A dor associada ao câncer pode ser causada não somente pelos efeitos diretos ou indiretos da patologia primária, mas também pelo tratamento quimioterápico. A Cisplatina é um dos medicamentos anti-neoplásicos mais efetivos e mais comumente usados no tratamento de tumores sólidos. Entretanto, um de seus principais efeitos colaterais é a neurotoxicidade periférica. Os mecanismos celulares e moleculares da dor crônica induzida por quimioterápico são ainda bastante obscuros. Investigamos o papel da proteína quinase dependente de RNA (PKR) nos diferentes mecanismos neurobiológicos associados à dor crônica induzida pelo quimioterápico Cisplatina. O presente estudo avaliou: (1) O desenvolvimento de alodínia mecânica e hipernocicepção térmica em camundongos PKR-/- e PKR+/+ submetidos à administração do quimioterápico Cisplatina; (2) O estado de fosforilação das MAPKs (Erk1,2, p38 e JNK/SAP) e o fator de transcrição STAT-3 nas células do gânglio da raiz dorsal de animais tratados com Cisplatina; (3) Alterações na resistência e força muscular dos camundongos PKR-/- e PKR+/+ submetidos a administração do quimioterápico Cisplatina; (4) A proteólise muscular em músculos EDL (glicolíticos) e soleus (oxidativos) de camundongos PKR-/- e PKR+/+ após tratamento com Cisplatina (5) A síntese proteica através de Western Blot das proteínas Akt, FoxO 1 e FoxO 4, S6k1 e a S6 em células C2C12 analisando temporalmente o efeito da Cisplatina (6h, 12h e 24h); (6) O estresse oxidativo mitocondrial em células do gânglio da raiz dorsal e do músculo Soleus de camundongos PKR-/- e PKR+/+ após tratamento com Cisplatina. Os resultados obtidos foram: (1) Que com o tratamento com cisplatina produziu hipernocicepção térmica e alodínia mecânica nos animais PKR+/+; (2) A reduz da fosforilação do p38 não justifica o quadro de hipernocicepção e a hipernocicepção pode ocorrer a partir do aumento da fosforilação de STAT 3; (3) Animais que não tem a PKR são menos vulneráveis à ação deletéria da cisplatina sobre o músculo pois todos os testes comportamentais para atividade motora; (4) Não houve diferença na proteólise total mas sim na síntese proteica em animais PKR tratados com cisplatina; (5) Há alteração na via Akt quando analisa temporalmente a ação da cisplatina; (6) animais PKR -/- apresentaram índices mais altos da respiração mitocondrial comparados aos PKR +/+. Este estudo combinou métodos de biologia celular e molecular com paradigmas comportamentais a fim de investigar os possíveis mecanismos de ação da PKR no desenvolvimento de hipersensibilidade sensorial após o tratamento com quimioterápico. Os resultados deste trabalho devem evidenciar novos mecanismos neurobiológicos que contribuam para o entendimento da fisiopatologia da dor crônica de origem neurotóxica e apontar novas estratégias terapêuticas para o tratamento da dor crônica causada por quimioterapia / Pain associated with cancer can be caused by only direct or indirect products of the primary pathology, but also by chemotherapeutic treatment. Cisplatin is one of the most effective and most common anti-neoplastic drugs without the treatment of solid tumors. However, one of its major side effects is peripheral neurotoxicity. The cellular and molecular mechanisms of chronic pain induced by chemotherapy are still rather obscure. Investigators of role of RNA-dependent protein kinase (PKR) in the different neurobiological mechanisms associated with chronic pain induced by the chemotherapeutic Cisplatin. The present study evaluated: (1) the development of mechanical allodynia and thermal hypernociception in mice PKR - / - and PKR + / + submitted to chemotherapy Cisplatin; (2) The state of phosphorylation of the MAPKs (Erk1,2, p38 and JNK / SAP) and the transcription factor STAT-3 in the cells dorsal root ganglion of Cisplatin-treated animals; (3) Changes in muscle strength and strength of mice PKR - / - and PKR + / + submitted to the administration of the chemotherapeutic Cisplatin; (4) Muscle protein in EDL (glycolytic) and soleus (oxidative) muscles of mice PKR - / - and PKR + / + after treatment with Cisplatin (5) Western Blot Synthesis Protein of Akt, FoxO 1 and FoxO 4 S6k1 proteins and S6 in C2C12 cells by temporarily analyzing the effect of Cisplatin (6h, 12h and 24h); (6) Mitochondrial oxidative stress in dorsal root ganglion and Soleil muscle cells of PKR - / - and PKR + / + mice after treatment with Cisplatin. The results obtained were: (1) That with the treatment with cisplatin produced thermal hypernociception and mechanical allodynia in the animals PKR + / +; (2) The