Resistance to 2-Hydroxy-Flutamide in Prostate Cancer Cells Is Associated with the Downregulation of Phosphatidylcholine Biosynthesis and Epigenetic Modifications.

In this study, we examined the metabolic adaptations of a chemoresistant prostate cancer cell line in comparison to a sensitive cell line. We utilized prostate cancer LNCaP cells and subjected them to a stepwise increase in the antiandrogen 2-hydroxy-flutamide (FLU) concentration to generate a FLU-resistant cell line (LN-FLU). These LN-FLU cells displayed characteristics of cancer stem cells, exhibited drug resistance, and showed a significantly reduced expression of Cyclin D1, along with the overexpression of p16, pointing to a proliferation arrest. In comparing the cancer stem-like LN-FLU cells to the LNCaP cells, we observed a decrease in the expression of CTP-choline cytidylyl transferase α (CCTα), as well as a decline in choline kinase, suggesting altogether a downregulation of the phosphatidylcholine biosynthetic pathway. In addition, we found decreased levels of the protein methyl transferase PRMT2 and the upregulation of the histone deacetylase Sirtuin1 (Sirt1). Analysis of the human prostate cancer samples revealed similar results in a population with high expressions of the stem cell markers Oct4 and ABCB1A1. Our findings suggest that the adaptation of prostate cancer cells to antiandrogens could induce reprogramming into stem cells that survive in a low phosphocholine metabolism and cell cycle arrest and display drug resistance.

Homeostasis: apoptosis and cell cycle in normal and pathological prostate.

Prostatic diseases such as hyperplasia and cancer are a consequence of glandular aging due to the loss of homeostasis. Glandular homeostasis is guaranteed by the delicate balance between production and cell death. Both cell renewal and apoptosis are part of this delicate balance. We will explore the predictive capacity for biochemical progression, following prostatectomy, of some members of the Bcl-2 family and of proteins involved in cell cycle inhibition in conjunction with established classical markers. The expression of Bcl-2, Bcl-xL, Mcl-1, Bax, Bim, Bad, PUMA, Noxa, p21, p27, Rb and p53 were analyzed by immunochemistry in 86 samples of radical prostatectomy and correlated with each of the markers established clinicopathological tests using statistical tests such as Sperman, Kaplan-Meier curves, unifactorial Cox, and multifactorial. The most relevant results are: (1) Positive correlation between: p27 with clinical T stage; and PUMA with pathological T stage; (2) Negative correlation between: Bcl-2 with clinical T stage, Bcl-xL with survival, Noxa and pRb with Gleason score.Our results suggest that the expression of Bcl-2, Bcl-xL, PUMA, Noxa, p27, and Rb were related to some of the classic markers established to predict biochemical progression after prostatectomy.

PI3K pathway and Bcl-2 family. Clinicopathological features in prostate cancer.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathways and Bcl-2 family play a central role in prostate cancer (PC). The aim was to determine influence in the biochemical progression in PC. To evaluate the association between clinic pathological and immunohistochemical variables, Spearman's test was performed. Log-rank test and Kaplan-Meier curves were used for survival comparisons. To explore the correlation of the studied immunohistochemical parameters and the established prognostic variables with biochemical progression, univariate and multivariate Cox proportional Hazard regression analyses were performed. Spearman analysis showed correlation between stroma expression and tumor expression of PI3K with biochemical progression (p = .009, p = .004), respectively, and tumor immunohistochemical score with biochemical progression (p = .051). In the multivariate Cox regression model, only PI3K was retained as independent predictors of biochemical progression. In stroma expression, PI3K is (HR 0.172, 95% CI 0.065-0.452, p = .000); tumor expression, PI3K is (HR 0.087, 95% CI 0.026-0.293, p = .000), and tumor immunohistochemical score (HR 0.382, 95% CI 0.209-0.697 p = .002). Our results suggest a role for prostatic expression of PI3K was prognostic markers for PC. PI3K/AKT/mTOR and Bcl-2 family are becoming an important therapeutic target and predictive biomarkers of onset and progression of PC.

