Introdução: O câncer de próstata (CaP) é o tumor sólido mais diagnosticado no homem atualmente, e a sexta ocorrência mais frequente de casos novos de neoplasia maligna no mundo, sendo a segunda causa de óbito por câncer. O câncer de bexiga (CaB) é a segunda neoplasia maligna mais comum e a segunda em causa de óbito entre os tumores genito-urinários. Mundialmente o CaB é responsável por aproximadamente 386.000 novos casos e 150.000 óbitos por ano. O conhecimento das alterações em processos celulares envolvidos na sua carcinogênese nos permite melhor compreensão da patogênese dessas neoplasias, subsidiando, assim, mais efetivamente, o planejamento de estratégias de prevenção, diagnóstico e tratamento. Micro RNA (miRNA) são pequenas sequências não codificantes de RNA que possuem grande papel no controle da expressão dos genes, inibindo a tradução da proteína ou promovendo a degradação do RNA mensageiro (RNAm). Os miRNA estão envolvidos em vários processos celulares fisiológicos e patológicos, incluindo o câncer, onde podem atuar como oncogenes (oncomiR) ou como supressores de tumor (tsmiR). Previamente demonstramos que níveis elevados de miR-100 estão relacionados a recidiva bioquímica pós-prostatectomia radical enquanto no carcinoma urotelial de bexiga de baixo grau ocorre subexpressão desse miRNA. Objetivo: O estudo pretende analisar o papel do miR-100 na regulação de seus supostos genes alvo SMARCA5, THAP2, BAZ2A, mTOR e FGFR3 em linhagens de CaB e CaP e sua relação com a proliferação, apoptose e ploidia de DNA Material e Métodos: As linhagens de CaB (RT4 e T24) e CaP (DU145 e PC3) foram transfectadas com pré-miR-100, antimiR-100 e seus respectivos controles negativos utilizando lipossomas. Após a transfecção o nível de expressão de RNAm e proteína dos genes alvos foi analisado pelas técnicas da cadeia da polimerase quantitativa em tempo real (qRT-PCR) e western blotting respectivamente. A proliferação celular, apoptose e instabilidade cromossômica foram analisadas por citometria de fluxo. Resultados: A transfecção de pré-miR 100, reduziu de modo significativo a expressão de RNAm dos genes mTOR(p=0,006), SMARCA5 (p=0,007) e BAZ2A (p=0,03) na linhagem RT4, mTOR (p=0,02) e SMARCA5 (p=0,01) na linhagem T24, mTOR (p=0,025), THAP2 (p=0,04), SMARCA5 (p=0,001) e BAZ2A (p=0,005) na linhagem DU145 e mTOR (p=0,01) na linhagem PC3. Quanto a expressão proteica houve diminuição global da expressão de todas as proteínas varável de 22,5% a 69% nas quatro linhagens estudadas. Na linhagem T24 miR-100 promoveu um aumento na proliferação e o antimiR-100 induziu a apoptose demonstrando o papel oncogênico desse miR no câncer de bexiga de alto grau. Na linhagem PC3, do mesmo modo, a exposição ao antimiR-100 promoveu um aumento de células em apoptose. Conclusões: Demonstramos que miR-100 controla a expressão gênica e proteica de seus genes alvos nas linhagens de CaP e CaB. Os genes mTOR e FGFR3 são proto-oncogenes envolvidos com o desenvolvimento e progressão de neoplasias, enquanto os genes BAZ2A, SMARCA5 e THAP2 estão relacionados a regulação da transcrição, estabilidade genômica e indução da apoptose. Desse modo podemos admitir que miR-100 tem um papel contraditório no câncer, podendo se comportar como um oncomiR ou como um tsmiR, o que o classificaria como um miRNA \"contexto dependente\". Demonstramos porém que miR-100 tem um papel oncogênico na linhagem T24 de carcinoma urotelial de alto grau de bexiga promovendo um aumento na proliferação e inibição da apoptose. Na linhagem PC3 também o papel oncogênico de miR-100 pode estar relacionado a inibição da apoptose. Dada a variação de ação dos miRNA nos diversos tecidos e estágios tumorais, a determinação do seu papel nos diversos tumores é fundamental pois existe a possibilidade de utiliza-los como marcadores diagnóstico, prognóstico e como alvos para terapias moleculares / Introduction: Prostate cancer (PC) is the most commonly diagnosed solid tumor in men today, and the sixth most