archives@tulane.edu / Vascular endothelial growth factor A (VEGFA) is a key contributor to the formation of new blood vessels and angiogenesis is commonly seen in wound healing, cancer, and inflammatory diseases. However, whether interleukin-17 (IL-17) can induce the expression of VEGFA in prostate cancer cells remains unknown. In this study, Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) analysis validated that expression of VEGFA in human prostate cancer LNCaP cells under IL-17 treatment was significantly higher than the untreated control group. The conditioned culture medium (CM) of LNCaP cells treated with IL-17 increased tube number, tube nodes, and tube length formed by human umbilical vein endothelial cells (HUVEC) in tube formation assays, compared with the control CM without IL-17 treatment. Collectively, these findings reveal that the expression of VEGFA is induced by IL-17 in LNCaP prostate cancer cells, which leads to increased angiogenesis of HUVEC cells. This study suggests that expression of VEGFA may be up-regulated by IL-17 in prostate cancer to enhance tumor angiogenesis. / 1 / Benyu Li

Orientador: Renato Atílio Jorge / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-17T02:06:37Z (GMT). No. of bitstreams: 1 Alves_JulianaMachadodaSilveira_M.pdf: 2543897 bytes, checksum: cfe682f5663f70da68e8234395efa7bc (MD5) Previous issue date: 2010 / Resumo: As porfirinas 5,10,15,20(3-hidroxifenil)porfirina (m-THPP), 5-hexil-10,20-bis(3hidrofenil)porfirina (hex-m-bisHPP) e 5-hexil-10,15,20-tris(3-hidroxifenil)porfirina (hex-m-triHPP), têm o mesmo rendimento quântico de oxigênio singleto e foram encapsuladas em nanopartículas (NPs) poli(láctico-co-glicólico) (PLGA) 50:50, preparadas através do método de emulsão e evaporação. As NPs obtidas têm diâmetros médios na faixa de submicrometros (<240 nm) e pequena dispersividade. O potencial zeta medido mostrou uma pequena variação (de -21.6 mV a -19.3 mV). A porcentagem de encapsulação das porfirinas obtidas foram: 93 ± 2; 97 ± 2 and 69 ± 1 para m-THPP, hex-m-bisHPP e hex-m-triHPP respectivamente. A eficácia fotodinâmica e a internalização das porfirinas foi investigada a 37oC utilizando células humanas cancerígenas de próstata (LNCaP). Após 2 horas de incubação com as NPs contendo a porfirina as porcentagens de internalização celular foram iguais para todas as porfirinas. As três porfirinas causam morte celular e dão a mesma viabilidade quando se varia o tempo de incubação (30-120 min), concentração da porfirina (2,5 a 7,5 mmol L) e dose de luz incidente (33 a 99 J cm). Análise das células por microscopia confocal mostrou que as porfirinas encapsuladas nas NPs foram localizadas no citoplasma, sempre na região perinuclear. Estes resultados mostram que porfirinas com estruturas semelhantes e anfifilicidades diferentes, com igual rendimento quântico de rendimento singleto e internalizadas nas células em concentrações iguais, tem a mesma eficácia fotodinâmica / Abstract: Porphyrins (5,10,15,20-tetra(3-hydroxyphenyl)porphyrin (m-THPP), 5-hexyl-10,20-bis(3-hydroxyphenyl) porphyrin (hex-m-bisHPP) and 5-hexyl-10,15,20-tris(3-hydroxyphenyl)porphyrin (hex-m-triHPP)) with different amphiphicities and equal singlet oxygen quantum yield in ethanol, were encapsulated into 50:50 poly(lactide-co-glycolide,) (PLGA) nanoparticles (NPs) prepared by the emulsion/evaporation technique. The NPs obtained had submicron average diameters (<240 nm) with low polydispersity. The zeta potential measurements showed slight variations in negativity (from -21.6 mV to -19.3 mV). Particle recovery (%) was determined with results of 93 ± 2; 97 ± 2 and 69 ± 1 for m-THPP, hex-m-bisHPP and hex-m-triHPP porphiryns, respectively. The photodynamic efficacy of the porphyrin-loaded nanoparticles and their cellular uptake at 37 oC was investigated with LNCaP prostate tumour cells. After 2 h incubation with porphyrin-loaded nanoparticles the percents of intracellular uptake were the same for all porphyrins. The three porphyrins cause cell death and gave the same cell viability with variations of incubation time (30¿120 min), drug concentration (2.5 to 7.5 mmol L) and incident light dose (33 to 99 J cm). Confocal laser scanning microscopy data showed that the porphyrin-loaded nanoparticles, were localized in the cells, always in the perinuclear region. These results show that porphyrins with similar structures and equal singlet oxygen quantum yields and internalized in equal concentrations in the cells, but different amphiphilicities, have equal photodynamic efficacy / Mestrado / Físico-Química / Mestre em Química

