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Caracterização de vesículas extracelulares liberadas por células de melanoma murino tratadas com quimioterápicos: possível papel modulador na sobrevivência das celulas tumorais? / Characterization of extracellular vesicles released by murine melanoma cells treated with chemotherapeutic agents: a possible modulating role in cell survival?Mariana Mari Ikoma 05 September 2017 (has links)
O Melanoma é um tipo de neoplasia que se origina de melanócitos normalmente presentes na epiderme. Uma das características do melanoma é a capacidade de adquirir resistência a terapias. As células de melanoma podem aumentar a liberação de vesículas extracelulares (VEs) em resposta ao tratamento com quimioterápicos. A cisplatina (CDDP) e a temozolomida (TMZ) são drogas utilizadas para o tratamento de tumores. Ambas as drogas formam adutos no DNA, mas as vias de sinalização que deflagram a morte celular são distintas. O objetivo desse estudo é investigar a morte celular da linhagem B16-F10 na presença de VEs oriundas de células B16-F10 tratadas com cisplatina CDDP ou TMZ. Inicialmente as VEs oriundas de células de melanoma murino, B16-F10, tratadas com CDDP ou TMZ e seus controles, foram isoladas por ultracentrifugações sucessivas. Para os experimentos in vitro, as células foram tratadas com as drogas em combinação com as respectivas VEs. As amostras foram realizados avaliações de ciclo celular e de morte e ensaio clonogênico. Para os experimentos in vivo, as células B16-F10 foram pré-tratadas com VEs, e posteriormente, as células foram inoculadas via subcutânea em camundongos C57BL/6 e os tumores foram mensurados diariamente. Em nosso estudo concluimos que a metodologia do isolamento de VEs é eficiente. Além disso, observamos que o tratamento com CDDP ou TMZ aumenta a liberação de VEs por células tumorais. Apesar do resultado contraditorio, as VEs liberadas por células tumorais tratadas com quimioterápicos aumentam a capacidade de sobrevivência das células de melanoma in vitro. VEs oriundas de células de melanoma não participam inicialmente da sensibilização à morte de células tumorais causada pelas mesmas drogas, mas a longo prazo, as VEs oriundas de células tratadas com a TMZ podem conferir uma resposta celular de sobrevivência às células tumorais in vitro. In vivo, o resultado é inconclusivo, uma vez que para confirmar se as VEs fazem parte da adaptação tumoral conferindo fenômenos de sobrevivência celular in vivo, é necessário avaliar em outros modelos celulares e animais / Melanoma is a neoplasm derived from melanocytes normally present in the skin specifically in the epidermis. One of the malignancies of melanoma is the ability to acquire chemoresistance. Cisplatin (CDDP) and temozolomide (TMZ) are drugs used for the treatment of tumors. Both drugs can form alkylating adducts in DNA, however, the pathways that trigger cell death are distinct. Tumor cells, including melanoma, may increase the release of extracellular vesicles (EVs) in response to chemotherapeutic treatment. The aim of this study is to investigate the cell death phenomenon in B16-F10 cell line in presence of EVs derived from chemotherapeutic-treated B16-F10 cells. For in vitro experiments, the cells were treated with CDDP or TMZ in combination with EVs from chemotherapictreated samples. For in vivo experiments, B16-F10 cells were exposed to EVs and inoculated subcutaneously in C57BL/6 mice. The growth was measured daily. In this work, we established and characterized VEs released by melanoma cells treated with chemotherapics and we established chemotherapics treatments to isolate EVs for next EVs isolation. Our results showed that CDDP or TMZ treatment increase the release of EVs by tumor cells. The EVs released by melanoma cells after CDDP or TMZ treatment seem to increase the survival capacity of melanoma cells. Thus, we concluded that EVs derived from melanoma cells do not participate in the cell death sensitization induced by CDDP or TMZ. However, EVs derived from TMZ treated cells may offer a survival effect to tumor cells in vitro a long term. In vivo, The result is inconclusive since to confirm how VEs are part of the tumor adaptation conferring cellular survival phenomena in vivo, it is necessary to evaluate in other cellular and animal models
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Effects of EF-24 and Cisplatin on Cancer, Renal, and Auditory CellsHodzic, Denis 01 April 2019 (has links)
Cisplatin is a chemotherapy drug effective against several forms of cancer, but can also cause serious side-effects, including nephrotoxicity and ototoxicity. Curcumin, a natural plant compound, can increase cisplatin’s anti-cancer activity and counteract cisplatin’s deleterious effect on the auditory and renal systems. Unfortunately, curcumin exhibits poor bioavailability, which has promoted interest in the development of synthetic curcumin analogs (curcuminoids) that are soluble, target cancer, and do not cause side effects. This study investigated whether the curcuminoid (3E,5E)-3,5-bis[(2-fluorophenyl) methylene]-4-piperidinone (EF-24) increases the anti-cancer effects of cisplatin against a human ovarian cancer cell line (A2780) and its cisplatin-resistant counterpart (A2780cis), while preventing cisplatin-mediated side effects in a human kidney cell line (HEK-293T) and a mouse auditory hybridoma cell line (HEI-OC1). The effect of cisplatin and EF-24 on cellular viability was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The expression and activity of signal transduction proteins in several apoptotic pathways was measured using caspase luminescence assays. Reactive oxygen species (ROS) production was also measured using flow cytometry. Our data suggest that cisplatin and EF-24 are effective against ovarian cancer cell lines, but both compounds may also have adverse effects on auditory and renal cells. This project provides relevant information that may improve our understanding of how these compounds function in different tissues, facilitating improved cancer treatment and circumvention of side effects commonly associated with cisplatin treatment.