Does [-2]Pro-Prostate Specific Antigen Meet the Criteria to Justify Its Inclusion in the Clinical Decision-Making Process?

INTRODUCTION: To assess whether [-2]pro-prostate-specific antigen (p2PSA) meets the criteria to justify its inclusion in a predictive model of prostate cancer (PCa) diagnosis and in the clinical decision-making process. MATERIALS AND METHODS: A total 172 men with total prostate-specific antigen of 2-10 ng/mL underwent measurement of free PSA and p2PSA before prostate biopsy in an observational and prospective study. From these measurements, the Prostate Health Index (PHI) was calculated. Clinical and analytical predictive models were created incorporating PHI. RESULTS: Of 172 men, 72 (42%) were diagnosed with PCa, 33 (46%) of whom were found to be with high-grade disease. PHI score was the most predictive of biopsy outcomes in terms of discriminative ability (area under the curve = 0.79), with an added gain in predictive accuracy of 17%. All the models that incorporated PHI worked better in terms of calibration close to 45° on the slope. In the decision curve analysis, at a threshold probability of 40% we could prevent 82 biopsies, missing only 16 tumors and 5 high-grade tumors. CONCLUSIONS: PHI score is a more discriminant biomarker, has superior calibration and superior net benefit, and provides a higher rate of avoided biopsies; thus, it can be useful for aiding in making a more informed decision for each patient.

Development of Castration Resistant Prostate Cancer can be Predicted by a DNA Hypermethylation Profile.

PURPOSE: Detection of DNA hypermethylation has emerged as a novel molecular biomarker for prostate cancer diagnosis and evaluation of prognosis. We sought to define whether a hypermethylation profile of patients with prostate cancer on androgen deprivation would predict castrate resistant prostate cancer. MATERIALS AND METHODS: Genome-wide methylation analysis was performed using a methylation cancer panel in 10 normal prostates and 45 tumor samples from patients placed on androgen deprivation who were followed until castrate resistant disease developed. Castrate resistant disease was defined according to EAU (European Association of Urology) guideline criteria. Two pathologists reviewed the Gleason score, Ki-67 index and neuroendocrine differentiation. Hierarchical clustering analysis was performed and relationships with outcome were investigated by Cox regression and log rank analysis. RESULTS: We found 61 genes that were significantly hypermethylated in greater than 20% of tumors analyzed. Three clusters of patients were characterized by a DNA methylation profile, including 1 at risk for earlier castrate resistant disease (log rank p = 0.019) and specific mortality (log rank p = 0.002). Hypermethylation of ETV1 (HR 3.75) and ZNF215 (HR 2.89) predicted disease progression despite androgen deprivation. Hypermethylation of IRAK3 (HR 13.72), ZNF215 (HR 4.81) and SEPT9 (HR 7.64) were independent markers of prognosis. Prostate specific antigen greater than 25 ng/ml, Gleason pattern 5, Ki-67 index greater than 12% and metastasis at diagnosis also predicted a negative response to androgen deprivation. Study limitations included the retrospective design and limited number of cases. CONCLUSIONS: Epigenetic silencing of the mentioned genes could be novel molecular markers for the prognosis of advanced prostate cancer. It might predict castrate resistance during hormone deprivation and, thus, disease specific mortality. Gene hypermethylation is associated with disease progression in patients who receive hormone therapy. It could serve as a marker of the treatment response.

A DNA Hypermethylation Profile Independently Predicts Biochemical Recurrence Following Radical Prostatectomy.

