ABSTRACT
Introduction and objective: Overexpression of MMPs has been related to biochemical recurrence after radical prostatectomy. TIMP1 and TIMP2 are controllers of MMPs and the aim of this study is to evaluate the expression levels of MMPs and their regulators using immunohistochemistry in tissue microarray of localized prostate cancer (PC). Materials and Methods: Immune-expression of MMP-9, MMP-2, TIMP1, TIMP-2, MMP-14 and IL8, were analyzed by immunohistochemistry in radical prostatectomy specimens of 40 patients with localized PC who underwent surgery between September 1997 and February 2000. Protein expression was considered as categorical variables, negative or positive. The results of the immune-expression were correlated to Gleason score (GS), pathological stage (TNM), pre-operatory PSA serum levels and biochemical recurrence in a mean follow up period of 92.5 months. Results: The loss of TIMP1 immune-expression was related to biochemical recurrence. When TIMP1 was negative, 56.3% patients recurred versus 22.2% of those whose TIMP1 was positive (p=0.042). MMP-9, MMP-2, IL8 and MMP-14 were positive in the majority of PC. TIMP-2 was negative in all cases. Conclusion: Negative immune-expression of TIMP1 is correlated with biochemical recurrence in patients with PC possibly by failing to control MMP-9, an important MMP related to cancer progression.
Subject(s)
Adult , Aged , Humans , Male , Middle Aged , Matrix Metalloproteinases/analysis , Neoplasm Recurrence, Local/pathology , Prostatic Neoplasms/pathology , Tissue Inhibitor of Metalloproteinase-1/analysis , /analysis , Biomarkers, Tumor/analysis , Disease Progression , Immunohistochemistry , /analysis , Kaplan-Meier Estimate , Neoplasm Grading , Neoplasm Staging , Neoplasm Recurrence, Local/chemistry , Prostatectomy , Prostate-Specific Antigen/blood , Prostatic Neoplasms/chemistry , Prostatic Neoplasms/surgery , Statistics, NonparametricABSTRACT
Purpose To analyze a possible correlation between a miRNA expression profile and important prognostic factors for pTa urothelial carcinomas (UC), including tumor size, multiplicity and episodes of recurrence. Materials and Methods Thirty low-grade non-invasive pTa bladder UC from patients submitted to transurethral resection were studied, in a mean follow-up of 17.7 months. As controls, we used normal bladder tissue from five patients submitted to retropubic prostatectomy to treat benign prostatic hyperplasia. Extraction, cDNA and amplification were performed for 14 miRNAs (miR-100, -10a, -21, -205, -let7c, -143, -145, -221, -223, -15a, -16, -199a and -452) using specific kits, and RNU-43 and -48 were used as endogenous controls. Statistical tests were used to compare tumor size, multiplicity and episodes of recurrence with miRNAs expression profiles. Results There was a marginal correlation between multiplicity and miR-let7c over-expression. For all others miRNA no correlation between their expression and prognostic factors was found. Conclusion We did not find differences for miRNAs expression profiles associated with prognostic factors in tumor group studied. The majority of miRNAs are down-regulated, except miR-10a, over-expressed in most of cases, seeming to have increased levels in tumor with more unfavorable prognostic factors. More studies are needed in order to find a miRNA profile able to provide prognosis in pTa UC to be used in clinical practice. .
Subject(s)
Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Carcinoma/genetics , MicroRNAs/analysis , Ureteral Neoplasms/genetics , Urinary Bladder Neoplasms/genetics , Analysis of Variance , Case-Control Studies , Carcinoma/pathology , Down-Regulation , Gene Expression , Gene Expression Profiling , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Prognosis , Reference Values , Statistics, Nonparametric , Tumor Burden/genetics , Biomarkers, Tumor/analysis , Ureteral Neoplasms/pathology , Urinary Bladder Neoplasms/pathologyABSTRACT
OBJECTIVE: MicroRNAs are noncoding RNA molecules involved in the development and progression of tumors. We have found that miRNA-100 is underexpressed in metastatic prostate cancer compared to localized disease. Conversely higher levels of miR-100 are related to biochemical recurrence after surgery. This suggests that miR-100 may be a context-dependent miRNA, acting as oncogene or tumor suppressor miRNA. Our aim is to demonstrate the role of miR-100 in the control of predicted target genes in prostate cancer cell lines. METHODS: Cell lines DU145 and PC3 were transfected with miR-100, antimiR-100 and after 24 h and 48 h of exposure, qRT-PCR and western blot were performed for mTOR, FGFR3, THAP2, SMARCA5 and BAZ2A. RESULTS: There was reduction in mTOR (p = 0.025), THAP2 (p = 0.038), SMARCA5 (p = 0.001) and BAZ2A (p = 0.006) mRNA expression in DU145 cells after exposure to miR-100. In PC3 cells, mTOR expression was decreased by miR-100 (p = 0.01). There was a reduction in the expression levels of proteins encoded by studied genes, ranging from 34% to 69%. CONCLUSIONS: We demonstrate that miR-100 is a context-dependent miRNA controlling BAZ2, mTOR, FGFR3, SMARCA5 and THAP2 that might be involved in PC progression. The elucidation of the roles of miRNAs in tumors is important because they can be used as therapeutic targets in the future. .