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Effect of Leaving Ligands of Platinum(II) Diamine Complexes on DNA and Protein ResiduesKolli, Ramya 01 May 2013 (has links)
Platinum compounds are widely used drugs in cancer treatments. Although DNA is the biological target, reaction of platinum compounds with proteins is also potentially significant. Our objective is to study the effects of leaving ligands on the relative reactivity between 5'-GMP (guanosine 5' phosphate), a key DNA target, and N-Acetyl - L-Methionine (N-AcMet), a key protein target. We have used NMR spectroscopy to monitor reactions with N-AcMet and 5'-GMP added to a platinum complex to see which products are formed preferentially. Previous research showed that both a non-bulky complex such as [Pt(en)(D2O)2]2+ [en=ethylenediamine], and a bulky complex such as [Pt(Me4en)(D2O)2]2+ [Me4en= N, N, N', N'-tetramethylethylenediamine] react more quickly with 5'-GMP than with N-AcMet. To improve the activity of platinum compounds in our current research, oxalates as leaving ligands are used. The results suggest that [Pt(en)(Ox)] [Ox= oxalate] reacts faster with N-AcMet than with 5'-GMP. Also, [Pt(Me4en)(Ox)] reacts slowly with 5'-GMP without N-AcMet and the reaction favors N-AcMet when both ligands are added simultaneously. Interestingly, the formation of the sulfur-oxygen chelate is slow enough to be observable in the oxalate reaction; but the mono product is not independently observed in the dinitrate complex.
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The Role and Regulation of p53-associated, Parkin-like Cytoplasmic Protein (PARC) in p53 Subcellular Trafficking and Chemosensitivity in Human Ovarian Cancer CellsWoo, Michael G. 26 March 2012 (has links)
Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.
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The Role and Regulation of p53-associated, Parkin-like Cytoplasmic Protein (PARC) in p53 Subcellular Trafficking and Chemosensitivity in Human Ovarian Cancer CellsWoo, Michael G. 26 March 2012 (has links)
Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.
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The Role and Regulation of p53-associated, Parkin-like Cytoplasmic Protein (PARC) in p53 Subcellular Trafficking and Chemosensitivity in Human Ovarian Cancer CellsWoo, Michael G. 26 March 2012 (has links)
Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.
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Analysis of the response of nucleotide excision repair genes in Dictyostelium discoideum /Yu, Sung-Lim, January 1997 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-130). Also available on the Internet.
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Analysis of the response of nucleotide excision repair genes in Dictyostelium discoideumYu, Sung-Lim, January 1997 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-130). Also available on the Internet.