PURPOSE: Detection of DNA hypermethylation is emerging as a novel molecular biomarker for different malignancies. We intend to define whether a hypermethylation profile of patients with prostate cancer (PCa) predicts biochemical recurrence (BCR) after radical prostatectomy (RP). MATERIAL AND METHODS: Genome-wide methylation analysis was performed using the GoldenGate Methylation Cancer Panel-I (Illumina, Inc.) on 10 normal prostate tissues and 58 tumor samples from patients treated by RP followed for prostate-specific antigen (PSA) failure (>0.4 ng/ml) and disease progression. Patients were classified on the basis of D'Amico criteria according to clinical staging, PSA at diagnosis and Gleason score after pathologist review. Hypermethylation status of 1505 CpGs present in the promoter region of 807 genes was studied. Hierarchical clustering analysis was performed and relationships with outcome were investigated using log-rank analysis and Cox regression model. RESULTS: We found 28 genes significantly hypermethylated in >20% of the tumors analyzed. Four clusters of patients were characterized by their DNA methylation profile, one at higher risk to develop BCR (p = 0.005). Multivariate analysis revealed patients in this cluster (HR 2.56), and high-risk patients (HR 4.34) according to D'Amico classification were independent predictors of BCR after prostatectomy. From the selected genes MT1A, ALOX12, GSTM2, APC, MYCL2 and RARB hypermethylation predicted BCR and GSTM2 (HR 3.78) and MYCL2 hypermethylation (HR 2.71) did so independently. CONCLUSION: Epigenetic silencing of GSTM2 and MYCL2 comprise novel molecular markers to predict BCR after surgery for medium- and high-risk localized PCa undergoing surgical treatment and hypermethylation of these genes could be incorporated to the clinical and pathological factors defining the patient at higher risk of PSA failure after prostatectomy. The limitation of the study is that no independent validation cohort is analysed.

A DNA hypermethylation profile reveals new potential biomarkers for prostate cancer diagnosis and prognosis.

BACKGROUND: DNA hypermethylation has emerged as a novel molecular biomarker for the evaluation of prostate cancer diagnosis and prognosis. Defining the specific gene hypermethylation profile for prostate cancer could involve groups of genes that specifically discriminate patients with indolent and aggressive tumors. METHODS: Genome-wide methylation analysis was performed on 83 tumor and 10 normal prostate samples using the GoldenGate Methylation Cancer Panel I (Illumina, Inc.). All clinical stages of disease were considered. RESULTS: We found 41 genes hypermethylated in more than 20% of the tumors analyzed (P < 0.01). Of these, we newly identified GSTM2 and PENK as being genes that are hypermethylated in prostate cancer and that were simultaneously methylated in 40.9% of the tumors analyzed. We also identified panels of genes that are more frequently methylated in tumor samples with clinico-pathological indicators of poor prognosis: a high Gleason score, elevated Ki-67, and advanced disease. Of these, we found simultaneous hypermethylation of CFTR and HTR1B to be common in patients with a high Gleason score and high Ki-67 levels; this might indicate the population at higher risk of therapeutic failure. The DNA hypermethylation profile was associated with cancer-specific mortality (log-rank test, P = 0.007) and biochemical recurrence-free survival (log-rank test, P = 0.0008). CONCLUSIONS: Our findings strongly indicate that epigenetic silencing of GSTM2 and PENK is a common event in prostate cancer that could be used as a molecular marker for prostate cancer diagnosis. In addition, simultaneous HTR1B and CFTR hypermethylation could help discriminate aggressive from indolent prostate tumors.

Do young patients with renal cell carcinoma feature a distinct outcome after surgery? A comparative analysis of patient age based on the multinational CORONA database.