Subject(s)
Humans , Male , MicroRNAs/physiology , Prostatic Neoplasms/genetics , Blotting, Western , Cell Line, Tumor , Disease Progression , Gene Expression Regulation, Neoplastic , Gene Targeting , MicroRNAs/pharmacology , Predictive Value of Tests , Prostatic Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
OBJECTIVE: The aim of this study is to verify the expression of proteins that are controlled by miR-let7c, 100 and 218 using immunohistochemistry in tissue microarray representative of localized and metastasized the lymph nodes and bone prostate cancer. METHODS: To verify the expression of proteins that are controlled by miR-let7c (C-MYC, BUB1, RAS) 100 (SMARCA5, RB) and 218 (LAMB3) and cell proliferation (Ki-67) we used immunohistochemistry and computerized image system ImageJ MacBiophotonics in three tissue microarrays representative of localized prostate cancer and lymph node and bone metastases. miRNA expression was evaluated by qRT-PCR using 60 paraffin blocks to construct the tissue microarray representative of localized disease. RESULTS: RAS expression was increased in localized prostate cancer and bone metastases compared to the lymph nodes (p=0.017). RB showed an increase in expression from localized prostate cancer to lymph node and bone metastasis (p=0.036). LAMB3 was highly expressed in localized and lymph node metastases (p<0.001). Cell proliferation evaluated by Ki-67 showed an increase from localized prostate cancer to metastases (p<0.001). We did not found any relationship between C-MYC (p=0.253), BUB1 (p=0.649) and SMARCA5 (p=0.315) protein expression with prognosis or tumor behavior. CONCLUSION: We found that the expression of RAS, RB, LAMB3 and Ki-67 changed in the different stages of prostate cancer. Furthermore, we confirmed the overexpression of the miRNAs let7c, 100 and 218 in localized prostate cancer but failed to show the control of protein expression by the putative controller miRNAs using immunohistochemistry. .
Subject(s)
Adult , Humans , Male , Middle Aged , Bone Neoplasms/secondary , MicroRNAs/metabolism , Neoplasm Proteins/physiology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Adenosine Triphosphatases/metabolism , Cell Adhesion Molecules/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Gene Expression Regulation, Neoplastic , Immunohistochemistry , /metabolism , Lymphatic Metastasis , MicroRNAs/genetics , MicroRNAs/physiology , Neoplasm Proteins/metabolism , Prognosis , Prostatic Neoplasms/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , /metabolism , Retinoblastoma Protein/metabolismABSTRACT
Purpose Bladder cancer (BC) is the second most common malignancy of the urinary tract, with high mortality. The knowledge of the molecular pathways associated with BC carcinogenesis is crucial to identify new diagnostic and prognostic biomarkers. MicroRNAs (miRNAs) are short non-coding RNA molecules that play important roles in the regulation of gene expression by acting directly on mRNAs. miR-145 has been considered as a tumor suppressor, which targets the c-MYC, MUC-1 and FSCN1 genes. Our aim was to evaluate the expression profile of miR-145 in low-grade non-invasive and high-grade invasive bladder urothelial carcinomas. Materials and Methods We studied 30 specimens of low-grade, non-invasive pTa and 30 of pT2/pT3 high-grade invasive UC obtained by transurethral resection or radical cystectomy, followed over a mean time of 16.1 months. Normal controls were represented by five samples of normal bladder biopsy from patients who underwent retropubic prostatectomy to treat BPH. miRNA extraction and cDNA generation were performed using commercial kits. Analysis was performed by qRT-PCR, and miR-145 expression was calculated using the 2-∆∆ct method; we used RNU-43 and RNU-48 as endogenous controls. Results miR-145 was under-expressed in 73.3% and 86.7% of pTa and pT2/pT3, respectively, with expression means of 1.61 for the former and 0.66 for the last. There were no significant differences in miR-145 expression and histological grade, tumor stage, angiolymphatic neoplastic invasion and tumor recurrence. Conclusion miR-145 is under-expressed in low-grade, non-invasive and high-grade invasive urothelial bladder carcinoma and may play an important role in the carcinogenesis pathway, being an interesting candidate diagnostic marker. .
