The involvement of miR-100 in bladder urothelial carcinogenesis changing the expression levels of mRNA and proteins of genes related to cell proliferation, survival, apoptosis and chromosomal stability.

INTRODUCTION: MicroRNAs (miRNA) are small non-coding RNAs that play an important role in the control of gene expression by inhibiting protein translation or promoting messenger RNA degradation. Today, miRNAs have been shown to be involved in various physiological and pathological cellular processes, including cancer, where they can act as oncogenes or tumor suppressor genes. Recently, lowered expression of miR-100, resulting in upregulation of FGFR3, has been correlated with low-grade, non-invasive bladder urothelial cancer, as an alternative oncogenesis pathway to the typical FGFR3 gene mutation. Our aim is to analyze the role of miR-100 in bladder cancer cell lines in controlling the expression of some of its possible target genes, including FGFR3 and its relationship with proliferation, apoptosis and DNA ploidy. METHODS: The bladder cancer cell lines RT4 and T24 were transfected with pre-miR 100, anti-miR 100 and their respective controls using a lipid-based formulation. After transfection mRNA and protein levels of its supposed target genes THAP2, BAZ2A, mTOR, SMARCA5 and FGFR3 were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. Cell proliferation, apoptosis and DNA ploidy were analyzed by flow cytometry. For statistical analysis, a t-test was applied, p < 0.05 was considered significant. RESULTS: After miR-100 transfection, there was a significant reduction in the mRNA of mTOR (p = 0.006), SMARCA5 (p = 0.007) and BAZ2A (p = 0.029) in RT4, mTOR (p = 0.023) and SMARCA5 (p = 0.015) in T24. There was a reduction in the expression of all proteins, variable from 22.5% to 57.1% in both cell lines. In T24 miR-100 promoted an increase in cell proliferation and anti-miR 100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a high-grade urothelial carcinoma. CONCLUSION: miR-100 transfection reduces expression of BAZ2A, mTOR and SMARCA5 mRNA and protein in BC cell lines. miR-100 would be classified as an oncomiR in T24 cells representative of high grade urothelial carcinoma promoting increase in cell proliferation and reduction in apoptosis. The knowledge of miRNA role in tumors will allow their use as tumor markers and targets for new therapies.

Stage, grade and behavior of bladder urothelial carcinoma defined by the microRNA expression profile.

PURPOSE: We identified miRNA expression profiles in urothelial carcinoma that are associated with grade, stage, and recurrence-free and disease specific survival. MATERIALS AND METHODS: The expression of 14 miRNAs was evaluated by quantitative reverse transcriptase-polymerase chain reaction in surgical specimens from 30 patients with low grade, noninvasive (pTa) and 30 with high grade, invasive (pT2-3) urothelial carcinoma. Controls were normal bladder tissue from 5 patients who underwent surgical treatment for benign prostatic hyperplasia. Endogenous controls were RNU-43 and RNU-48. miRNA profiles were compared and Kaplan-Meier curves were constructed to analyze disease-free and disease specific survival. RESULTS: miR-100 was under expressed in 100% of low grade pTa specimens (p <0.001) and miR-10a was over expressed in 73.3% (p <0.001). miR-21 and miR-205 were over expressed in high grade pT2-3 disease (p = 0.02 and <0.001, respectively). The other miRNAs were present at levels similar to those of normal bladder tissue or under expressed in each tumor group. miR-21 over expression (greater than 1.08) was related to shorter disease-free survival in patients with low grade pTa urothelial carcinoma. Higher miR-10a levels (greater than 2.30) were associated with shorter disease-free and disease specific survival in patients with high grade pT2-3 urothelial carcinoma. CONCLUSIONS: Four miRNAs were differentially expressed in the 2 urothelial carcinoma groups. miR-100 and miR-10a showed under expression and over expression, respectively, in low grade pTa tumors. miR-21 and miR-205 were over expressed in pT2-3 disease. In addition, miR-10a and miR-21 over expression was associated with shorter disease-free and disease specific survival. miRNAs could be incorporated into the urothelial carcinoma molecular pathway. These miRNAs could also serve as new diagnostic or prognostic markers and new target drugs.

miR-21 may acts as an oncomir by targeting RECK, a matrix metalloproteinase regulator, in prostate cancer.

