Wine polyphenols exert antineoplasic effect on androgen resistant PC-3 cell line through the inhibition of the transcriptional activity of COX-2 promoter mediated by NF-kß.

OBJECTIVE: Mediterranean diet may play a role in the prevention of prostate cancer (PCa) development and progression. Cyclooxygenase-2 (COX-2) expression is associated with increased cellular proliferation, prevents apoptosis and favors tumor invasion. We intend to clarify whether resveratrol and other polyphenols effectively inhibit COX-2 activity and induce apoptosis in hormone-resistant PC-3 cell line. MATERIAL AND METHOD: PC-3 cells were cultured and treated with different concentrations of gallic acid, tannic acid, quercetin, and resveratrol in presence of phorbol myristate acetate (PMA; 50 µg/ml) that induces COX-2 expression. Total RNA was extracted and COX-2 expression was analyzed by relative quantification real-time PCR (ΔΔCt method). COX-2 activity was determined by PGE-2 detection using ELISA. Caspase 3/7 luminescence assay was used to disclose apoptosis. Transitory transfection with short human COX-2 (phPES2 -327/+59) and p5xNF-kß-Luc plasmids determined COX-2 promoter activity and specifically that dependant of NF-kß. RESULTS: COX-2 expression was not modified in media devoid of PMA. However, under PMA induction tannic acid (2.08 ±.21), gallic acid (2.46 ±.16), quercetin (1.78 ±.14) and resveratrol (1.15 ±.16) significantly inhibited COX-2 mRNA with respect to control (3.14 ±.07), what means a 34%, 23%, 46% and 61% reduction, respectively. The inhibition in the levels of PGE-2 followed a similar pattern. All compounds studied induced apoptosis at 48 h, although at a different rate. PMA caused a rise in activity 7.4 ±.23 times phPES2 -327/+59 and 2.0 ±.1 times p5xNF-kß-Luc at 6h compared to basal. Resveratrol suppressed these effects 17.1 ±.21 and 32.4 ±.18 times, respectively. Similarly, but to a lesser extent, the rest of evaluated polyphenols diminished PMA inductor effect on the activity of both promoters. CONCLUSIONS: Polyphenols inhibit transcriptional activity of COX-2 promoter mediated by NF-kß. This effect could explain, at least in part, the induction of apoptosis in vitro by these substances in castration resistant PCa.

Effects of resveratrol and other wine polyphenols on the proliferation, apoptosis and androgen receptor expression in LNCaP cells.

PURPOSE: To address the effect of resveratrol and other red wine polyphenols on cell proliferation, apoptosis and androgen receptor (AR) expression in human prostate cancer LNCaP cells. MATERIALS AND METHODS: LNCaP cells (5 × 102) were cultured in microtiter plate modules and treated with gallic acid, tannic acid and quercetin (1, 5 and 10 µM), rutin and morin (25, 50 and 75 µM) and resveratrol (5, 10 and 25 µM). To address the extent of proliferation at 24, 48, 72 and 96 hours, a colorimetric immunoassay method was used. An activity caspase 3/7 detection assay was used to disclose apoptosis at 24, 48 and 72 hours. AR mARN levels were determined by real time RT-PCR. RESULTS: All polyphenols studied significantly inhibited (P<.05) cell proliferation compared to control. However, there were moderate differences between them. Resveratrol was the strongest inhibitor at different times and doses. Also, caspase-3 and caspase-7 activity was significantly higher (P<.05) than control in the presence of all the compounds, but the earlier response was achieved by resveratrol. Resveratrol, quercetin and morin were the only nutrients that significantly inhibited AR mRNA expression. Again resveratrol produced the highest inhibition (90-250 times less than control), followed by morin (67-100 times) and quercetin (55-91 times). CONCLUSIONS: All polyphenols studied showed important antiproliferative effects and induced apoptosis when added to LNCaP cells culture. We confirm that resveratrol, morin and quercetin may achieve such effect through reduced expression of AR. The synergistic effects of these compounds and their potential to prevent progression of hormone-dependent prostate cancer merit further study.

The role of matrix metalloproteinase MMP-9 and TIMP-2 tissue inhibitor of metalloproteinases as serum markers of bladder cancer.