PURPOSE: We analyzed the distinct clinicopathological features and prognosis of patients with renal cell carcinoma age 40 years or less compared to a reference group of patients 60 to 70 years old. MATERIALS AND METHODS: Overall 2,572 patients retrieved from a multicenter international database comprised of 6,234 patients with surgically treated renal cell carcinoma were included in this retrospective study. Clinical and histopathological features of 297 patients 40 years old or younger (4.8%) were compared to those of 2,275 patients (36.5%) 60 to 70 years old, who served as the reference group. Median followup was 59 months. The impact of young age and further parameters on disease specific mortality and all cause mortality was evaluated by multivariate Cox proportional hazards regression analyses. RESULTS: Young patients more frequently underwent nephron sparing surgery (27% vs 20%, p = 0.008) and regional lymph node dissection compared to older patients (38% vs 32%, p = 0.025). Organ confined tumor stage (81% vs 70%, p <0.001), smaller tumor diameter (4.5 vs 4.7 cm, p = 0.014) and chromophobe subtype (10% vs 4%, p <0.001) were significantly more frequent in young patients. On multivariate analysis older patients had a higher disease specific (HR 2.21, p <0.001) and all cause mortality (HR 3.05, p <0.001). The c indices for the Cox models were 0.87 and 0.78, respectively. However, integration of the variable age group did not significantly increase the predictive accuracy of the disease specific and all cause mortality models. CONCLUSIONS: Young patients with renal cell carcinoma (40 years old or younger) have significantly different frequencies of clinical and histopathological features, and a significantly lower all cause and disease specific mortality compared to patients 60 to 70 years old.

Vasoactive intestinal peptide (VIP) inhibits human renal cell carcinoma proliferation.

Clear renal cell carcinoma (cRCC) is an aggressive and fatal neoplasm. The present work was undertaken to investigate the antiproliferative potential of vasoactive intestinal peptide (VIP) exposure on non-tumoral (HK2) and tumoral (A498, cRCC) human proximal tubular epithelial cell lines. Reverse transcription and semiquantitative PCR was used at the VIP mRNA level whereas enzyme immunoanalysis was performed at the protein level. Both renal cell lines expressed VIP as well as VIP/pituitary adenylate cyclase-activating peptide (VPAC) receptors whereas only HK2 cells expressed formyl peptide receptor-like 1 (FPRL-1). Receptors were functional, as shown by VIP stimulation of adenylyl cyclase activity. Treatment with 0.1µM VIP (24h) inhibited proliferation of A498 but not HK2 cells as based on a reduction in the incorporation of [(3)H]-thymidine and BrdU (5'-Br-2'-deoxyuridine), PCNA (proliferating-cell nuclear antigen) expression and STAT3 (signal transducer and activator of transcription 3) expression and activation. VPAC(1)-receptor participation was established using JV-1-53 antagonist and siRNA transfection. Growth-inhibitory response to VIP was related to the cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP (EPAC)/phosphoinositide 3-kinase (PI3-K) signaling systems as shown by studies on adenylate cyclase stimulation, and using the EPAC-specific compound 8CPT-2Me-cAMP and specific kinase inhibitors such as H89, wortmannin and PD98059. The efficacy of VIP on the prevention of tumor progression was confirmed in vivo using xenografted athymic mouse. These actions support a potential role of this peptide and its agonists in new therapies for cRCC.

Clinical significance of both tumor and stromal expression of components of the IL-1 and TNF-α signaling pathways in prostate cancer.

IL-1 and TNF-α, the two major proinflammatory cytokines, have been involved in initiation and progression of several malignancies. They could influence the biological behavior of prostatic tumors and patient outcome, and could be useful as prognostic factors. This study evaluated the prognostic capability for biochemical progression after radical prostatectomy of expression of IL-1, TNF-α and related signaling components, in the tumor and surrounding stroma, as well as its correlation with other clinicopathological features. Expression of IL-1α, IL-1ß, IL-1Ra, IL-1RI, IL-1RII, IRAK-1, TRAF6, TNF-α, TNFRI and TRAF2 was analyzed by immunohistochemistry in radical prostatectomy samples from 93 prostate cancer patients. Spearman's test, Kaplan-Meier curves, and univariate and multivariate Cox proportional hazard regression analyses were performed. Expression of TNF-α, TNFRI, TRAF2, ILRI, IRAK-1 and TRAF6 correlated with at least one clinicopathological feature (clinical T stage, pathological T stage, preoperative serum PSA or Gleason score). Increased tumor expression of TNF-α, TNFRI and IL-1RI, and reduced tumor expression of IRAK-1 were significantly correlated with a poor prognosis in univariate analysis. Reduced stromal expression of IL-1ß and IL-1RII, and increased stromal expression of IRAK-1 were also adverse prognostic factors in univariate analysis. Remarkably, tumor IL-1ß and stromal IL-1RII and IRAK-1 remained as independent prognostic factors after adjustment for preoperative serum PSA, pathological T stage and Gleason score in multivariate Cox models. Our results suggest that prostatic expression of TNF-α, IL-1ß and related signaling proteins (TNFRI, IL-1RI, IL-1RII and IRAK-1) predicts clinical outcome in prostate cancer, and support the involvement of TNF-α and IL-1ß signaling in prostate cancer progression.