BACKGROUND: Prognosis of prostate cancer (PCa) is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA) mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP) inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line. MATERIALS AND METHODS: To determine if RECK is a target of miR-21 in prostate cancer we performed an in vitro assay with PCa cell line DU-145 transfected with pre-miR-21 and anti-miR-21. To determine miR-21 and RECK expression levels in PCa samples we performed quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The in vitro assays showed a decrease in expression levels of RECK after transfection with pre-miR-21, and an increase of MMP9 that is regulated by RECK compared to PCa cells treated with anti-miR-21. We defined three profiles to compare the prognostic factors. The first was characterized by miR-21 and RECK underexpression (N = 25) the second was characterized by miR-21 overexpression and RECK underexpression (N = 12), and the third was characterized by miR-21 underexpression and RECK overexpression (N = 16). From men who presented the second profile (miR-21 overexpression and RECK underexpression) 91.7% were staged pT3. For the other two groups 48.0%, and 46.7% of patients were staged pT3 (p = 0.025). CONCLUSIONS: Our results demonstrate RECK as a target of miR-21. We believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion.

Underexpression of MMP-2 and its regulators, TIMP2, MT1-MMP and IL-8, is associated with prostate cancer.

OBJECTIVE: Extracellular matrix homeostasis is strictly maintained by a coordinated balance between the expression of metalloproteinases (MMPs) and their regulators. The purpose of this study was to investigate whether MMP-2 and its specifi c regulators, TIMP-2, MT1-MMP and IL-8, are expressed in a reproducible, specific pattern and if the profiles are related to prognosis and clinical outcome of prostate cancer (PCa). MATERIALS AND METHODS: MMP-2, TIMP-2, MT1-MMP and IL-8 expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in freshly frozen malignant and benign tissue specimens collected from 79 patients with clinically localized PCa who underwent radical prostatectomies. The control group consisted of 11 patients with benign prostate hyperplasia (BPH). The expression profile of the MMP-2 and its regulators were compared using Gleason scores, pathological stage, pre-operative PSA levels and the fi nal outcome of the PCa. RESULTS: The analysis of 79 specimens of PCa revealed that MMP-2, TIMP-2, MT1-MMP and IL-8 were underexpressed at 60.0%, 72.2%, 62.0% and 65.8%, respectively, in malignant prostatic tissue in relation to BPH samples. Considering the prognostic parameters, we demonstrated that high Gleason score tumors (≥ 7) overexpressed MMP-2 (p = 0.048) and TIMP-2 (p = 0.021), compared to low Gleason score tumors (< 7). CONCLUSION: We have demonstrated that MMP-2 and its regulators are underexpressed in PCa. Alternatively, overexpression of MMP-2 and TIMP-2 was related to higher Gleason score tumors. We postulate that alterations in metalloproteinase expression may be important in the control of tissue homeostasis related to prostate carcinogenesis and tumor behavior.

An evaluation of the anti-neoplastic activity of curcumin in prostate cancer cell lines.

OBJECTIVE: The aim of our study is to investigate the anti-neoplastic effect of curcumin in prostate cancer cell lines. Specifically, we are using the LNCaP cell line and another prostate cell line developed in our laboratory, PcBra1. The PcBra1 cells were derived from a localized, obstructive prostate cancer with a Gleason score of 9 (4+5). MATERIALS AND METHODS: A prostate cancer cell line was isolated from a localized, obstructive prostate cancer with a Gleason score of 9 (4+5), and it was characterized using immunohistochemistry. After six passages, the new cell line was treated with varying doses of curcumin: 10 microM, 25 microM or 50 microM. Apoptosis was detected by flow cytometry using Annexin V FITC. For comparison, the same experiment was performed using the well-established metastatic prostate cancer cell line, LNCaP. RESULTS: Increasing concentrations of curcumin promoted more apoptosis in the PcBra1 cells. Exposure to 10 and 25 microM curcumin induced apoptosis in 31.9% and 52.2% of cells, respectively. Late apoptosis was induced in 37% of cells after treatment with 10 microM curcumin and 35% of cells with a 25 microM treatment. Necrosis accounted for less than 10% of the death in these cells at those two concentrations. When curcumin was used at 50 microM, apoptosis was observed in 64.3% of the cells. Including late apoptosis and necrosis, 98.6% of the cells died in response to 50 microM curcumin. Results with the LNCaP cells were similar although late apoptosis was the main phenomenon at 25 microM. CONCLUSION: We have shown that curcumin acts on localized prostate cancer to induce apoptosis and may therefore be an option as a future therapeutic agent.