INTRODUCTION: The diagnosis and molecular staging of bladder cancer based on the detection of gelatinases mRNA (MMP-2 and MMP-9) in peripheral blood circulating and mononuclear cells have shown promising results. We analyze if the determination of the corresponding protein synthesis products makes it possible to diagnose and characterize patients with bladder cancer. MATERIAL AND METHOD: Quantification of the serum levels of MMP-2, MMP-9 and TIMP-2 in a series of 42 individuals (31 patients with bladder cancer in different stages and 11 healthy controls) using the ELISA technique was carried out. The determinations were compared between cases and controls (Mann-Whitney U) and between different groups of tumors (Mann-Whitney U or Kruskal-Wallis), according to the clinical-pathological characteristics (age, gender, T category, M category or grade). Diagnostic yield of these markers was evaluated by analysis of the ROC curves. RESULTS: There is a correlation between the determinations of MMP-2 and TIMP-2 (R=.699; P>.0001) and MMP-9 and TIMP-2 (R=.305; P=.049). Patients with bladder cancer have higher levels of MMP-9 (p<0.0001) and TIMP-2 (P=.047) than the controls. Furthermore, the MMP-9/TIMP-2 ratio is also superior in cancer patients (P<.001). Differences were not detected between cancer and controls regarding age (P=.64) or gender (P=.64). Differences were also not detected regarding MMP-2 (P=.35) or MMP-2/TIMP-2 rate (P=.45). Within the cancer patient population, the MMP-2 and MMP-9 values differ according to T category (P=.022 and P=.038, respectively) and those of the TIMP-2 according to M category (P=.036). ROC curve analysis showed that both MMP-9 and the MMP-9/TIMP-2 ratio discriminate patients with cancer and controls, with equivalent diagnostic accuracy (ABC 0.953) and cut offs of 3.93 ng/mL (S 90%; Sp 81%) and 0.053 ng/mL (S 96%; Sp 84%), respectively. CONCLUSIONS: The results obtained suggest that both serum MMP-9 and TIMP-2 would have an application in the prediction of the development and progression of bladder cancer, and a potential utility as clinical markers of the disease. Multicenter, prospective studies that confirm their preliminary results are necessary.

Lung histopathological findings in fatal pandemic influenza A (H1N1).

OBJECTIVE: To describe the lung pathological changes in influenza A (H1N1) viral pneumonia. We studied morphological changes, nitro-oxidative stress and the presence of viral proteins in lung tissue. METHODS AND PATIENTS: Light microscopy was used to examine lung tissue from 6 fatal cases of pandemic influenza A (H1N1) viral pneumonia. Fluorescence for oxidized dihydroethydium, nitrotyrosine, inducible NO synthase (NOS2) and human influenza A nucleoprotein (NP) (for analysis under confocal microscopy) was also studied in lung tissue specimens. RESULTS: Age ranged from 15 to 50 years. Three patients were women, and 5 had preexisting medical conditions. Diffuse alveolar damage (DAD) was present in 5 cases (as evidenced by hyaline membrane formation, alveolo-capillary wall thickening and PMN infiltrates), and interstitial fibrosis in one case. In the fluorescence studies there were signs of oxygen radical generation, increased NOS2 protein and protein nitration in lung tissue samples, regardless of the duration of ICU admission. Viral NP was found in lung tissue samples from three patients. Type I pneumocytes and macrophages harbored viral NP, as evidenced by confocal immunofluorescence microscopy. CONCLUSIONS: Lung tissue from patients with pandemic influenza A (H1N1) viral pneumonia shows histological findings consistent with DAD. Prolonged nitro-oxidative stress is present despite antiviral treatment. Viral proteins may remain in lung tissue for prolonged periods of time, lodged in macrophages and type I pneumocytes.

[Detection and molecular staging of bladder cancer using real-time RT-PCR for gelatinases (MMP-2, MMP-9) and TIMP-2 in peripheral blood]. / Detección y estadificación molecular del cáncer vesical mediante RT-PCR a tiempo real para gelatinasas (MMP-2, MMP-9) y TIMP-2 en sangre periférica.

INTRODUCTION: Molecular staging of bladder cancer based on the detection of mRNA of urothelial specific genes in circulating cancer cells has been inconclusive. We analyze whether real-time RT-PCR evaluation of gelatinases (MMP-9, MMP-2) and TIMP-2 in peripheral blood to diagnose and characterize patients with bladder neoplasm. MATERIAL AND METHOD: Total RNA is extracted from circulating blood cells in 42 individuals (11 healthy controls, 31 patients with bladder cancer of different stages) and real-time RT-PCR performed using specific primers for MMP-9, MMP-2, TIMP-2 and ribosomal 18S. The quantification values of mRNA are described as relative to 18S mRNA (ΔΔCt method) and the results are blindly compared with data obtained from histological diagnosis and clinical staging. RESULTS: Normalized levels of MMP-9 and MMP-2 mRNA are higher in patients with cancer than controls (1.82±0.6-fold and 2.7±0.6-fold, respectively; P<.05). Patients with metastatic disease also have increased MMP-9, MMP-2 and TIMP-2 mRNA levels (9.6±0,20-fold, 5.22±0.26-fold and 1,97±0,22-fold, respectively; P<.05). MMP-9 and MMP-2 are also associated with advanced clinical stage and grade (P<.05). A ratio between variables that increases the ability to segregate patients with Ta, T1, T2-4M0 and T2-4M1 tumours is proposed. CONCLUSIONS: Both non-invasive bladder tumor recognition and molecular staging of the disease is possible using real-time RT-PCR-based detection of gelatinases and TIMP-2 in peripheral blood. The ability to distinguish metastatic disease is higher for MMP-9 but MMP-2 discriminates better levels of tumour invasion. Further investigation in this field could yield promising results regarding molecular evaluation of bladder neoplasia.