frequent occurrence of new cases of malignancy in the world, being the second cause of death by cancer in men. Bladder cancer (BC) is the second most common malignancy and the second cause of death among genitourinary tumors. Globally BC is responsible for approximately 386.000 new cases and 150.000 deaths per year. The knowledge of cellular processes involved in carcinogenesis allows us to better understand the etiology and pathogenesis of these neoplasms, supporting thus more effectively, planning strategies for prevention and treatment. Micro RNAs (miRNA) are small non-coding RNA sequences that have a large role in the control of gene expression by inhibiting protein translation or promoting the degradation of messenger RNA (RNAm). The miRNA are currently involved in various physiological and pathological cellular processes, including cancer where they can act as oncogenes (oncomiR) or tumor suppressors (tsmiR). Previously we demonstrated that high levels of miR-100 are associated with biochemical recurrence after radical prostatectomy while in low-grade bladder urothelial carcinoma it is persistently underexpressed. Objective: The study aims to examine the role of miR-100 in the regulation of its supposed target genes SMARCA5, THAP2, BAZ2A, mTOR and FGFR3 in BC and PC cell lines and its relationship with proliferation, apoptosis and chromosomal instability. Material and Methods: The BC (RT4 and T24) and PC cell lines (DU145 and PC3) were transfected with pre-miR-100, antimiR-100 and their respective controls using liposomes. After transfection RNAm and protein levels of its supposed target genes 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. Results: After transfection of pre-miR 100, there was a significant reduction in the RNAm expression of mTOR (p=0.006), SMARCA5 (p=0.007) and BAZ2A (p=0.03) in RT4, mTOR (p=0.02) and SMARCA5 (p=0.01) in T24, mTOR (p=0.025), THAP2 (p=0.04), SMARCA5 (p=0.001) and BAZ2A (p=0.005) in DU145 and mTOR (p=0.01) in PC3. There was a reduction in the expression of all proteins, variable from 22.5% to 69% in all cell lines. In T24 miR-100 promoted an increase in cell proliferation and antimiR-100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a right grade urothelial carcinoma. Also in PC3 antimiR-100 promoted an increase in apoptosis. Conclusions: We have shown that miR-100 controls the expression of gene and protein of its supposed target genes in PC and BC cell lines. mTOR and FGFR3 are proto-oncogenes related to the tumor development and progression, while BAZ2A, SMARCA5 and THAP2 are related to the DNA transcription regulation, chromossomic stability and apoptosis induction. We can conclude that miR-100 has a contradictory role in cancer, behaving as an oncomir or tsmiR depending the type and stage of a specific neoplasia, classifying it as a \"context depending\" miRNA. In T24 cell line however miR-100 acts as an oncomiR increasing cell proliferation and inhibiting apoptosis. In PC3 cell line miR-100 also acts as an oncomiR inhibiting apoptosis. Due to the variation of roles of miRNAs in different tissues and stage of tumors, the characterization of their role in neoplasm is very important because of the possibility to use them as diagnostic or prognostic markers, even as targets for the development of new drugs

<p>Radioterapija (RT) je jedan od najvažnijih modaliteta lečenja solidnih malignih tumora i koristi je vi&scaron;e od 50% pacijenata (52,3%) sa malignim tumorima. Nauka koja proučava efekte elektromagnetnog zračenja na biolo&scaron;ke sisteme naziva se radiobiologija. Radiobiologija se fokusira na odgovor ćelija, tkiva i organizma kao celine na jonizujuće zračenje i proučava mehanizme radiobiolo&scaron;kog odgovora. Izlaganje ćelija niskim dozama JZ koje su nakon određenog vremenskog intervala praćene uobičajenim radioterapijskim dozama naziva se radioadaptivno zračenje. Adaptivni odgovor u sebi može da sadrži nekoliko fenomena: hiperradiosenzitaciju/radiorezistenciju, &ldquo;bystander&rdquo; efekat i radioadaptivni efekat u užem smislu. O molekularnim mehanizmima koji stoje iza navedenih efekata ne zna se dovoljno. U ovom radu ispitivan je odgovor malignih i zdravih ćelija na različite modalitete jonizujućeg zračenja u cilju boljeg poznavanja puteva ćelijske smrti i preživljavanja. Potpuno razumevanje molekularnih puteva koji vode u apoptozu ili u preživljavanje ćelija nakon izlaganja jonizujućem zračenju moglo bi koristiti u iznalaženju novih i efikasnijih strategija i modaliteta lečenja malignih tumora u cilju njihove potpune eredikacije. U istraživanju su kori&scaron;tene dve humane ćelijske linije ćelijska linija humanog kolorektalnog karcinoma HT-29 i ćelijska linija humanih fetalnih fibroblasta pluća MRC-5. Ćelije su zračene u dva režima različitim pre-iradijacionim dozama(0,03; 0,05 i 0,07Gy) i istom kurativnom dozom (2Gy) tokom 4 dana. Vi&scaron;ekratna primena niskih doza JZ nije značajno smanjila vijabilnost HT-29 ćelija, dok su dve radioadaptivne doze (0,05+2Gy i 0,07+2Gy), adekvatne doze JZ za radioterapijski postulat po&scaron;tede zdravih ćelija i bolji antitumorski efekat u odnosu na neradioadaptivno zračenje od 2Gy u toku 4 dana. Pokazana je mogućnost modulisanja ćelijskog odgovora na JZ uz pomoć niskih doza JZ koje su praćene dozom od 2Gy (radioadaptivni tip zračenja) u oba dizajnirana režima zračenja. Stepen o&scaron;tećenja hromozoma za većinu isporučenih doza pokazao dozno zavisni trend. Dozno-zavisno o&scaron;tećenje naslednog materijala izazvano radioadaptivnim zračenjem potvrđuje hipotezu da je stepen o&scaron;tećenja zdravih, MRC-5, ćelija manji nego u ćelijama kolorektalnog adenokarcinoma. Fragmentacija DNK je zabeležena za pojedine doze JZ u obe ćelijske linije, a uočena je i razlika u odgovoru zdrave i tumorske ćelijske linije. Detekcijom mutacija primarne sekvence fragmenta p53 gena pokazano je da se broj mutacija povećava sa povećanjem doze JZ. Oba režima radioadaptivnog zračenja, u obe ćelijske linije izazivaju vi&scaron;i nivo ekspresije p53. Ekspresija p38 MAPK proteina u HT-29 ćelijama bila je niža za sve isporučene doze JZ u odnosu na nezračene ćelije. U MRC-5 ćelijama, povi&scaron;ena ekspresija p38 MAPK utvrđena je samo u uzorcima koji su jednokratno primili samo niske doze JZ i dozu od 2Gy dnevno tokom 4 dana, u odnosu na nezračenu kontrolu. Razlike u ekspresiji ispitivanih proteina dobijene nakon primene dva režima radioadaptivnog zračenja posledica su delovanja niskih pre-iradijacionih doza JZ na modulisanje radiobiolo&scaron;kog odgovora obe ćelijske linije. Nivo ekspresije Bcl-2 i Bax proteina i njihov međusobni odnos, u obe ćelijske linije, su odraz različitog radiobiolo&scaron;kog odgovora ispitivanih ćelija koji zavisi od primenjenog režima zračenja.</p> / <p>Radiotherapy (RT) is one of the most important treatment modality for solid malignant tumors and it is applied in more than 50% of the patients (52.3%). Radiobiology id scientific discipline which studies the effects of electromagnetic irradiation on biological systems. Radiobiology focuses on the response of the cells, tissues and the organism as a whole to ionizing radiation and studying the mechanisms of radiobiological response. Exposure of cells to low-dose irradiation (priming dose) followed by challenging doses is called radioadaptive radiation. Adaptive response is described as several phenomena: hyperradiosensitivity / radiorezistence, &quot;bystander&quot; effect and radioadaptive effect in sensu strict. Molecular mechanisms underlying the above effects are not sufficiently known. In this study, the response of malignant and healthy cells on various modalities of ionizing radiation is explored in order to improve knowledge of pathways of cell death and survival. Fully understanding the molecular pathways leading to apoptosis or cell survival after exposure to ionizing radiation may be used in finding new and more effective strategies and modalities