reduced phosphorylation of p38 does not justify the hyperencouragement and hypernociception can occur from increased STAT 3 phosphorylation; (3) Animals that do not have a PKR are less vulnerable to the deleterious action of cisplatin on muscle for all behavioral tests for motor activities; (4) There was no difference in total proteolysis but in protein synthesis in PKR animals treated with cisplatin; (5) There is alteration in the Akt pathway when the action of cisplatin is temporarily analyzed; (6) Animals PKR - / - had higher mitochondrial respiration rates compared to PKR + / +. This study combined methods of cellular and molecular biology with behavioral paradigms to investigate the possible mechanisms of action of PKR in the development of sensory hypersensitivity after treatment with chemotherapy. The results of this program are mandatory, which are responsible for the development of medicines for health and human health

Orientador: Edgard Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T18:26:23Z (GMT). No. of bitstreams: 1 Moreira_FernandadosSantos_M.pdf: 1478167 bytes, checksum: 57f6f26580823a79292af5a274beaa2d (MD5) Previous issue date: 2014 / Resumo: A ácido graxo sintase (FASN) é a principal enzima envolvida na lipogênese neoplásica e apontada como uma oncoproteína metabólica por favorecer o crescimento e sobrevivência das células tumorais, nas quais sua expressão é em geral elevada. Vários são os compostos capazes de inibir a atividade de FASN, dentre eles o orlistat (Xenical®), que possui efeitos antineoplásicos em cânceres de mama e próstata e nos melanomas. O carcinoma espinocelular (CEC) representa aproximadamente 90% de todas as neoplasias malignas que acometem a cavidade oral, sendo diagnosticado em estágios avançados em grande parte dos casos. Nestes pacientes, geralmente com metástases, é realizada uma abordagem sistêmica com agentes quimioterápicos como a cisplatina, o 5-fluorouracil (5-FU) e o paclitaxel, de maneira isolada ou combinada. Este trabalho tem como objetivo investigar in vitro se cisplatina, 5-FU ou paclitaxel potencializam o efeito antitumoral do orlistat no CEC oral. Para isto, células de CEC de língua altamente metastáticas (SCC-9 LN1) foram tratadas com estas drogas isoladamente ou combinadas com orlistat e avaliadas com relação às taxas de apoptose e necrose, progressão do ciclo celular e secreção de VEGFA e VEGFA165b. As maiores taxas de apoptose foram encontradas com o uso da combinação de orlistat com paclitaxel, após 48 horas de tratamento. Com relação ao ciclo celular, houve acúmulo de células na fase S com a combinação de orlistat com cisplatina e na fase G2 com a combinação de orlistat com paclitaxel. O tratamento das células SCC-9 LN1 com os agentes quimioterápicos reduziu a secreção dos fatores VEGFA e VEGFA165b, em comparação ao tratamento com orlistat isolado ou em combinação. Em conjunto, estes resultados mostram a existência de sinergismo na combinação de orlistat com paclitaxel com evidente potencialização do efeito pró-apoptótico nas células derivadas de CEC oral metastático / Abstract: Fatty acid synthase (FASN) is the main enzyme involved in the neoplastic lipogenesis. The expression of FASN is elevated in many tumor cells, suggesting a role as a metabolic oncoprotein, with the ability to promote growth and survival of tumor cells. There are several compounds that inhibit FASN activity, including orlistat. Orlistat has evident antineoplastic effects in breast and prostate cancers, as well as melanomas. Oral squamous cell carcinoma (OSCC), corresponds to approximately 90% of all cancers that affect the oral cavity, and is diagnosed in advanced stages in most cases. Distant metastases of OSCC are systemically treated with chemotherapeutic agents, such as cisplatin, 5-fluorouracil (5-FU) and paclitaxel. This study aims to investigate the in vitro antitumor effect of orlistat combined with cisplatin, 5-FU and/or paclitaxel in OSSC cells. Highly metastatic tongues squamous cell carcinoma cells (SCC-9-LN1) were treated with these drugs separately or combined with orlistat, in concentrations close to their respective IC50. Next, the cultures were evaluated regarding the rates of cell death (apoptosis and necrosis), cell cycle progression and the secretion VEGFA and VEGFA165b. The highest rate of apoptosis was observed with the combination of orlistat and paclitaxel, after 48 hours of treatment. Cell cycle analysis assay demonstrate as an accumulation of cells SCC-9 LN1 in the S phase, after incubation with the combination of orlistat with cisplatin, and in the G2 phase with orlistat plus paclitaxel. The 5-FU alone, promoted accumulation of cells in the G1/S. Additionally, secretion of VEGFA and was VEGFA165b was inhibited in SCC-9-LN1cells by the combined treatments. These results demonstrate the synergism existence of the mixture orlistat and paclitaxel with potentiation of their pro-apoptotic effects in SCC-9 LN1 cells / Mestrado / Estomatologia / Mestra em Estomatopatologia