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Carcinoma, Transitional Cell/genetics , MicroRNAs/analysis , Urinary Bladder Neoplasms/genetics , Analysis of Variance , Carcinogens/metabolism , Carcinoma, Transitional Cell/metabolism , Gene Expression , Neoplasm Grading , Neoplasm Recurrence, Local , Statistics, Nonparametric , Biomarkers, Tumor/analysis , Urinary Bladder Neoplasms/metabolismABSTRACT
OBJECTIVES: Bladder cancer represents 3% of all carcinomas in the Brazilian population and ranks second in incidence among urological tumors, after prostate cancer. The loss of p53 function is the main genetic alteration related to the development of high-grade muscle-invasive disease. Prima-1 is a small molecule that restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Our aim was to investigate the ability of Prima-1 to induce apoptosis after DNA damage in bladder cancer cell lines. METHOD: The therapeutic effect of Prima-1 was studied in two bladder cancer cell lines: T24, which is characterized by a p53 mutation, and RT4, which is the wild-type for the p53 gene. Morphological features of apoptosis induced by p53, including mitochondrial membrane potential changes and the expression of thirteen genes involved in apoptosis, were assessed by microscopic observation and quantitative real-time PCR (qRT-PCR). RESULTS: Prima-1 was able to reactivate p53 function in the T24 (p53 mt) bladder cancer cell line and promote apoptosis via the induction of Bax and Puma expression, activation of the caspase cascade and disruption of the mitochondrial membrane in a BAK-independent manner. CONCLUSION: Prima-1 is able to restore the transcriptional activity of p53. Experimental studies in vivo may be conducted to test this molecule as a new therapeutic agent for urothelial carcinomas of the bladder, which characteristically harbor p53 mutations.
Subject(s)
Humans , Apoptosis Regulatory Proteins/physiology , Carcinoma/metabolism , /genetics , /metabolism , Urinary Bladder Neoplasms/metabolism , Apoptosis Regulatory Proteins/genetics , Carcinoma/pathology , Cell Line, Tumor/metabolism , Gene Expression/genetics , Mutation/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Real-Time Polymerase Chain Reaction , /genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
OBJETIVO: Analisar os efeitos da membrana de hialuronato de sódio e carboximetilcelulose, na formação de colágeno e fibroblastos na colorrafia de ratos. MÉTODOS: Foram utilizados 48 ratos machos da linhagem Wistar, com peso entre 250 e 343g, distribuídos em dois grupos: grupo I colorrafia sem aplicação de membrana bioabsorvível e grupo II colorrafia com aplicação de membrana bioabsorvível; tendo sido divididos em subgrupos de 3, 14 e 30 dias de observação, com 8 animais em cada um dos subgrupos, todos submetidos à eutanásia após o período correspondente. RESULTADOS: Não foi observada morbimortalidade. A quantidade de colágeno no grupo I foi de 23,4 por cento, 72,1 por cento e 67,6 por cento; no grupo II 22,5 por cento, 52,5 por cento e 51,6 por cento, ambos os grupos nos 3, 14 e 30 dias, respectivamente, tendo na comparação intergrupos demonstrado que os subgrupos 14 (p=0,0013) e 30 (p=0,0587) obtiveram variância significativa, apresentando zona de colágeno maior nos animais nos quais não se usou a membrana; entretanto, em relação à quantidade de fibroblastos, o grupo I obteve 2 por cento, 13 por cento e 8 por cento; o grupo II obteve 2 por cento, 10 por cento e 8 por cento, ambos os grupos nos 3 (p=1,0), 14 (p=0,3184) e 30 (p=0,5995) dias, respectivamente, demonstrando não haver variância significativa. CONCLUSAO: A utilização da membrana bioabsorvível determinou diminuição da formação de colágeno e não alterou o número de fibroblastos na cicatrização de colorrafias, sem apresentar morbi-mortalidade, em ratos.