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HISTOLOGIA, FUNÇÃO COCLEAR E GENOTOXICIDADE EM COBAIAS TRATADAS COM CISPLATINA / HISTOLOGY, COCHLEAR FUNCTION AND GENOTOXICITY IN GUINEA PIG TREATED WITH CISPLATINFranceschi, Cacineli Marion de 02 March 2011 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The present work aims to investigate the cisplatin influence on the cochlea and the deoxiribonucleic acid (DNA) of guinea pigs. An experimental study was executed with 12 guinea pigs (Cavia porcellus). The inclusion criterion for guinea pigs in the sample was the presence of Preyer's reflex (contraction of the pinna when facing sound stimulus) and distortion product otoacoustic emissions (DPOAEs). Guinea pigs were divided into two groups: Control Group (CG) - composed by six guinea pigs, to which it was administrated physiological solution of sodium chloride 0.9% during six consecutive days; Study Group (SG) - composed by six guinea pigs to which it was
administrated cisplatin in six consecutive doses of 3mg/kg/day intraperitoneally. Twenty-four hours after the last cisplatin injection, guinea pigs were sacrificed, the
blood sample was collected to perform the Comet Essay, and the cochleas were removed to histological analysis. When comparing the SG guinea pigs before and after the cisplatin administration, it was verified a statistically significant reduction of the amplitude of DPOAEs, mainly in the frequencies of 1000Hz to 3998Hz. After the
cisplatin administration, it was certified that the amplitude of the DPOAEs frequencies of 2830Hz and 5657Hz from the SG guinea pigs suffered a statistically significant reduction compared to the CG guinea pigs. After the Cisplatin administration there were not detected any identifiable DNA changes in the Comet essay, the histological analysis showed alterations in the organ of Corti and in the spiral ganglion. Cisplatin causes alterations in the function and cochlear morphology, however, any damage to the DNA was detected. / O presente trabalho tem como objetivo verificar a influência da cisplatina sobre a cóclea e o ácido desoxirribonucleico (DNA) de cobaias. Estudo experimental executado com 12 cobaias (Cavia porcellus). O critério de inclusão de cobaias na
amostra foi a presença de reflexo de Preyer (contração do pavilhão auricular frente a estímulo sonoro) e emissões otoacústicas produto de distorção (EOAPDs). As cobaias foram dividas em dois grupos: Grupo controle (GC) - composto de seis cobaias, às quais foi administrada solução fisiológica de cloreto de sódio a 0,9% por seis dias consecutivos; Grupo estudo (GE) - composto por seis cobaias, às quais foi
administrada cisplatina em seis doses consecutivas de 3mg/kg/dia via intraperitoneal. Vinte e quatro horas após a última aplicação de cisplatina as cobaias foram sacrificadas, foi coletada amostra sanguínea para realização do Ensaio
Cometa, e as cócleas foram removidas para análise histológica. Ao comparar-se as cobaias do GE antes e após a administração de cisplatina verificou-se redução estatisticamente significante da amplitude das EOAPDs principalmente nas frequências de 1000Hz à 3998Hz. Após a administração de cisplatina constatou-se que a amplitude das EOAPDs nas frequências de 2830Hz e 5657Hz, das cobaias do
GE, sofreram redução estatisticamente significante quando comparado com as cobaias do GC. Após a administração de cisplatina não foram detectados danos genotóxicos identificáveis no Ensaio Cometa, a análise histológica mostrou
alterações no órgão de Corti e gânglio espiral. A cisplatina provoca alterações na função e morfologia coclear, no entanto não foi detectado dano genotóxico.
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OTOPROTEÇÃO DA N-ACETILCISTEÍNA E VIA DE MODULAÇÃO DA APOPTOSE EM CÉLULAS CILIADAS DE RATOS TRATADOS COM CISPLATINA / OTOPROTECTION BY N-ACETYLCYSTEINE AND THE APOPTOSIS MODULATION PATHWAY IN HAIR CELLS OF RATS TREATED WITH CISPLATINGonçalves, Maiara Santos 10 March 2015 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This paper aimed to investigate the apoptosis modulation pathway and the otoprotection mechanism of N-acetylcysteine (NAC) through the analysis of the glutathione peroxidase (GSH-Px) enzyme and the Bcl-2 protein expression in outer hair cells (OHCs) of rats treated with cisplatin. The listening function was also assessed in mice under the effect of different doses of cisplatin and NAC. Two experiments were performed, named A and B, the first being over an experimental period of five days, and the second during three days. Each experiment comprised four groups, under the following protocols: group A1 (negative control): intraperitoneally saline solution 0,9%, in the same volume corresponding to cisplatin dose; group A2 (positive control): 100mg/kg/day of NAC, oral administration by gavage; group A3 (ototoxic): 3mg/kg/day of intraperitoneally cisplatin; group A4 (ototoxic