Orientador: Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T23:45:07Z (GMT). No. of bitstreams: 1 Costa_ErickaFrancislaineDiasCosta_M.pdf: 2497812 bytes, checksum: e6a45e68bd7bd5e6338b3a0c6353c3d4 (MD5) Previous issue date: 2014 / Resumo: A cisplatina (CDDP) associada à radioterapia (RT) é utilizada no tratamento de portadores de carcinoma de células escamosas de cabeça e pescoço (CCECP). Já é conhecido que tanto a resposta ao tratamento como seus efeitos colaterais variam de indivíduo para indivíduo. Uma possível explicação para o fato pode ser a variabilidade genética no metabolismo da CDDP. O objetivo deste estudo é verificar se as habilidades herdadas para induzir apoptose de células danificadas, mediadas pelas enzimas P53, CASP3 e CASP9, alteram os efeitos colaterais, a concentração de CDDP urinária e a taxa de resposta à terapêutica em pacientes com CCECP. Foram avaliados, de forma prospectiva, 90 pacientes consecutivos com CCECP do Ambulatório de Oncologia Clínica do Hospital de Clínicas da UNICAMP, que receberam CDDP associada à RT como tratamento neoadjuvante, definitivo ou paliativo da doença. Os genótipos dos polimorfismos P53 Arg72Pro, CASP9 A-1263G, CASP9 C-712T e CASP3 A-928G foram analisados por meio da reação em cadeia da polimerase (PCR) e digestão enzimática em DNA de sangue periférico. Os efeitos colaterais ao tratamento foram graduados por meio de questionário e exames laboratoriais, de acordo com os critérios do National Cancer Institute. As toxicidades auditiva e renal foram avaliadas, respectivamente, por meio de audiometria tonal, clearance de creatinina estimado e taxa de filtração glomerular (TFG) com EDTA-51Cr, realizados antes e após o tratamento. As dosagens urinárias da CDDP foram realizadas por cromatografia líquida de alta eficiência. A resposta ao tratamento foi avaliada por tomografia computadorizada de pescoço, de acordo com os critérios Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) e por tomografia computadorizada por emissão de fóton único (SPECT-TC). O significado estatístico das diferenças entre grupos foi calculado pelo teste da probabilidade exata de Fisher ou qui-quadrado. A regressão logística múltipla foi feita para obter a razão das chances, e a ANOVA por transformação em postos foi realizada para medidas repetidas. O genótipo P53 72ProPro esteve associado com maior ocorrência de vômitos dos graus 2, 3 e 4 e os genótipos CASP3 -928 AA e AG estiveram associados com menor perda auditiva unilateral e menor nefrotoxicidade após terapêutica em nossos casos. Concluímos que os polimorfismos P53 Arg72Pro e CASP3 A-928G modulam os efeitos colaterais do tratamento de pacientes com CCECP com CDDP e RT, como vômitos, acuidade auditiva e nefrotoxicidade. Acreditamos que estes resultados podem contribuir para definir o tratamento personalizado futuro de pacientes com CCECP / Abstract: Cisplatin (CDDP) associated with radiotherapy (RT) is used in treatment of patients with squamous cell head and neck carcinoma (HNSCC). It is well known that both response to treatment and side effects vary among individuals. A possible explanation for this may be genetic variability in CDDP metabolism. The aim of this study was to assess if inherited ability of inducing damaged cells to apoptosis, mediated by P53, CASP3 and CASP9 enzymes, change side effects, urinary concentration of CDDP, and rate of response to therapy in HNSCC patients. We evaluated prospectively, 90 HNSCC patients of Outpatient Oncology Clinic of UNICAMP¿s Clinical Hospital, who received CDDP associated to RT as neoadjuvant, definitive or palliative treatment. Genotypes of P53 Arg72Pro, CASP9 A-1263G, CASP9 C-712T and CASP3 A-928G, polymorphisms were analyzed by polymerase chain reaction (PCR) and restriction enzyme digestion of peripheral blood DNA. Treatment side effects were ranked by questionnaire and laboratory tests, according to criteria of National Cancer Institute. Hearing and renal toxicities were assessed using, respectively, audiometry, estimated creatinine clearance and EDTA-51Cr glomerular filtration rate (GFR), measured before and