The LNCaP cell line is a versatile and useful model that is suitable for the study of human prostate cancer in vitro. The elevation of LNCaP intracellular cAMP levels through the addition of membrane permeable cAMP analogues, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal transduction pathway can induce reversible neuroendocrine differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights for the molecular mechanisms governing neuroendocrine differentiation, early detection of prostate cancer, and/or potential targets for gene therapy. In this study, differential display PCR was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and cloned. DNA sequencing and database comparisons were performed. Among the differentially expressed genes, the human ribosomal S3a gene was identified as down regulated in response to LNCaP differentiation. In order to better ascertain the mechanism by which HRS3a gene expression is decreased during differentiation, the promoter region for this gene was analyzed. Electrophoretic mobility shift assay, antibody supershift assays, site-directed mutagenesis, and luciferase reporter gene analysis were employed to authenticate the roles of several transcription factors in the regulation of the HRS3a gene. Two cyclic AMP response elements, a Sp1 element and a GA-binding protein element, were involved in the regulation of HRS3a gene expression. In order to ascertain the effect of HRS3a down regulation in LNCaP cells, antisense phosphorothioate oligonucleotides were designed to inhibit HRS3a gene expression. Treatment of LNCaP cells with antisense HRS3a oligonucleotides did not influence cAMP induced neuroendocrine differentiation but antisense treatment did result in a decrease in LNCaP cell growth. In addition, it was determined that morphological changes associated with cAMP induced differentiation of LNCaP cells from the epithelial to the neuroendocrine state may not require alterations in gene expression nor the expression of novel proteins. / Ph. D.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2016-09-22T19:02:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2016-09-29T19:20:07Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Made available in DSpace on 2016-09-29T19:20:08Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) Previous issue date: 2016-09 / O câncer é apontado como a segunda maior causa de morte em todo o mundo, com previsão de em breve ser tornar a primeira. O câncer de próstata está entre os 5 tipos de câncer mais diagnosticados em homens, sendo que o câncer hepático está em segundo lugar em taxa de mortalidade entre homens e mulheres. Indícios apontam para uma ativação das vias da inflamação associados a uma inibição das vias de morte celular no processo de carcinogênese. A regulação destas vias torna-se alvo importante e complementar no controle do câncer, sendo estimulada a busca de biomoléculas com este potencial. As plantas são importante fonte de descoberta de novas biomoléculas com ampla utilização para o tratamento de diversas patologias. A família Myrtaceae possui diversas espécies que são apontadas como fortes candidatos em potencial nesta busca, incluindo as do gênero Myrciaria. A espécie Myrciaria plinioides não possui estudos referentes suas propriedades terapêuticas ou a atuação em vias de sinalização envolvidas na inflamação ou na carcinogênese. Neste contexto, este estudo teve por objetivo avaliar a atividade do extrato etanólico de M. plinioides em células de carcinoma hepatocelular (HepG2) e próstata (LNCaP) , através da análise de expressão dos marcadores p38-α, pp38-α, NF-κB e caspase-3, envolvidos na carcinogênese, e o efeito sobre a viabilidade celular através do método de MTT. A viabilidade das células foi alterada significativamente, em ambas as linhagens celulares quando tratadas com o extrato etanólico. A análise da expressão proteica demonstra significativa inibição da expressão de p38-α e caspase-3 nas células LNCaP, quando tratadas com extrato etanólico de M. plinioides seguido de LPS. Em células HepG2, somente houve alteração na expressão da caspase-3 na concentração de 200 μg/mL, com ou sem adição de LPS após tratamento com extrato. Os resultados deste estudo demonstraram redução da viabilidade celular nas duas linhagens tumorais, expressão diferenciada de proteínas envolvidas em apoptose, o que leva a indícios da ativação de mecanismos distintos pelo extrato em cada tipo celular. Estudos futuros para averiguar o mecanismo celular e a indução de morte em células tumorais de câncer de próstata e de fígado podem contribuir para a identificação e elucidação de novas biomoléculas com potencial antitumoral. / Cancer is touted as the second leading cause of death worldwide, forecast to soon be making the first. Prostate cancer is among the five most cancers diagnosed in men, and liver cancer is second in mortality between men and women. Evidence points to the activation of pathways of inflammation associated with an inhibition of cell death pathways in carcinogenesis. The regulation of these pathways becomes important and complementary target in cancer control, and stimulated the search for biomolecules with this potential. The plants are important source of discovery of new biomolecules with wide use for the treatment of various diseases. The Myrtaceae family has many species that are identified as potential candidates strong in this search, including the Myrciaria genre. The species Myrciaria plinioides not have studies on its therapeutic properties or performance in signaling pathways involved in inflammation or carcinogenesis. In this context, this study aimed to evaluate the activity of the ethanol extract of M. plinioides in hepatocellular carcinoma cells (HepG2) and prostate (LNCaP) by expression analysis of p38-α markers, PP38-α, NF-kB and caspase-3, involved in carcinogenesis, and the effect on cell viability by the MTT method. The viability of cells was significantly altered in both cell lines when treated with ethanolic extract. Protein expression analysis demonstrates significant inhibition of p38-α expression and caspase-3 in LNCaP cells, when treated with ethanolic extract of M. plinioides followed by LPS. In HepG2 cells there was only a change in the expression of caspase-3 at a concentration of 200 / ml, with or without addition of LPS after treatment with extract. The results showed reduction of cell viability in both tumor lines, differential expression of proteins involved in apoptosis, leading to evidence of activation by distinct mechanisms in each extract cell type. Further studies to investigate the cellular mechanism, and induction of death in tumor cells of prostate and liver cancer may contribute to the identification and elucidation of new biomolecules with antitumor potential.