Assessing the accuracy and generalizability of the preoperative and postoperative Karakiewicz nomograms for renal cell carcinoma: results from a multicentre European and US study.

OBJECTIVE: To assess the accuracy and generalizability of the pre- and postoperative Karakiewicz nomograms for predicting cancer-specific survival (CSS) in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: This retrospective study included 3231 patients from European and US centres, who were treated by radical or partial nephrectomy for RCC between 1992 and 2010. Prognostic scores for each patient were calculated and the primary endpoint was CSS. Discriminating ability was assessed by Harrell's c-index for censored data. The 'validation by calibration' method proposed by Van Houwelingen was used for checking the calibration of covariate effects. Calibration was graphically explored. RESULTS: Local and systemic symptoms were present in 23.2% and 9.1% of the patients, respectively. The median follow-up (FU) was 49 months. At the last FU, 408 cancer-related deaths were recorded, Kaplan-Meier estimates of CSS (with 95% confidence intervals [CIs]) at 5 and 10 years were 0.86 (0.84-0.87) and 0.77 (0.75-0.80), respectively. Both nomograms discriminated well. Stratified c-indices for CSS were 0.784 (95% CI 0.753-0.814) for the preoperative nomogram, and 0.842 (95% CI 0.816-0.867) for the postoperative one, with a significant difference between the two values (P < 0.001). The covariate-based predictions on our data for both nomograms were valid. The calibration plots showed no relevant departures from ideal predictions. CONCLUSIONS: The results suggest that the postoperative Karakiewicz nomogram discriminates substantially better than the preoperative one. These nomogram-based predictions may be used as benchmark data for pretreatment and postoperative decision-making in patients at various stages of RCC.

Gender differences in clinicopathological features and survival in surgically treated patients with renal cell carcinoma: an analysis of the multicenter CORONA database.

PURPOSE: To investigate gender differences in clinicopathological features and to analyze the prognostic impact of gender in renal cell carcinoma (RCC) patients undergoing surgery. METHODS: A total of 6,234 patients (eleven centers; Europe and USA) treated by radical or partial nephrectomy were included in this retrospective study (median follow-up 59 months; IQR 30-106). Gender differences in clinicopathological parameters were assessed. Multivariable Cox regression models were applied to determine the influence of parameters on disease-specific survival (DSS) and overall survival (OS). RESULTS: A total of 3,751 patients of the study group were male patients (60.2 %), who were significantly younger at diagnosis and received more frequently NSS than women. Significantly, more often high-grade tumors and simultaneous metastasis were present in men. Whereas tumor size and pTN stages did not differ between genders, clear-cell and chromophobe RCC was diagnosed less frequently, but papillary RCC more often in men. Gender also independently influenced DSS (HR 0.75, p < 0.001) and OS (HR 0.80, p < 0.001) with a benefit for women. However, inclusion of gender in multivariable models did not significantly gain predictive accuracies (PA) for DSS (0.868-0.870, p = 0.628) and OS (0.775-0.777, p = 0.522). Furthermore, no significantly different DSS and OS rates were found in patients undergoing NSS. CONCLUSIONS: This study demonstrates important gender differences in clinicopathological features and outcome of RCC patients with improved DSS and OS for women compared to men, even if solely patients with clear-cell RCC or M0-stage are taken into evaluation. However, inclusion of gender in multivariable models does not significantly gain PA of multivariable models.

A Highly Sensitive Immunoassay for Determination of Immune Response to SARS-CoV-2 in Capillary Blood Samples.