after treatment. CDDP Urinary dosages were measured by high performance liquid chromatography. Treatment response was assessed by computed tomography of neck, according to Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) and by Single Photon Emission Computed Tomography (SPECT-CT). Statistical significance of differences between groups was calculated by Fisher's exact probability test or chi-square. Multiple logistic regression was performed to obtain odds ratio, and ANOVA with rank-transform method was performed for repeated measures. P53 72ProPro genotype was associated with vomiting of grades 2, 3 and 4 and CASP3 -928 AA and AG genotypes were associated with lower unilateral hearing loss and lower nephrotoxicity after therapy in our cases. We conclude that P53 Arg72Pro and CASP3 A-928G polymorphisms modulate the side effects, such as vomiting, hearing thresholds decrease and nephrotoxicity, in HNSCC treated with CDDP and RT. We believe these results may contribute to definition of future personalized treatment of HNSCC patients / Mestrado / Clinica Medica / Mestra em Clínica Médica

Orientador: Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T03:53:51Z (GMT). No. of bitstreams: 1 Nogueira_GuilhermeAugustodaSilva_M.pdf: 2742030 bytes, checksum: f23fb1e053f1302891b66643feebad7b (MD5) Previous issue date: 2014 / Resumo: A cisplatina (CDDP) associada à radioterapia (RT) é utilizada no tratamento de portadores de células escamosas de cabeça e pescoço (CCECP). Já é conhecido que tanto a resposta ao tratamento como seus efeitos colaterais variam de indivíduo para indivíduo. Uma possível explicação para o fato pode ser a variabilidade genética no metabolismo da CDDP. O objetivo deste estudo foi o de verificar se habilidades herdadas no reparo de lesões do DNA, mediadas pelas enzimas MLH1, MSH2, MSH3, e EXO1, alteram os efeitos terapêuticos, colaterais e a concentração de CDDP urinária em pacientes com CCECP. Foram avaliados, de forma prospectiva, 90 pacientes consecutivos com CCECP do Hospital de Clínicas da UNICAMP, que receberam CDDP associada à RT como tratamento neoadjuvante, definitivo ou paliativo da doença. Os genótipos dos polimorfismos MLH1 G-93A, MSH2 IVS1+9G>C, MSH3 Ala1045Thr, EXO1 Pro757Leu e EXO1 Glu589Lys foram analisados por meio da reação em cadeia da polimerase (PCR) e digestão enzimática ou PCR em tempo real em DNA de sangue periférico. A resposta ao tratamento foi avaliada por tomografia computadorizada do pescoço, de acordo com os critérios Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1). Os efeitos colaterais ao tratamento foram graduados por meio de questionário e exames laboratoriais, de acordo com os critérios do National Cancer Institute 4.0. As toxicidades renal e auditiva foram avaliadas por meio do clearance de creatinina estimado, da taxa de filtração glomerular com EDTA-51Cr e de audiometria tonal, respectivamente, medidas antes e após o tratamento. As dosagens urinárias da CDDP foram realizadas por cromatografia líquida de alta eficiência. O significado estatístico das diferenças entre grupos foi calculado pelo teste da probabilidade exata de Fisher ou qui-quadrado e regressão logística múltipla. O genótipo MLH1 GG+GA esteve associado com menor ocorrência de náuseas. O genótipo MSH2 CC esteve associado com melhora da acuidade auditiva e tendência a menor excreção de CDDP na urina. Os genótipos MSH3 AlaAla+AlaThr e AlaAla estiveram associados com maior ototoxicidade e maior nefrotoxicidade, e piora da acuidade auditiva, respectivamente. O genótipo EXO1 ProLeu esteve associado com maior ototoxicidade e piora da acuidade aditiva. Os genótipos EXO1 GluLys+LysLys e LysLys estiveram associados com menor resposta completa, e piora da acuidade auditiva e tendência a nefrotoxicidade, respectivamente. Concluímos que os referidos polimorfismos, relacionados a anormalidades herdadas no reparo de DNA, podem alterar a taxa de resposta, os efeitos colaterais e a eliminação urinária de CDDP em pacientes com CCECP tratados com CDDP e RT. Acreditamos que estes resultados possam contribuir para o tratamento personalizado futuro de pacientes com o tumor / Abstract: Cisplatin (CDDP) associated with radiotherapy (RT) is used in treatment of patients with head and neck squamous cell carcinoma (HNSCC). It is well known that both response to treatment and side effects vary among individuals. A possible explanation for this may be the genetic variability in metabolism of CDDP. The aim of this study was to acess if inherited ability to repair DNA damage, mediated by MLH1, MSH2, MSH3 