Prima-1 induces apoptosis in bladder cancer cell lines by activating p53.

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.

Estilo de vida e perfil socioeconômico de pacientes hipertensos / Lifestyle and socioeconomic profile of hypertensive patients

Objetivo: identificar o perfil socioeconômico e estilo de vida de pacientes hipertensos cadastrados noprograma Hiperdia da unidade da Estratégia de Saúde da Família. Método: trata-se de um estudoquantitativo, descritivo, de base populacional. Foram identificados 253 hipertensos. Destes, 134 consentiramem participar (52,9%). Resultados: no perfil socioeconômico, prevaleceu o sexo feminino (57%); idade entre60 e 69 anos (33%); escolaridade de quatro a seis anos (56,25%); com situação conjugal “com parceiro” (73%);raça branca (73%) e classe social C1 (45%), com dois moradores em casa (56%) e renda per capita de meio aum salário mínimo (68%). Conclusão: Quanto ao estilo de vida, identificou-se que, quanto à prática deexercícios físicos (92%), os participantes mostraram-se sedentários. A percepção do peso foi de “um poucoacima” (43%) e, quanto ao hábito de tabagismo, 5% o possuem. Quanto ao consumo de álcool, 9% oconsomem.

Avaliação do perfil sociodemográfico e nutricional na diferença entre homens e mulheres idosos ingressantes no programa Universidade Aberta para a Maturidade / Evaluation of the sociodemographic and nutritional profile in the difference between men and women old ingressants in the University Program Open for Maturity

Objetivo: avaliar o perfil sociodemográfico e nutricional na diferença entre homens e mulheres idosos no programa Universidade Aberta para a Maturidade. Método: estudo descritivo, de abordagem quantitativa, com 67 idosos selecionados por amostragem não probabilística intencional, ingressantes em 2013-2014, por meio de um formulário estruturado. Resultados: verificou-se predominância de mulheres entre 60 a 65 anos; homens casados e mulheres viúvas; escolaridade - ensino fundamental em ambos. Dentre as variáveis antropométricas, os valores de IMC e CC apresentam-se acima das normalidades nos homens; nas variáveis bioquímicas, o aumento da glicemia está mais presente nos homens, já o colesterol e o triglicérides encontra-se com valores alterados nas mulheres. Conclusão: conhecer as características sociodemográficas, concomitante às informações antropométricas e bioquímicas, permite trabalhar o envelhecimento no sentido de prevenir as doenças crônicas não transmissíveis e acrescentar qualidade de vida ao idoso.

Intervenção nutricional sobre medidas antropométricas, percentual de gordura corporal e parâmetros bioquímicos / Nutritional intervention on anthropometric, percentage of body fat and biochemical parameters

Objetivo: Avaliar a eficácia da intervenção nutricional sobre medidas antropométricas, percentual de gordura corporal (%G) e parâmetros bioquímicos de pacientes, maiores de 18 anos, atendidos em uma Clínica de Nutrição do município de Passos, MG. Método: Trata-se de um estudo de intervenção realizado com pacientes que procuraram o Centro de Atendimento Nutricional (CAN) da Universidade Estadual de Minas Gerais, unidade de Passos, e possuíam desvios nutricionais. Como instrumento de medida, utilizou-se a anamnese alimentar ou história dietética. Analisou- se, antes e após a intervenção nutricional, peso, índice de massa corporal, %G, circunferência abdominal, triglicérides, colesterol total e glicose de jejum. Realizou-se análise estatística descritiva e o teste T Student pareado. O nível de significância adotado foi de 5%. Participaram do estudo 19 indivíduos, com média de idade de 35±17 anos, sendo 84% do sexo feminino. Resultados: Verificou-se redução significativa no %G (t=2,469; p=0,024), triglicérides (t=2,551; p=0,020) e colesterol total (t=2,526; p=0,021) após a intervenção nutricional. Conclusões: Os resultados sugerem que a intervenção nutricional contribuiu para a redução do %G, dos parâmetros bioquí- micos e para qualidade de vida dos pacientes.(AU)

Objective: To evaluate the effectiveness of nutritional intervention on anthropometric measure- ments, body fat and biochemical parameters of patients, older than 18 years-old, attended in a Nutrition Clinic in Passos (Minas Gerais). Methods: This is an intervention study of patients who sought the Service Center Nutrition at the State University of Minas Gerais, Passos unit, and had nutritional problems. The measurement instrument was used food history or diet history analyzed before and after nutritional intervention, weight, body mass index, %G, waist circumference, triglycerides, total cholesterol and fasting glucose. A descriptive statistical analysis and paired Student T test. The study included 19 subjects with a mean age of 35±17 years, 84% female. Results: It was found significant reduction in% body fat, triglycerides and total cholesterol after nutritional intervention. Conclusions: The results suggest that dietary intervention contributed to the reduction in body fat C, biochemical parameters and quality of life of patients.(AU)

Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model.