Throughout the pandemic, serological assays have been revealed as crucial for detecting previous exposures to the virus and determining the timing of antibody maintenance after vaccination or natural infection. This study aimed to develop an optimized enzyme-linked immunosorbent assay (ELISA)-based serology, which could be used in case of reagent shortages, such as that occurred in the beginning of this health emergency. As a result, we present a high-sensitive immunoassay for the determination of IgG levels in venous serum samples, using 2 µg/mL antigen (receptor-binding domain of the spike protein S1) for coating the plate and utilizing human samples at a dilution 1:1000. This method showed non-inferiority features versus a commercial kit, is less expensive, and has a higher spectrophotometric range that allows for a better quantification of the antibody titers. The optical density values before and after heating venous serum samples at 56 °C during 30 min was quite similar, showing that heat inactivation can be used to reduce the biohazardous risks while handling samples. Furthermore, we show that finger-stick capillary blood samples can also serve as a suitable source for IgG detection, bypassing the need for serum isolation and being suitable for point-of-care application (Pearson's coefficient correlation with capillary serum was 0.95, being statistically significant).

Increase in Ischemia-Modified Albumin and Pregnancy-Associated Plasma Protein-A in COVID-19 Patients.

This study was undertaken due to the urgent need to explore reliable biomarkers for early SARS-CoV-2 infection. We performed a retrospective study analyzing the serum levels of the cardiovascular biomarkers IL-6, TNF-α, N-terminal pro-B natriuretic peptide, cardiac troponin T (cTnT), ischemia-modified albumin (IMA) and pregnancy-associated plasma protein-A (PAPP-A) in 84 patients with COVID-19.Patients were divided into three groups according to their RT-qPCR and IgG values: acute infection (n = 35), early infection (n = 25) or control subjects (n = 24). Levels of biomarkers were analyzed in patient serum samples using commercially available ELISA kits. Results showed a significant increase in IMA and PAPP-A levels in the early infected patients. Moreover, multivariate analysis and receiver operating characteristic (ROC) curve showed that IMA and PAPP-A had excellent discrimination value for the early stage of COVID-19. For IMA, the area under the ROC curve (AUC) had a value of 0.94 (95% confidence interval (CI): 0.881-0.999). Likewise, the serum level of PAPP-A was significantly higher in patients with early infection than in the control subjects (AUC = 0.801 (95% CI: 0.673-0.929)). The combined use of IMA and PAPP-A enhanced the sensitivity for total SARS-CoV-2-infected patients to 93%. These results suggest that the increased levels of PAPP-A and IMA shed light on underlying mechanisms of COVID-19 physiopathology and might be used as efficient biomarkers with high sensitivity and specificity for the early stage of COVID-19. Importantly, when monitoring pregnancy and cardiovascular diseases using PAPP-A or IMA levels, a SARS-CoV-2 infection should be discarded for proper interpretation of the results.

Regulation of HER expression and transactivation in human prostate cancer cells by a targeted cytotoxic bombesin analog (AN-215) and a bombesin antagonist (RC-3095).

Bombesin (BN) and gastrin-releasing peptide (GRP) have been shown to stimulate the growth of human prostate cancer in vivo and in vitro by mechanisms initiated by binding of the peptide to BN/GRP receptor (GRPR). GRPR is overexpressed in a variety of human cancers, including human prostatic carcinoma. This led us to evaluate the effectiveness of blocking GRPR and of chemotherapy targeted to GRPR in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cells, which exhibit different features of disease progression. Thus, we used a cytotoxic BN/GRP analog, AN-215, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to BN-like carrier peptide, and a BN/GRP receptor antagonist, RC-3095. Semiquantitative RT-PCR and Western blotting revealed that mRNA and protein levels for GRPR increased in prostate cancer cells as compared with nonneoplastic RWPE-1 cells. Immunofluorocytochemistry and Western blot assays revealed that AN-215 was the most effective analog decreasing both the expression of epidermal growth factor receptor family members and the activation of epidermal growth factor receptor and HER-2, which are associated to a poor prognosis. Furthermore, analogs targeted to BN/GRP receptors, AN-215 and RC-3095, blocked the effect of BN on cell growth in RWPE-1, LNCaP and PC-3 cells. These findings shed light on the mechanisms of action of these analogs and support the view that the use of AN-215 and RC-3095 for blocking BN/GRP receptors for targeted therapy may be of benefit for treatment of advanced prostate cancer.