for the treatment of malignant tumors. The study used two human cell lines: human colorectal cancer HT-29 cell line and the human fetal lung fibroblast MRC-5. The cells were irradiated in two modalities using different pre-irradiation doses (0.03, 0.05 and 0,07Gy) and the same challenging dose (2Gy) for 4 days. Everyday use of low-dose did not significantly reduce the viability of HT-29 cells, while two radioadaptive doses (0.05 + 2Gy and 0.07+2Gy), are adequate doses for sparing healthy cells with better anti-tumor effects. The possibility of modulating the cellular response to the ionizing radiation was shown using low-doses followed by 2Gy (radioadaptive radiation) in both designed regimes of radiation. The level of chromosomal damage showed a dose-dependent trend. Dose-dependent damage to the genetic material caused by radiation confirms the hypothesis that the degree of damage to MRC-5 cells is smaller than the HT29 cells. DNA fragmentation differed between HT29 and MRC-5 cells. Detection of mutations in p53 gene fragment sequence increased with increasing doses. Both irradiation modalities, in both cell lines induce a higher level of p53 expression. Expression of p38 MAPK protein in the HT-29 cells was lower for all delivered doses compared to nonirradiated. In MRC-5 cells, increased expression of the p38 MAPK was found only in the samples that had only received on first day low-doses compared to the control nonirradiated cells. Differences in the expression of the tested proteins reflect different molecular mechanisms activated in normal and tumor cells. The level of Bcl.2 and Bax expression also reflected different radiobiological responses between normal and tumor cells, which depended on the applied irradiation regime.</p>

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 181 pages. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 149 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 155 pages. Includes vita. Includes bibliographical references.

Introdução: O câncer de próstata (CaP) é o tumor sólido mais diagnosticado no homem atualmente, e a sexta ocorrência mais frequente de casos novos de neoplasia maligna no mundo, sendo a segunda causa de óbito por câncer. O câncer de bexiga (CaB) é a segunda neoplasia maligna mais comum e a segunda em causa de óbito entre os tumores genito-urinários. Mundialmente o CaB é responsável por aproximadamente 386.000 novos casos e 150.000 óbitos por ano. O conhecimento das alterações em processos celulares envolvidos na sua carcinogênese nos permite melhor compreensão da patogênese dessas neoplasias, subsidiando, assim, mais efetivamente, o planejamento de estratégias de prevenção, diagnóstico e tratamento. Micro RNA (miRNA) são pequenas sequências não codificantes de RNA que possuem grande papel no controle da expressão dos genes, inibindo a tradução da proteína ou promovendo a degradação do RNA mensageiro (RNAm). Os miRNA estão envolvidos em vários processos celulares fisiológicos e patológicos, incluindo o câncer, onde podem atuar como oncogenes (oncomiR) ou como supressores de tumor (tsmiR). Previamente demonstramos que níveis elevados de miR-100 estão relacionados a recidiva bioquímica pós-prostatectomia radical enquanto no carcinoma urotelial de bexiga de baixo grau ocorre subexpressão desse miRNA. Objetivo: O estudo pretende analisar o papel do miR-100 na regulação de seus supostos genes alvo SMARCA5, THAP2, BAZ2A, mTOR e FGFR3 em linhagens de CaB e CaP e sua relação com a proliferação, apoptose e ploidia de DNA Material e Métodos: As linhagens de CaB (RT4 e T24) e CaP (DU145 e PC3) foram transfectadas com pré-miR-100, antimiR-100 e seus respectivos controles negativos utilizando lipossomas. Após a transfecção o nível de expressão de RNAm e proteína dos genes alvos foi analisado pelas técnicas da cadeia da polimerase quantitativa em tempo real (qRT-PCR) e western blotting respectivamente. A proliferação celular, apoptose e instabilidade cromossômica