OBJECTIVE: Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer. METHODS: Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA. RESULTS: Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model. CONCLUSION: Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer.

Incidência de transmissão vertical do HIV entre gestantes soropositivas cadastradas em um serviço de referência regional / Incidencia de transmisión vertical del VIH entre las gestantes seropositivas registradas en un servicio de referencia regional / Incidence of HIV vertical transmission among HIV-positive pregnant women treated at a regional reference service

surveying and to characterizing the incidence of HIV vertical transmission, from 2004 to 2013, among pregnant women accompanied by a referral service in STD, HIV/AIDS and Viral Hepatitis in the countryside of Minas Gerais. Method: it is a quantitative descriptive study. Data were obtained from the medical records of women infected by HIV registered in the unit. For analysis, there was used the simple descriptive statistics. It was approved by the Research Ethics Committee of the Higher Education Foundation of Passos (FESP), with CAAE: 28399314.8.0000.5112. Results: of the 33 pregnancies treated at the service, 60,6% (20) knew the diagnosis before pregnancy and 39,4% (13) of them obtained it during the prenatal period. In the first group there was no vertical transmission, while in the second group, there was one case, 8%. Conclusion: it is recommended that pregnant women do the test still in the first three months of pregnancy, because late diagnosis makes difficult doing prophylaxis...

levantar e caracterizar a incidência de transmissão vertical do HIV, no período de 2004 a 2013, entre gestantes acompanhadas por serviço de referência em DST, HIV/AIDS e Hepatites Virais no interior de Minas Gerais. Método: estudo quantitativo descritivo. Os dados foram obtidos em prontuários de mulheres soropositivas para o HIV. Para análise utilizou-se a estatística descritiva simples. Aprovado pela Comissão de Ética em Pesquisa da Fundação de Ensino Superior de Passos (FESP), com CAAE: 28399314.8.0000.5112. Resultados: das 33 gestações acompanhadas no serviço, 60,6% (20) conheciam o diagnóstico antes da gravidez e 39,4% (13) o obtiveram durante o pré-natal. No primeiro grupo não houve transmissão vertical, enquanto que no segundo houve um caso, 8%. Conclusão: recomenda-se realização de teste ainda no primeiro trimestre gestacional, pois o diagnóstico tardio dificulta a profilaxia...

conocer y caracterizar la incidencia de la transmisión vertical del VIH, en el período 2004-2013, entre mujeres embarazadas acompañadas en un servicio de referencia de enfermedades de transmisión sexual, VIH/SIDA y la hepatitis viral en Minas Gerais. Método: este es un estudio cuantitativo descriptivo. Los datos se obtuvieron de los registros médicos de mujeres infectadas por el VIH registradas en la unidad. Para el análisis, se utilizó la estadística descriptiva simple. Aprobado por el Comité de Ética en Investigación de la Fundación de la Educación Superior de Passos (FESP), CAEE: 28399314.8.0000.5112. Resultados: de las 33 gestaciones atendidas en el servicio, el 60,6% (20) fueron de mujeres que conocían el diagnóstico antes del embarazo y el 39,4% (13) de mujeres que sólo lo obtuvieron durante el período prenatal. En el primer grupo no hubo ninguna transmisión vertical, mientras que en el segundo hubo un caso, 8%. Conclusión: se recomienda la prueba en el primer trimestre del embarazo, ya que el diagnóstico tardío dificulta el profilaxis...

miRNA analysis of prostate cancer by quantitative real time PCR: comparison between formalin-fixed paraffin embedded and fresh-frozen tissue.