Vasoactive intestinal peptide behaves as a pro-metastatic factor in human prostate cancer cells.

BACKGROUND: There is little known on the involvement of vasoactive intestinal peptide (VIP) in the metastatic cascade of human prostate cancer, that is, cell proliferation, cell-cell adhesion, extracellular-matrix degradation, and migration/invasion. Here we evaluated the expression of related biomarker proteins (cyclin D1, metalloproteinases MMP-2 and MMP-9, and E-cadherin) in human androgen-dependent (LNCaP) and independent (PC3) prostate cancer cells. METHODS: Reverse transcriptase (RT)-polymerase chain reaction (PCR), gelatin zymography, Western blotting, confocal immunofluorescence microscopy, and assays on cell proliferation, adhesion, wound-healing, migration and random homing were performed. RESULTS: VIP increased cell proliferation and cyclin D1 expression whereas it decreased cell adhesion and E-cadherin expression in LNCaP and PC3 cells. VIP enhanced the gelatinolytic activity of MMP-2 and MMP-9. Semiquantitative RT-PCR assays showed that VIP stimulated mRNA levels of these MMPs and suppressed mRNA levels of its inhibitory protein RECK. VIP promoted cell invasion and migration, and the responses were faster according to the most aggressive status in cancer progression (androgen-independence). The involvement of nuclear factor-kappaB (NF-kappaB) was demonstrated since the anti-inflammatory agent curcumin blocked VIP effects on the above biomarkers in both cell lines. CONCLUSIONS: Taken together, these results and the presence of kappaB sites on gene promoter of cyclin D1, MMPs and, possibly, E-cadherin suggest that VIP may act as a cytokine in an early metastatic stage of human prostate cancer through the NF-kappaB/MMPs-RECK/E-cadherin system. Our findings may help to define novel targets and agents with potential usefulness in prostate cancer therapy.

Vasoactive intestinal peptide induces cyclooxygenase-2 expression through nuclear factor-kappaB in human prostate cell lines Differential time-dependent responses in cancer progression.

The effect of vasoactive intestinal peptide (VIP) on cyclooxygenase-2 (COX-2) expression was analyzed in human prostate non-neoplastic (RWPE-1) as well as cancer androgen-dependent (LNCaP) and independent (PC3) cells. The three cell lines expressed VIP mRNA and VIP peptide, as measured by RT-PCR and immunochemistry, which supports an autocrine/paracrine action of VIP in the prostate gland. VIP levels were progressively higher from non-neoplastic to androgen-dependent and independent cells. Real-time RT-PCR and Western-blotting showed that VIP stimulated both COX-2 mRNA and protein expression in a faster manner as prostate cancer stage progressed (i.e. RWPE1

Transactivation of HER2 by vasoactive intestinal peptide in experimental prostate cancer: Antagonistic action of an analog of growth-hormone-releasing hormone.

Receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) are potent promoters of cell proliferation, survival, migration, adhesion and differentiation in prostate cancer cell lines. In this study, we analyzed the cross-talk between both classes of receptors through the regulation of HER2 transactivation and expression by VIP. Three growth-hormone-releasing hormone analogs endowed with antagonistic activity for VIP receptors (JV-1-51, -52, and -53) abrogated the autocrine/paracrine stimuli of VIP on androgen-independent PC3 cells in the absence or the presence of 10% fetal bovine serum. Semiquantitative and real-time quantitative RT-PCR together with Western blotting showed increased expression levels of both mRNA and proteins for HER2 and HER3 in PC3 and androgen-dependent LNCaP prostate cancer cells as compared to non-neoplastic RWPE-1 cells. VIP (100 nM) stimulated the expression levels of both HER2 and HER3 in PC3 cells in a time-dependent manner. Whereas these effects were relatively slow, VIP rapidly (0.5 min) increased HER2 tyrosine phosphorylation. This pattern of HER transactivation was blocked by H89, a protein kinase A (PKA) inhibitor, as well as by the specific VIP antagonist JV-1-53, indicating the involvement of VIP receptors and PKA activity in phosphorylated HER2 formation. These findings support the merit of further studies on the potential usefulness of VIP receptor antagonists and both HER2 antibodies and tyrosine kinase inhibitors for prostate cancer therapy.