Die Cathepsine B, H und L sind lysosomale Enzyme, die zur Gruppe der Cysteinproteasen gehören. Erhöhte Aktivitäten dieser Proteasen fand man in Gewebeproben verschiedener Tumore, vermutlich Hinweis auf eine Beteiligung an Invasion und Metastasierung. Unter physiologischen Bedingungen werden die Cathepsine von endogenen Inhibitoren kontrolliert, eine Verminderung dieser Cysteinprotease-Inhibitoren (CPI) würde die proteolytische Dysbalance verstärken und zur Ausbreitung des malignen Prozesses beitragen. Für das Prostatakarzinom gab es bisher keine Untersuchungen. Die Aktivität der Cathepsine wurde mit Hilfe spezifischer Substrate bestimmt, die zur Bildung fluorogener Produkte führten. Zur Bestimmung der inhibitorischen Aktivität der CPI wurden die Proben nach einer Hitzeaktivierung gegen reines Cathepsin B getestet. Untersucht wurden Gewebeproben verschiedener Patientengruppen, Primärzellkulturen, die aus normalem und maligne veränderten Prostatagewebe angezüchtet wurden und vergleichend dazu die drei immortalisierten Zellinien LNCaP, PC3 und DU145. Die Ergebnisse der Gewebeproben zeigten höhere Aktivitäten der Cathepsine B und L im nichterkrankten Gewebe, nicht wie erwartet im Tumorgewebe der Prostata. Hingegen waren bei den Primärzellkulturen alle drei Cathepsine und der Quotient Cathepsine/CPI in den Tumorproben erhöht. Die immortalisierten Zellinien zeigten die gleiche Verteilung bei allen Cathepsinen, DU145 mit der höchsten Aktivität, gefolgt von LNCaP und PC3. Anhand der Ergebnisse schlußfolgern wir, daß die Untersuchung von Gewebe-proben der Prostata hinsichtlich der Beteiligung der Cathepsine am Tumor-geschehen keine eindeutigen Erkenntnisse erbringt. Dies ist vermutlich auf die Heterogenität des Gewebes zurückzuführen, das nicht nur epitheliale, sondern auch stromale Zellen enthält. Die aus dem Gewebe angezüchteten Primär-zellkulturen scheinen ein genaueres Bild des Verhältnisses von Cathepsinen und den Inhibitoren zu geben und sind unserer Meinung nach den Bestimmungen in Gewebeproben vorzuziehen. / The cathepsins B, H and L (CB, CH, CL) are lysosomal proteolytic enzymes belonging to the cysteine protease family. Elevated cathepsin levels and decreased concentration of their endogenous inhibitors have been demonstrated in a variety of tumors, suggesting a contribution to invasion and metastasis. The situation for prostate cancer was so far unknown. Using fluorimetric assays, catalytic activities of the cathepsins B, H, L were measured in prostatic tissue samples obtained from different groups of patients, in primary cell cultures established from human prostate and in the immortalized cell lines LNCaP, PC3 and DU145. Inhibitory activities of cysteine protease inhibitors (CPI) were tested against purified cathepsin B. Comparing matched pairs of normal and cancerous tissue samples from the prostate, significantly decreased levels of CB and CL were found in malignant samples. In contrast, primary cell cultures from malignant tissue showed elevated levels of all three cathepsins and increased ratios of cathepsins to CPI when compared to cell cultures from non-malignant prostate. The permanent cell lines showed a similiar distribution of cathepsin levels, DU145 with the highest activity, followed by LNCaP and PC3. These results suggest that elevated cathepsin activities and increased ratios of cathepsins to CPI in malignant cell cultures compared to non-malignant samples may be an indication for a cellular proteolytic imbalance in prostatic cancer cells. Regarding different results, determinations in primary cell cultures should be preferred to tissue samples.

No description available.