OBJECTIVE: Micro RNA (miRNA) is a class of small noncoding RNA that plays a major role in the regulation of gene expression, which has been related to cancer behavior. The possibility of analyzing miRNA from the archives of pathology laboratories is exciting, as it allows for large retrospective studies. Formalin is the most common fixative used in the surgical pathology routine, and its promotion of nucleic acid degradation is well known. Our aim is to compare miRNA profiles from formalin-fixed paraffin embedded (FFPE) tissues with fresh-frozen prostate cancer tissues. METHODS: The expression of 14 miRNAs was determined by quantitative real time polymerase chain reaction (qRT-PCR) in 5 paired fresh-frozen and FFPE tissues, which were representative of prostate carcinoma. RESULTS: There was a very good correlation of the miRNA expression of miR-let7c and miR-32 between the fresh-frozen and FFPE tissues, with Pearson's correlation coefficients of 0.927 (P = 0.023) and 0.960 (P = 0.010), respectively. For the remaining miRNAs, the correlation was good with Spearman correlation coefficient of 0.638 (P < 0.001). CONCLUSION: Analysis of miRNAs from routinely processed and stored FFPE prostate tissue is feasible for some miRNAs using qRT-PCR. Further studies should be conducted to confirm the reliability of using stock tissues for miRNA expression determination.

Establishment and characterization of androgen-independent human prostate cancer cell lines, PcBra1, PcBra2, and PcBra3.

One of the main obstacles for understanding biological events involved in cancer is the lack of experimental models for in vitro studies especially for prostate cancer (PC). There are a limited number of PC cell lines being the majority originated from metastatic tumors mostly acquired from American Tissue Cell Culture which demands importation an expensive and bureaucratic process. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors and the research based on cell lines derived from Brazilians should be interesting. Our aim was to develop tumor cell lines from primary PC.

Establishment and characterization of human bladder cancer cell lines BexBra1, BexBra2, and BexBra4.

Bladder cancer (BC) is the fourth most common cancer in the USA. In Brazil, BC represents 3% of the total existing carcinomas in the population and represents the second highest incidence among urological tumors. The majority of bladder cancer cell lines available were derived from Caucasians and established in the seventies or eighties. Thus, neoplasia development in these cells likely occurred in environment conditions vastly different than today. In the present study, we report the establishment and characterization of three Brazilian bladder cancer cell lines (BexBra1, BexBra2, and BexBra4). These cell lines may be helpful for dissecting the genetic and epigenetic aspects that trigger the progression of BC. Moreover, the development of a Brazilian representative of the disease will allow us to investigate the potential inter-racial differences of malignancy-associated phenotypes in bladder cancer.

Prima-1 induces apoptosis in bladder cancer cell lines by activating p53

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.

Underexpression of MMP-2 and its regulators, TIMP2, MT1-MMP and IL-8, is associated with prostate cancer

OBJECTIVE: Extracellular matrix homeostasis is strictly maintained by a coordinated balance between the expression of metalloproteinases (MMPs) and their regulators. The purpose of this study was to investigate whether MMP-2 and its specific regulators, TIMP-2, MT1-MMP and IL-8, are expressed in a reproducible, specific pattern and if the profiles are related to prognosis and clinical outcome of prostate cancer (PCa). MATERIALS AND METHODS: MMP-2, TIMP-2, MT1-MMP and IL-8 expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in freshly frozen malignant and benign tissue specimens collected from 79 patients with clinically localized PCa who underwent radical prostatectomies. The control group consisted of 11 patients with benign prostate hyperplasia (BPH). The expression profile of the MMP-2 and its regulators were compared using Gleason scores, pathological stage, pre-operative PSA levels and the final outcome of the PCa. RESULTS: The analysis of 79 specimens of PCa revealed that MMP-2, TIMP-2, MT1-MMP and IL-8 were underexpressed at 60.0%, 72.2%, 62.0% and 65.8%, respectively, in malignant prostatic tissue in relation to BPH samples. Considering the prognostic parameters, we demonstrated that high Gleason score tumors (> 7) overexpressed MMP-2 (p = 0.048) and TIMP-2 (p = 0.021), compared to low Gleason score tumors (< 7). CONCLUSION: We have demonstrated that MMP-2 and its regulators are underexpressed in PCa. Alternatively, overexpression of MMP-2 and TIMP-2 was related to higher Gleason score tumors. We postulate that alterations in metalloproteinase expression may be important in the control of tissue homeostasis related to prostate carcinogenesis and tumor behavior.

Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model

OBJECTIVE: Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer. METHODS: Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA. RESULTS: Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model. CONCLUSION: Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer.