Quantitative real-time reverse transcription: polymerase chain reaction of prostate-specific antigen (PSA) for detection of circulating prostatic cells in patients with clinically localized prostate cancer.

OBJECTIVE: To evaluate the clinical utility of using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify prostate-specific antigen (PSA) mRNA in peripheral blood samples from patients with prostate cancer as a predictor of extraprostatic extension of the disease and to assess any correlations with known predictive markers of this condition. METHODS: Immediately before radical prostatectomy, peripheral blood samples were taken from 42 men with clinically localized prostate cancer and analysed for PSA and 18S ribosomal (endogenous control) genes using real-time RT-PCR (with gene expression assays and the comparative CT-cycle threshold-method for quantifying). A total of 30 healthy male blood donors aged <50 y was taken as a control group. The relationships between PSA mRNA values, pathological and clinical features were analysed. PSA mRNA value, PSA level and biopsy Gleason score were then compared as predictors of extraprostatic extension. RESULTS: PSA gene expression was 3.73 times significantly higher in patients with clinically localized prostate cancer than in healthy men (P<0.05). There was no relationship between PSA real-time RT-PCR values and pathological stage pT2 or pT3 (P=0.5), and no association between PSA mRNA value and serum PSA level (P=0.9) or the Gleason score of the preoperative biopsy (P=0.9). CONCLUSION: There was no significant advantage in using the real-time RT-PCR assay of PSA mRNA before surgery to stage prostate cancer and to discriminate between organ-confined and extraprostatic extension.

Prostate apoptosis after doxazosin treatment in the spontaneous hypertensive rat model.

OBJECTIVE: To validate the spontaneous hypertensive rat (SHR) model for basic research into benign prostate hyperplasia (BPH), and to assess doxazosin-induced changes in prostatic structure and apoptotic status. MATERIALS AND METHODS: Four groups of rats were assessed: group 1, 15 SHRs treated with doxazosin; group 2, 14 SHRs with unilateral excision of the major pelvic ganglion; group 3, 14 untreated SHRs; and group 4, 16 intact Wistar-Kyoto (WKY) rats. The doxazosin mesylate (0.03 mg daily) was given compacted in rat food for 3 months. The prostatic ventral lobe (VL) was excised and weighed. Stereological light microscopy, multiplex reverse transcription-polymerase chain reaction of prostate caspases, and caspase-3 activity (cellular enzymatic assay) were assessed. RESULTS: There was more development of the glandular epithelium (P < 0.001) in SHR rats than in controls, which was even greater after doxazosin exposure (P = 0.027). SHR animals had higher caspase expression (P < 0.05) and activity (P = 0.008) than WKY rats, but both were reduced after doxazosin therapy (P < 0.01 and 0.028, respectively). CONCLUSIONS: This study confirmed prostate hyperplasia in the SHR model. Doxazosin exposure did not reduce the volume of glandular epithelium and contributed to protecting against caspase-induced apoptosis. The SHR model may be not a valid option to study doxazosin-induced apoptosis in BPH.

The clinical utility of the prostate specific membrane antigen reverse-transcription/polymerase chain reaction to detect circulating prostate cells: an analysis in healthy men and women.

OBJECTIVE: To evaluate the overall specificity of nested reverse transcriptase-polymerase chain reaction (RT-PCR) to detect prostate-specific membrane antigen (PSM) mRNA in peripheral blood samples of healthy donors. SUBJECTS AND METHODS: Peripheral blood samples were taken from 60 healthy blood-donors (30 men and 30 women aged < 50 years) and analysed for PSM-mRNA using nested RT-PCR (in 'hot-start' conditions and confirmed using nested EcoRI restriction enzyme). Intron-spanning primer pairs specific for human PSM were deduced from the GenBank sequence (M99487) using gene software. The outer primer pair for PSM was: fwd: 1368 5'-TCACCGGGACTCATGGGTGT-3'; reverse: 1860 5'-GCCTGAAGCAATTCCAAGTCGG-3'. Inner primer pair for PSM was: fwd: 1480 5'-AAGGAAGGGTGGAGACCTAG-3'; reverse: 5-ACTGAACTCTGGGGAAGGAC-3'. The integrity of cDNAs was checked using primer pairs specific for the housekeeping gene beta-actin. The specificity and false-positive rate were calculated assuming that the underlying prostate cancer incidence was nil. RESULTS: The first PCR was negative for all samples (100% specificity; 0% false-positive rate). The nested PCR detected 23 positive samples (23/60, 38%) with an overall specificity of 62% (false positive rate, 38%). CONCLUSION: Nested RT-PCR of PSM-mRNA in peripheral blood is highly unspecific. Its clinical utility in the management of prostate cancer must be low. Further development is needed of quantitative RT-PCR, primers that identify prostatic PSM or another prostate-specific marker gene to differentiate PSM mRNA from circulating prostate cells and from non-prostatic tissues.