L’HBP et le cancer de la prostate constituent les deux maladies prostatiques les plus répandues chez l’homme âgé. La compréhension de leur étiologie et de leur pathogenèse est nécessaire afin de permettre la prévention mais aussi la recherche et le développement de nouveaux agents thérapeutiques. Notre but est d’étudier l’implication du stress oxydatif dans l’HBP et d’étudier l’effet de l’extrait de propolis sur le cancer de la prostate in vivo chez le rat Wistar et in vitro sur les cellules LNCaP. Nous avons montré que : 1) Le stress oxydatif joue le rôle de promoteur dans le développement de l’HBP, 2) Le benzo(a)pyrène administré aux rats Wistar induit le développement du cancer de la prostate, 3) La propolis induit la diminution de la prolifération, de l’expression du Ki-67 (-49 %) et de l’expression de AhR chez le rat Wistar, 4) La propolis possède un effet antiprolifératif sur les cellules LNCaP via le blocage de la signalisation de AR. / BPH and prostate cancer are the two most prevalent prostatic diseases in elders. Understanding their etiology and pathogenesis is necessary in order to prevent them and to enhance both research and development of new therapeutic agents. Our goal is to study the implication of oxidative stress in BPH and to study the effect of propolis extract on prostate cancer in vivo in Wistar rats and in vitro on LNCaP cells. We have shown that: 1) Oxidative stress acts as a promoter in the development of BPH, 2) Benzo(a)pyrene administered to Wistar rats induces the development of prostate cancer, 3) Propolis induces a decrease in proliferation, Ki-67 expression (-49%) and AhR expression in the Wistar rat, (4) Propolis has an antiproliferative effect on LNCaP cells via AR signaling blockade.

A simple method is reported to synthesize an anticancer drug, NCX 4040, conveniently in mild conditions using silicon chemistry. A starting material, 4-hydroxybenzyl alcohol, was silylated selectively first to give t-butyldimethylsilyl 4-hydroxybenzyl ether, which was then converted to NCX 4040 by esterification, desilylation, hydrochlorination and nitration.

Prostate cancer is the most frequently diagnosed cancer in American men and the second leading cause of cancer deaths. Supplementation with Se has reduced the incidence of prostate cancer and Se status is inversely correlated with prostate cancer risk. One molecular mechanism by which high Se concentrations may affect cancer risk is by catalyzing disulfide bond formation or otherwise complexing with reactive sulfhydryl groups in cellular proteins. The estrogen receptor (ER) contains cysteines in zinc (Zn) fingers that are susceptible to oxidation and internal disulfide formation, which can prevent DNA binding. We examined ER binding to its DNA response element and gene expression levels for estrogen-regulated genes in human prostate cancer cells (LNCaP) treated with control (50 nM) or high (5 ìM) concentrations of Se. High Se treatment resulted in a non-significant 16 % decrease in ER binding to the estrogen response element (ERE), and no significant changes were found in expression levels of estrogen-regulated genes for either run-on nuclear transcripts or total mRNA. The well documented reaction of Se with reactive sulfhydryl groups, if it occurs in the ER in vivo, has a minimal effect on the binding of ER to DNA and its regulation of gene expression.

A major problem in prostate cancer (PCa) management is the development of drug resistance. It is known that there are changes in PCa biology upon prolonged treatment with drugs, including anti-androgen drugs that alter cellular signaling processes leading to the development of castration resistant PCa. MicroRNAs (miRNAs) are regulatory molecules that modulate gene expression through inhibition of protein translation and modulate cellular functions. Altered expression of miRNAs is often noted in drug resistant cancer including PCa. Studies from our laboratory have identified a number of down-regulated miRNAs in PCa, including miR-l 7-92a miRNAs. Frabin (FGD4) is a target of the miR-l 7-92a cluster that was found to be up-regulated in PCa cells. For this paper’s investigation, an FGD4 knockdown approach was used to identify the effects on cell viability, cell cycle progression, cell migration and drug sensitivity. Two PCa cells lines, LNCaP-104S (androgen sensitive) and PC-3 (androgen independent), were used for our studies. MTS assays for both cell lines showed significant reduction in cell viability following knockdown of FGD4 compared to transfection with control siRNAs. Cell cycle analysis revealed an arrest in the G2/M phase of the cells that were transfected with FGD4 siRNAs. Cell migration assays revealed a decrease in migration rate of PC-3 cells after knockdown, which supports the involvement of FGD4 in actin- cytoskeleton rearrangement. Treatments with anti-mitotic drug Docetaxel (PC-3) or androgen receptor antagonist bicalutamide/Casodex (LNCaP-104S) showed improved sensitivity of the FGD4 siRNA treated cells to these drugs. Our results suggest the potential for FGD4 knockdown to be used in combination with currently used drugs, increasing the effectiveness of frontline chemotherapeutics.