and EXO1 enzymes change the therapeutic side effects and urinary concentration of CDDP in HNSCC patients. We evaluated prospectively, 90 consecutive HNSCC patients of UNICAMP¿s Hospital, who received CDDP-associated RT as neoadjuvant, definitive or palliative treatment. Genotypes of MLH1 G-93A, MSH2 IVS1+9G>C, MSH3 Ala1045Thr, EXO1 P757L and EXO1 K589E polymorphisms were analyzed by polymerase chain reaction (PCR) and restriction enzyme digestion or real-time PCR in DNA of peripheral blood. Treatment response was assessed by computed tomography of the neck, according to Response Evaluation Criteria in Solid Tumors 1.1. Treatment side effects were ranked through questionnaire and laboratory tests, according to the National Cancer Institute 4.0. Renal and hearing toxicities were assessed using, respectively, estimated creatinine clearance and glomerular filtration 51Cr-EDTA, and audiometry, measured before and after treatment. Urinary doses of CDDP were performed by high performance liquid chromatography. Statistical significance of differences between groups was calculated by Fisher's exact probability test or chi-square, and multiple logistic regression. MLH1 GG+GA genotype was associated with lower incidence of nausea. MSH2 CC genotype was associated with improvement of hearing acuity and tendency of lower CDDP urinary excretion. MSH3 AlaAla+AlaThr and AlaAla genotypes were associated with high ototoxicity and high nephrotoxicity, and worsening of hearing acuity, respectively. EXO1 ProLeu genotype was associated with high ototoxicity and worsening of hearing acuity. EXO1 GluLys+LysLys and LysLys genotypes were associated with lower complete response, and worsening of hearing acuity and and tendency to high nephrotoxicity, respectively. We conclude that the referred polymorphisms, related to inherited abnormalities in DNA repair, may change rate of complete response, side effects, and CDDP urinary concentration in patients with HNSCC treated with CDDP and RT. We believe that these results may contribute to the future personalized treatment of HNSCC patients / Mestrado / Fisiopatologia Médica / Mestre em Ciências

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Magister Scientiae - MSc / Apoptosis is a process in which a cell programmes its own death. It is a highly organized physiological mechanism in which injured or damaged cells are destroyed. Apart from physiological stimuli however, exogenous factors can induce apoptosis. Many anti-cancer drugs work by activating apoptosis in cancer cells. Natural substances have been found to have the ability to induce apoptosis in various tumour cells and these substances have been used as templates for the construction of novel lead compounds in anticancer treatment. On the other hand, alkylating agents such as cisplatin, cis- [PtCl2 (NH3) 2] have been widely used as antineoplastic agents for a wide variety of cancers including testicular, ovarian, neck and head cancers, amongst others. However, the use of cisplatin as an anticancer agent is limited due to toxicity and resistance problems. The aim of this present study was to screen the leaves of Rhus laevigata, a South African indigenous plant, for the presence of pro-apoptotic and anti-proliferative natural compounds and also to screen newly synthesised palladium based complexes (15 and 57) and a platinum based complex (58) for their antineoplastic activities tested against a panel of cell lines. Results. The results showed that crude methanol extracts from Rhus laevigata as well as the newly synthesised palladium based complexes (15 and 57) and a platinum based complex (58) induced apoptosis in the cell lines tested, as demonstrated by the externalization of phosphatidylserine, mitochondrial membrane permeabilization, caspase-3 activation, and DNA fragmentation. Caski (cervical cancer) and H157 (non small cell lung carcinoma) cell lines treated with the methanol extract from Rhus laevigata however, were more resistant to apoptosis induction. Among the metallocomplexes, complexes 15 and 57, palladium based complexes, were the most active. Conclusion The methanol extract from the leaves of Rhus laevigata contain pro-apoptotic and antiproliferative natural compound(s), which need to be characterised and elucidated as they could provide the much-needed lead compounds in the fight against cancer. On the other hand the newly synthesized palladium complexes also need further evaluation to see if they can be used as anticancer agents that can overcome the problems associated with cisplatin.