Polyphenols in red wine inhibit the proliferation and induce apoptosis of LNCaP cells.

OBJECTIVE: To assess the effect of five polyphenol constituents of red wine (quercetin, morin, rutin, gallic acid and tannic acid) on the proliferation of LNCaP cells, and to quantify the extent of apoptosis with each polyphenol. MATERIALS AND METHODS: LNCaP cells (500) were cultured in microtitre plates and treated with gallic acid, tannic acid, quercetin (1, 5 and 10 micromol/L), rutin and morin (25, 50 and 75 micromol/L). A colorimetric immunoassay was then used to determine the extent of proliferation at 24, 48, 72 and 96 h, and a cell-death detection assay to assess apoptosis at 24, 48 and 72 h. RESULTS: Gallic and tannic acid (5 and 10 micromol/L), morin (50 and 75 micromol/L), quercetin (5 and 10 micromol/L) and rutin (50 and 75 micromol/L) all significantly inhibited (P<0.05) cell proliferation compared with the control. Apoptotic indexes were significantly greater (P<0.01) in the presence of gallic (5 and 10 micromol/L) and tannic acid (5 and 10 micromol/L), and rutin (75 micromol/L, P<0.05) than in the control. The apoptotic effect of morin (75 micromol/L), although significant (P<0.01), only appeared at 72 h. Conversely, while significant (P<0.05) quercetin (5 and 10 micromol/L) had a transient (first 48 h) apoptotic effect compared with the control. CONCLUSION: Quercetin, rutin, morin, gallic acid and tannic acid inhibited the growth of LNCaP cells at different concentrations, and induced apoptosis. The results provide a strong rationale for studying the in vivo effects of these compounds.

Detecting circulating prostate cells in patients with clinically localized prostate cancer: clinical implications for molecular staging.

OBJECTIVE: To evaluate the clinical utility of using the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect prostate-specific antigen (PSA) mRNA in peripheral blood samples from patients with prostate cancer, as a predictor of extraprostatic disease, and to assess any correlations with known predictive markers of this condition. PATIENTS AND METHODS: Immediately before radical prostatectomy, peripheral blood samples were taken from 25 men with clinically localized prostate cancer and analysed for PSA mRNA using RT-PCR (in 'hot-start' conditions and confirmed using ClaI restriction enzyme). The relationships between PSA mRNA positivity, pathological and clinical features were analysed; PSA mRNA positivity, PSA level and biopsy Gleason score were then compared as predictors of extraprostatic disease. RESULTS: There was no relationship between PSA mRNA positivity and pathological stage (pT2 or pT3), and no association between PSA mRNA positivity and serum PSA level, PSA density, the findings on a digital rectal examination or transrectal ultrasonography, and perineural invasion in the prostatic biopsy. However, there was a significant correlation between the Gleason score of the preoperative biopsy and PSA mRNA positivity. The best predictors of extraprostatic disease were the biopsy Gleason score and the PSA level. CONCLUSION: There was no significant advantage in using the RT-PCR assay of PSA mRNA before surgery to stage prostate cancer and to discriminate between organ-confined and extraprostatic neoplasms.

Induction of apoptosis in p53-null HL-60 cells by inhibition of lanosterol 14-alpha demethylase.

To determine the role of cholesterol deprivation in cell proliferation and, eventually, in apoptosis, HL-60 promyelocytic cells were incubated in a cholesterol-depleted medium in the presence of SKF 104976, a specific inhibitor of lanosterol 14-alpha demethylase. As expected, SKF 104976 efficiently blocked the [14C]-acetate incorporation into cholesterol, whereas it induced the accumulation of both lanosterol and, especially, dihydrolanosterol. As a consequence, cell proliferation was greatly depressed at 24 h of treatment with the drug, and clear signs of apoptosis--annexin V binding, condensed and fragmented nuclei and DNA ladder--were observed thereafter. Provided that the HL-60 cell line does not express p53, it may be concluded that apoptosis induced by cholesterol deprivation is not dependent on this tumor suppressor protein. Supplementing the incubation medium with LDL-cholesterol or pure free cholesterol, fully prevented cell growth inhibition and apoptosis induction, whereas mevalonate was ineffective. These results indicate that cholesterol plays a specific role in cell proliferation, a function that is not shared by its precursors lanosterol and dihydrolanosterol.