foram analisadas por citometria de fluxo. Resultados: A transfecção de pré-miR 100, reduziu de modo significativo a expressão de RNAm dos genes mTOR(p=0,006), SMARCA5 (p=0,007) e BAZ2A (p=0,03) na linhagem RT4, mTOR (p=0,02) e SMARCA5 (p=0,01) na linhagem T24, mTOR (p=0,025), THAP2 (p=0,04), SMARCA5 (p=0,001) e BAZ2A (p=0,005) na linhagem DU145 e mTOR (p=0,01) na linhagem PC3. Quanto a expressão proteica houve diminuição global da expressão de todas as proteínas varável de 22,5% a 69% nas quatro linhagens estudadas. Na linhagem T24 miR-100 promoveu um aumento na proliferação e o antimiR-100 induziu a apoptose demonstrando o papel oncogênico desse miR no câncer de bexiga de alto grau. Na linhagem PC3, do mesmo modo, a exposição ao antimiR-100 promoveu um aumento de células em apoptose. Conclusões: Demonstramos que miR-100 controla a expressão gênica e proteica de seus genes alvos nas linhagens de CaP e CaB. Os genes mTOR e FGFR3 são proto-oncogenes envolvidos com o desenvolvimento e progressão de neoplasias, enquanto os genes BAZ2A, SMARCA5 e THAP2 estão relacionados a regulação da transcrição, estabilidade genômica e indução da apoptose. Desse modo podemos admitir que miR-100 tem um papel contraditório no câncer, podendo se comportar como um oncomiR ou como um tsmiR, o que o classificaria como um miRNA \"contexto dependente\". Demonstramos porém que miR-100 tem um papel oncogênico na linhagem T24 de carcinoma urotelial de alto grau de bexiga promovendo um aumento na proliferação e inibição da apoptose. Na linhagem PC3 também o papel oncogênico de miR-100 pode estar relacionado a inibição da apoptose. Dada a variação de ação dos miRNA nos diversos tecidos e estágios tumorais, a determinação do seu papel nos diversos tumores é fundamental pois existe a possibilidade de utiliza-los como marcadores diagnóstico, prognóstico e como alvos para terapias moleculares / Introduction: Prostate cancer (PC) is the most commonly diagnosed solid tumor in men today, and the sixth most frequent occurrence of new cases of malignancy in the world, being the second cause of death by cancer in men. Bladder cancer (BC) is the second most common malignancy and the second cause of death among genitourinary tumors. Globally BC is responsible for approximately 386.000 new cases and 150.000 deaths per year. The knowledge of cellular processes involved in carcinogenesis allows us to better understand the etiology and pathogenesis of these neoplasms, supporting thus more effectively, planning strategies for prevention and treatment. Micro RNAs (miRNA) are small non-coding RNA sequences that have a large role in the control of gene expression by inhibiting protein translation or promoting the degradation of messenger RNA (RNAm). The miRNA are currently involved in various physiological and pathological cellular processes, including cancer where they can act as oncogenes (oncomiR) or tumor suppressors (tsmiR). Previously we demonstrated that high levels of miR-100 are associated with biochemical recurrence after radical prostatectomy while in low-grade bladder urothelial carcinoma it is persistently underexpressed. Objective: The study aims to examine the role of miR-100 in the regulation of its supposed target genes SMARCA5, THAP2, BAZ2A, mTOR and FGFR3 in BC and PC cell lines and its relationship with proliferation, apoptosis and chromosomal instability. Material and Methods: The BC (RT4 and T24) and PC cell lines (DU145 and PC3) were transfected with pre-miR-100, antimiR-100 and their respective controls using liposomes. After transfection RNAm and protein levels of its supposed target genes 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. Results: After transfection of pre-miR 100, there was a significant reduction in the RNAm expression of mTOR (p=0.006), SMARCA5 (p=0.007) and BAZ2A (p=0.03) in RT4, mTOR (p=0.02) and SMARCA5 (p=0.01) in T24, mTOR (p=0.025), THAP2 (p=0.04), SMARCA5 (p=0.001) and BAZ2A (p=0.005) in DU145 and mTOR (p=0.01) in PC3. There was a reduction in the expression of all proteins, variable from 22.5% to 69% in all cell lines. In T24 miR-100 promoted an increase in cell proliferation and antimiR-100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a right grade urothelial carcinoma. Also in PC3 antimiR-100 promoted an increase in apoptosis. Conclusions: We have shown that miR-100 controls the expression of gene and protein of its supposed target genes in PC and BC cell lines. mTOR and FGFR3 are proto-oncogenes related to the tumor development and progression, while BAZ2A, SMARCA5 and THAP2 are related to the DNA transcription regulation, chromossomic stability and apoptosis induction. We can conclude that miR-100 has a contradictory role in cancer, behaving as an oncomir or tsmiR depending the type and stage of a specific neoplasia, classifying it as a \"context depending\" miRNA. In T24 cell line however miR-100 acts as an oncomiR increasing cell proliferation and inhibiting apoptosis. In PC3 cell line miR-100 also acts as an oncomiR inhibiting apoptosis. Due to the variation of roles of miRNAs in different tissues and stage of tumors, the characterization of their role in neoplasm is very important because of the possibility to use them as diagnostic or prognostic markers, even as targets for the development of new drugs

AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy. / Dissertation

<p>&Scaron;tavelj (Rumex crispus, Polygonaceae) je vi&scaron;egodi&scaron;nja zeljasta biljka, koja predstavlja bogat izvor fenolnih komponenti. Iako se smatra invazivnim korovom, mlado li&scaron;će &scaron;tavelja je jestivo i često se koristi kao salata. Dalje, upotreba plodova &scaron;tavelja opisana je u srpskoj i turskoj narodnoj medicini u lečenju gastrointestinalnih tegoba. Cilj ovog rada bio je procena in vitro i in vivo antioksidantne/prooksidantne i citotoksične aktivnosti, i određivanje eventualnog in vitro antiinflamatornog efekta vodenog ekstrakta ploda Rumex crispus. Ukupan sadržaj flavonoida određen je spektrofotometrijskom metodom. Kvalifikacija i kvantifikacija flavonoida potvrđena je visokoefikasnom tečnom hromatografijom (HPLC). Antioksidantna aktivnost vodenog ekstrakta ploda &scaron;tavelja procenjena je na osnovu in vitro testova: Ferric-reducing antioxidant power (FRAP), sposobnosti ekstrakta da neutrali&scaron;e slobodne radikale NO&bull;, OH&bull; i DPPH&bull; i uticaja na lipidnu peroksidaciju u lipozomima. Citotoksičnost ispitivanog ekstrakta je određena in vitro na tumorskim ćelijskim linijama: humani karcinom cerviksa (HeLa), adenokarcinom (HT-29) i adenokarcinom dojke (MCF7). Takođe, moguća in vivo hepatoprotektivna i antioksidantna svojstva ekstrakta određena su kod oksidativnog stresa izazvanog CCl4 kod eksperimentalnih životinja. Pored toga, proverena je hipoteza u kojoj testiran ekstrakt pokazuje in vivo antiproliferativnu aktivnost kod Ehrlich-ovih (EAC) i Hepatoma AS30D ćelija, merenjem zapremine ascitesa, procenta vijabilnih ćelija i nivoa nekoliko antioksidantnih enzima. Optimizovan in vitro test za određivanje potencijala inhibicije ciklooksigenaze-1 (COX-1) i 12-lipooksigenaze (12-LOX) preduzet je u svrhu procene antiinflamatornog efekta vodenog ekstrakta ploda R. crispus. HPLC analiza otkrila je da je mikvelianin najdominantniji flavonoidni konstituent ekstrakta. Testirani ekstrakt pokazao je potencijalnu antioksidantnu aktivnost rezultujući velikom moći u neutralizaciji slobodnih radikala, i sposobno&scaron;ću da smanji lipidnu peroksidaciju u lipozomima. Rezultati su ukazali na tkivno-selektivnu citotoksičnost ekstrakta ploda R. crispus in vitro. Najizraženija antitumorska aktivnost primećena je prema HeLa i MCF7 ćelijskim linijama. Podaci sugeri&scaron;u da bi se ispitivani ekstrakt mogao smatrati potencijalnim in vivo hepatoprotektivnim i antioksidantnim agensom, sprečavajući oksidativna o&scaron;tećenja jetre. S druge strane, pomenuti ekstrakt može pokazati in vivo prooksidantna