A DNA hypermethylation profile reveals new potential biomarkers for the evaluation of prognosis in urothelial bladder cancer.

DNA hypermethylation has emerged as a molecular biomarker for the evaluation of cancer diagnosis and prognosis. We define a methylation signature of bladder cancer and evaluate whether this profile assesses prognosis of patients. Genome-wide methylation analysis was performed on 70 tumor and 10 normal bladder samples. Hypermethylation status of 1505 CpGs present in the promoter region of 807 genes was studied. Thirty-three genes were significantly hypermethylated in ≥10% of the tumors. Three clusters of patients were characterized by their DNA methylation profile, one at higher risk of dead of disease (p = 0.0012). Association between cluster distribution and stage (p = 0.02) or grade (p = 0.02) was demonstrated. Hypermethylation of JAK3 and absence of hypermethylation of EYA4, GAT6, and SOX1 were associated with low-grade non-invasive disease. On the other hand, in high-grade invasive disease hypermethylation of CSPG2, HOXA11, HOXA9, HS3ST2, SOX1, and TWIST1 was associated with muscle invasiveness. A panel of hypermethylated genes including APC, CSPG2, EPHA5, EYA4, HOXA9, IPF1, ISL1, JAK3, PITX2, SOX1, and TWIST1 predicted cancer-specific survival and SOX1 (HR = 3.46), PITX2 (HR = 4.17), CSPG2 (HR = 5.35), and JAK3 hypermethylation (HR = 0.19) did so independently. Silencing of genes by hypermethylation is a common event in bladder cancer and could be used to develop diagnostic and prognostic markers. Combined hypermethylation of SOX1, PITX2, or CSPG2 signals patients at higher risk of death from bladder cancer.

Does obesity modify prostate cancer detection in a European cohort?

INTRODUCTION: To investigate prostate-specific antigen (PSA) accuracy and digital rectal examination (DRE) accuracy in detecting prostate cancer according to body mass index (BMI) in Spanish men with an indication of the first prostate biopsy. MATERIAL AND METHODS: We reviewed the clinical and histopathological data of 1,319 patients who underwent transrectal ultrasound-guided prostate needle biopsy. The patients were categorised according to the BMI as follows: <25 kg/m2 (normal weight); 25-29.9 kg/m2 (overweight); and ≥30 kg/m2 (obese). Receiver operator characteristic curves were used to assess PSA accuracy and DRE accuracy by calculating the area under the curve. RESULTS: The obesity rate of the cohort was 14%. PSA accuracy for predicting prostate cancer in each BMI category was 0.52, 0.58 and 0.62, respectively (p = 0.01). After stratification by DRE findings, there was no difference in the performance accuracy of PSA in predicting the presence of cancer across BMI groups in abnormal DRE (p = 0.90). Serum PSA, DRE and BMI were strong predictors of prostate cancer diagnosis (odds ratio 1.07, 2.02 and 1.4, respectively; p <0.001). When the DRE was abnormal, a BMI ≥30 increased the risk of prostate cancer twice. With the addition of BMI to the model, the area under the curve of the combined PSA and DRE for diagnosing prostate cancer improved from 0.60 to 0.63. CONCLUSIONS: The predictive value of PSA in predicting prostate cancer is not poorer in the obese population and the predictive value of an abnormal DRE in cancer detection is significantly modified by the patient's BMI.