An evaluation of the anti-neoplastic activity of curcumin in prostate cancer cell lines

OBJECTIVE: The aim of our study is to investigate the anti-neoplastic effect of curcumin in prostate cancer cell lines. Specifically, we are using the LNCaP cell line and another prostate cell line developed in our laboratory, PcBra1. The PcBra1 cells were derived from a localized, obstructive prostate cancer with a Gleason score of 9 (4+5). MATERIAL AND METHODS: A prostate cancer cell line was isolated from a localized, obstructive prostate cancer with a Gleason score of 9 (4+5), and it was characterized using immunohistochemistry. After six passages, the new cell line was treated with varying doses of curcumin: 10 µM, 25 µM or 50 µM. Apoptosis was detected by flow cytometry using Annexin V FITC. For comparison, the same experiment was performed using the well-established metastatic prostate cancer cell line, LNCaP. RESULTS: Increasing concentrations of curcumin promoted more apoptosis in the PcBra1 cells. Exposure to 10 and 25 µM curcumin induced apoptosis in 31.9 percent and 52.2 percent of cells, respectively. Late apoptosis was induced in 37 percent of cells after treatment with 10 µM curcumin and 35 percent of cells with a 25 µM treatment. Necrosis accounted for less than 10 percent of the death in these cells at those two concentrations. When curcumin was used at 50 µM, apoptosis was observed in 64.3 percent of the cells. Including late apoptosis and necrosis, 98.6 percent of the cells died in response to 50 µM curcumin. Results with the LNCaP cells were similar although late apoptosis was the main phenomenon at 25 µM. CONCLUSION: We have shown that curcumin acts on localized prostate cancer to induce apoptosis and may therefore be an option as a future therapeutic agent.

Análise do efeito do Prima-1 na expressão dos genes envolvidos na morte celular programada em linhagens de câncer de bexiga / Analysis of the effect of Prima-1 in the expression of genes involved in programmed cell death in bladder cancer cell lines

Introdução: O carcinoma urotelial de bexiga (CUB) é o segundo tumor mais frequente do trato urinário. A perda da função do p53 é a alteração mais conhecida do carcinoma urotelial de alto grau invasivo. Prima-1 é uma pequena molécula, a qual restaura a função do p53 mutado promovendo a morte celular em vários tipos celulares. O objetivo do nosso estudo foi analisar o efeito do Prima-1 na indução da apoptose mediada por p53 após indução do dano ao DNA em linhagens de CUB. Material e métodos: O mecanismo de ação do Prima-1 foi avaliado em duas linhagens celulares de câncer de bexiga, T24 que tem como característica a mutação do p53 e RT4 que possui p53 intacto. Características morfológicas da apoptose, alterações no potencial de membrana mitocondrial e análise da expressão de treze genes envolvidos na apoptose mediada por p53 foram avaliados através de observação microscópica e reação em cadeia da polimerase em tempo real quantitativa (qRT-PCR). Resultados: Prima-1 foi capaz de reativar a função da P53 na linhagem de câncer de bexiga com p53 mt, promovendo a apoptose através da expressão de Bax e Puma, ativação da cascata das caspases e ruptura da membrana mitocondrial, independente de Bax, na linhagem celular T24 (p53 mt). Conclusão: Prima-1 foi capaz de restaurar a atividade transcricional de p53. Estudos experimentais in vivo poderiam ser conduzidos no intuito de testar essa molécula como um novo agente terapêutico do CUB de alto grau, invasivo, que apresenta caracteristicamente mutação de p53...

Introduction: Urothelial carcinoma of the bladder is the second most common tumor of the urinary tract. 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 cell death in various cancer types. Our aim is to investigate the ability of Prima-1 to induce apoptosis after DNA damage in BC cancer cell lines. Material and Methods: The therapeutic effect of Prima-1 was studied in two BC cell lines, T24, characterized by p53 mutation, and RT4, with no mutation in the p53 gene. Morphological features of apoptosis, mitochondrial membrane potential changes and expression of thirteen genes involved in p53-induced apoptosis were assessed by microscopic observation and quantitative real-time PCR (qRT-PCR) Results: Prima-1 is able to reactivate P53 function in p53-mutated bladder cancer cell line promote apoptosis through the induction of Bax and Puma expression, activating the caspase cascade and disruption of mitochondrial membrane, independent of Bak, in T24 cell line (p53 mt). Conclusion: Prima-1 is able to restore the transcriptional activity of p53. Experimental studies in vivo could be conducted in order to test this molecule as a new therapeutic agent of the urothelial carcinoma of the bladder, which characteristically presents p53 mutation...