svojstva, uzrokujući oksidativni stres u maligno transformisanim EAC i AS30D ćelijama i smanjujući zapreminu ascitesa i udeo vijabilnih ćelija, u poređenju sa kontrolnom grupom. Promene u aktivnosti antioksidantnih enzima su verovatno posledica indukovanog oksidativnog stresa u EAC i AS30D ćelijama, naročito kod pretretiranih životinja. Vodeni ekstrakt ploda &scaron;tavelja pokazao je COX-1, kao i 12-LOX inhibitornu aktivnost, navodeći da bi ispitivani ekstrakt mogao biti antiinflamatorni agens. Vodeni ekstrakt ploda R. crispus ima potencijalnu antioksidantnu, citotoksičnu i antiinflamatornu aktivnost. Ispoljavanje prooksidantnih svojstava predstavlja mogući mehanizam antiproliferativnog efekta ekstrakta.</p> / <p>Curly dock (Rumex crispus, Polygonaceae) is a wild perennial herbaceous plant, which products are described as a rich source of phenolic compounds. Apart from being considered a seriously invasive weed, young leaves of curly dock are edible and often used as salad. Furthermore, the use of its fruits has been described in Serbian and Turkish traditional medicine against stomach complaints. The objectives of this study were to evaluate in vitro and in vivo antioxidant/prooxidant and cytotoxic activities, and to determine an eventual in vitro anti-inflammatory effect of the aqueous extract of Rumex crispus fruits. Total flavonoid content was determined by spectrophotometric method. Qualification and quantification of flavonoids were confirmed using High performance liquid chromatography (HPLC). The aqueous extract of curly dock fruits was evaluated for its antioxidant activity by in vitro assays for Ferric-reducing antioxidant power (FRAP), NO&bull;, OH&bull; and DPPH&bull;-free radical scavenging activities and the influence on lipid peroxidation in liposomes. The cytotoxicity of tested extract was examined in vitro in human cervix carcinoma (HeLa), colon adenocarcinoma (HT-29) and breast adenocarcinoma (MCF7). Also, the potential in vivo hepatoprotective and antioxidant properties of investigated extract were determined on CCl4-induced oxidative stress in experimental animals. Furthermore, the hypothesis that the examined extract might show in vivo antiproliferative activity in Ehrlich carcinoma (EAC) and Hepatoma AS30D cells was tested by measuring volume of ascites, percentage of viable cells and level of several antioxidant enzymes. The optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken in order to estimate an anti-inflammatory effect of aqueous extract of R. crispus fruits. HPLC analysis revealed miquelianin as the most abundant flavonoid constituent of the extract. The tested extract might have an antioxidant activity resulting in scavenging of free radicals and ability to decrease lipid peroxidation in liposomes. The results could indicate tissue-selective cytotoxicity of R. crispus fruit extract in vitro. The most prominent antitumor activity was observed towards HeLa and MCF7 cell lines. The data suggested that investigated extract may be considered as potential in vivo hepatoprotective and antioxidant agent due to prevention of the liver injuries induced by oxidative damage. On the other hand, mentioned extract could exhibit in vivo prooxidant property, causing the oxidative stress in malignant transformed EAC and AS30D cells and reducing volume of ascites and percentage of viable cells, in comparison with control group. Changes in activities of antioxidant enzymes might be the results of induced oxidative stress in EAC and AS30D cells, especially in the pretreated animals. The aqueous extract of curly dock fruits showed COX-1, as well as 12-LOX inhibitory activity, suggesting that tested extract might be an anti-inflammatory agent. It could be concluded that aqueous fruit extract of R. crispus might have antioxidant, cytotoxic and anti-inflammatory activities. The prooxidant properties of examined extract could be the mechanism of potential antiproliferative effect of extract.</p>