with otoprotection): 100 mg/kg/day of NAC oral administration by gavage, one hour before the administration of 3 mg/kg/day of intraperitoneally cisplatin; group B1 (negative control): intraperitoneally saline solution 0,9% in the same volume corresponding to the cisplatin dose (8mg/kg/day); group B2 (positive control): 300 mg/kg/day of NAC, oral administration by gavage; group B3 (ototoxic): 8 mg/kg/day of intraperitoneally cisplatin; group B4 (ototoxic with otoprotection): 300 mg/kg/day of NAC orally by gavage, one hour before the administration of 8 mg/Kg/day of intraperitoneally cisplatin. The animals in experiment A underwent otoscopy, distortion-product otoacustic emissions (DPOAEs) and brainstem auditory evoked potential (BAEP), before and after the administration of drugs. The animals in experiment B underwent the same testing in pre- and post-treatment, their tympanic bulla being removed ant their cochleae prepared for anatomical assessment with scanning electron microscopy and immunofluorescence for labeling the GSH-Px enzyme and the Bcl-2 protein. In experiment A, it was verified that there was no significant decrease in the signal-to-noise ratio of DPOEAs, but there was a significant increase in the electrophysiologic threshold obtained through BAEP in groups A3 e A4. In experiment B, it was verified that: there was no significant increase in the electrophysiologic threshold obtained through BAEP; the OHCs remained anatomically intact; the GSH-Px enzyme showed immunostaining absent in group B1 and immunostaining present in groups B2, B3 and B4; the Bcl-2 protein showed immunostaining absent in all groups. From the results, it was concluded that the listening function was more impaired by the exposure to a subdose of cisplatin over a longer period, the apoptosis modulation pathway in outer hair cells of mice treated with cisplatin is related to the expression of the GSH-Px enzyme and not expression of the Bcl-2 protein. / Este trabalho teve o objetivo de investigar o mecanismo de otoproteção da N-acetilcisteína (NAC) e a via de modulação da apoptose por meio da análise da expressão da enzima glutationa peroxidase (GSH-Px) e da proteína Bcl-2 em células ciliadas externas (CCEs) de ratos tratados com cisplatina. Também foi avaliada a função auditiva de ratos sob efeito de diferentes doses de cisplatina e NAC. Foram realizados dois experimentos, denominados de A e B, sendo o primeiro com um período experimental de cinco dias e o segundo de três dias. Cada experimento foi composto por quatro grupos, submetidos aos seguintes protocolos: grupo A1 (controle negativo): solução fisiológica 0,9%, via intraperitoneal, no mesmo volume correspondente à dose de cisplatina; grupo A2 (controle positivo): 100mg/Kg/dia de NAC, via oral por gavagem; grupo A3 (ototóxico): 3mg/Kg/dia de cisplatina via intraperitoneal; grupo A4 (ototóxico com otoproteção): 100 mg/Kg/dia de NAC via oral por gavagem, uma hora antes da administração de 3 mg/Kg/dia de cisplatina via intraperitoneal; grupo B1 (controle negativo): solução fisiológica 0,9% via intraperitoneal no mesmo volume correspondente à dose de cisplatina (8mg/Kg/dia); grupo B2 (controle positivo): 300 mg/Kg/dia de NAC via oral por gavagem; grupo B3 (ototóxico): 8 mg/Kg/dia de cisplatina via intraperitoneal; grupo B4 (ototóxico com otoproteção): 300 mg/Kg/dia de NAC via oral por gavagem, uma hora antes da administração via intraperitoneal de 8 mg/Kg/dia de cisplatina. Os animais do experimento A realizaram otoscopia, emissões otoacústicas produto de distorção (EOAPD) e potencial evocado auditivo de tronco encefálico (PEATE), antes e depois da administração das drogas. Os animais do experimento B realizaram estas mesmas avaliações também no pré e pós-tratamento, além de terem suas bulas timpânicas removidas e suas cócleas preparadas para a avaliação anatômica com microscopia eletrônica de varredura e imunofluorescência para a marcação da enzima GSH-Px e da proteína Bcl-2. No experimento A, verificou-se que não houve diminuição significativa da relação sinal-ruído das EOAPD, porém houve aumento significativo do limiar eletrofisiológico obtido por PEATE nos grupos A3 e A4. No experimento B, verificou-se que: não houve aumento significativo do limiar eletrofisiológico obtido por PEATE; as CCEs mantiveram-se anatomicamente íntegras; a enzima GSH-Px apresentou imunomarcação ausente no grupo B1 e imunomarcação presente nos grupos B2, B3 e B4; a proteína Bcl-2 apresentou imunomarcação ausente em todos os grupos estudados. A partir dos resultados, concluiu-se que a função auditiva foi mais prejudicada com a exposição de uma subdose de cisplatina durante um período mais prolongado, a via de modulação da apoptose nas células ciliadas externas de ratos tratados com cisplatina está relacionada com a expressão da enzima GSH-Px e não expressão da proteína Bcl-2.
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