1 levels, and maintained its location in the cell membrane and cytoplasm. SLUG induced no change in ID­SDC­1 location. At the protein level, an association between SNAIL and nuclear ID­SDC­1 was observed. In conclusion, the results of the present study demonstrated that nuclear ID­SDC­1 localization was associated with SNAIL expression in PCa cell lines.

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.

Regulation of cell survival by resveratrol involves inhibition of NF kappa B-regulated gene expression in prostate cancer cells.

BACKGROUND: Polyphenols have been proposed as antitumoral agents. We have shown that resveratrol (RES) induced cell cycle arrest and promoted apoptosis in prostate cancer cells by inhibition of the PI3K pathway. The RES effects on NF kappaB activity in LNCaP cells (inducible NF kappaB), and PC-3 cells (constitutive NF kappaB) are reported. METHODS: Cells were treated with 1-150 microM of RES during 36 hr. NF kappaB subcellular localization was analyzed by western blot and immunofluorescence. I kappaB alpha was evaluated by immunoprecipitation followed by Western blot. Specific DNA binding of NF kappaB was determined by EMSA assays and NF kappaB-mediated transcriptional activity by transient transfection with a luciferase gene reporter system. RESULTS: RES induced a dose-dependent cytoplasmic retention of NF kappaB mediated by I kappaB alpha in PC-3 cells but not in LNCaP. RES-induced inhibition of NF kappaB specific binding to DNA was more significant in PC-3 cells. NF kappaB-mediated transcriptional activity induced by EGF and TNFalpha were inhibited by RES in both cell lines. LY294002 mimicked RES effects on NF kappaB activity. CONCLUSION: Antiproliferative and apoptotic effects of RES on human prostate cancer cells may be mediated by the inhibition of NF kappaB activity. This mechanism seems to be associated to RES-induced PI3K inhibition. RES could have therapeutic potential for prostate cancer treatment.

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.

The transcription factor ZEB1 promotes chemoresistance in prostate cancer cell lines.

One of the factors promoting tumoral progress is the abnormal activation of the epithelial-mesenchymal transition (EMT) program which has been associated with chemoresistance in tumoral cells. The transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), a key EMT activator, has recently been related to docetaxel resistance, the main chemotherapeutic used in advanced prostate cancer treatment. The mechanisms involved in this protective effect are still unclear. In a previous work, we demonstrated that ZEB1 expression induced an EMT-like phenotype in prostate cancer cell lines. In this work, we used prostate cancer cell lines 22Rv1 and DU145 to study the effect of ZEB1 modulation on docetaxel resistance and its possible mechanisms. The results showed that ZEB1 overexpression conferred to 22Rv1 cell resistance to docetaxel while its silencing made DU145 cells more sensitive to it. Analysis of resistance markers showed no presence of ATP-binding cassette subfamily B member 1 (MDR1) and no changes in breast cancer resistance protein (BCRP) or ATP-binding cassette subfamily C member 10 (MRP7). However, a correlation between ZEB1, multidrug resistance-associated protein 1 (MRP1), and ATP-binding cassette subfamily C member 4 (MRP4) expression was observed. MRP4 inhibition, using MK571, resensitized cells with ZEB1 overexpression to docetaxel treatment. In addition, modulation of ZEB1 and subsequent change in MRP4 expression correlated with a lower apoptotic response to docetaxel, characterized by lower B-cell lymphoma 2 (Bcl2), high BCL2-associated X protein (Bax), and high active caspase 3 expression. The response to docetaxel in our model seems to be mediated mainly by activation of the apoptotic death program. Our results showed that modulation of MRP4 could be a mediator of ZEB1-related resistance to docetaxel in prostate cancer, making it a possible marker for chemotherapy response in patients who do not express MDR1.