Magister Scientiae - MSc / Background and objectives: Apoptosis is a process in which a cell programmes its own death. It is a highly organized physiological mechanism in which injured or damaged cells are destroyed. Apart from physiological stimuli however, exogenous factors can induce apoptosis. Many anti-cancer drugs work by activating apoptosis in cancer cells. Natural substances have been found to have the ability to induce apoptosis in various tumour cells and these substances have been used as templates for the construction of novel lead compounds in anticancer treatment. On the other hand, alkylating agents such as cisplatin, cis- [PtCl2 (NH3) 2] have been widely used as antineoplastic agents for a wide variety of cancers including testicular, ovarian, neck and head cancers, amongst others. However, the use of cisplatin as an anticancer agent is limited due to toxicity and resistance problems. The aim of this present study was to screen the leaves of Rhus laevigata, a South African indigenous plant, for the presence of pro-apoptotic and anti-proliferative natural compounds and also to screen newly synthesised palladium based complexes (15 and 57) and a platinum based complex (58) for their antineoplastic activities tested against a panel of cell lines. Results. The results showed that crude methanol extracts from Rhus laevigata as well as the newly synthesised palladium based complexes (15 and 57) and a platinum based complex (58) induced apoptosis in the cell lines tested, as demonstrated by the externalization of phosphatidylserine, mitochondrial membrane permeabilization,caspase-3 activation, and DNA fragmentation. Caski (cervical cancer) and H157 (non small cell lung carcinoma) cell lines treated with the methanol extract from Rhus laevigata however, were more resistant to apoptosis induction. Among the metallocomplexes, complexes 15 and 57, palladium based complexes, were the most active. Conclusion: The methanol extract from the leaves of Rhus laevigata contain pro-apoptotic and antiproliferative natural compound(s), which need to be characterised and elucidated as they could provide the much-needed lead compounds in the fight against cancer. On the other hand the newly synthesized palladium complexes also need further evaluation to see if they can be used as anticancer agents that can overcome the problems associated with cisplatin. / South Africa

Orientador: Carmen Silvia Passos Lima / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T18:37:49Z (GMT). No. of bitstreams: 1 Pincinato_EderdeCarvalho_D.pdf: 3229024 bytes, checksum: a92cc669d0c70abe36aab0ff261da986 (MD5) Previous issue date: 2015 / Resumo: A cisplatina (CDDP) associada à radioterapia (RT) é utilizada no tratamento de portadores de carcinoma de células escamosas de cabeça e pescoço (CCECP). A resposta ao tratamento bem como seus efeitos colaterais variam de indivíduo para indivíduo e tal fato pode ser explicado pela variabilidade genética no metabolismo da CDDP. O objetivo deste estudo foi o de verificar se habilidades herdadas na detoxificação da CDDP, mediadas pelas enzimas GSTM1, GSTT1 e GSTP1, alteram os efeitos terapêuticos, colaterais e a concentração de CDDP urinária em pacientes com CCECP. Foram avaliados, de forma prospectiva, 90 pacientes com CCECP tratados com CDDP associada à RT. Os genótipos dos polimorfismos GSTM1, GSTT1 e GSTP1 Ile105Val foram analisados por meio da reação em cadeia da polimerase (PCR) multiplex e PCR seguida de digestão enzimática, respectivamente, em DNA de sangue periférico. A resposta ao tratamento foi avaliada por exame clínico e tomografia computadorizada do pescoço. Os efeitos colaterais ao tratamento foram graduados por questionários e exames laboratoriais. As toxicidades renal e auditiva foram avaliadas por clearance de creatinina estimado (ClCrea), taxa de filtração glomerular com EDTA-51Cr (TFG) e audiometria tonal limiar. As concentrações urinárias da CDDP foram realizadas por cromatografia líquida de alta eficiência (HPLC). Pacientes com a deleção homozigótica do gene GSTT1 estiveram sob risco 0,09 (IC 95%: 0,02-0,41) vezes menor de ocorrência de vômitos, 0,22 (IC 95%: 0,05-0,89) vezes menor de ototoxicidade e menor redução de ClCrea (81,69 ± 21,40 para 84,13 ± 25,69 versus 93,16 ± 28,94 para 77,52 ± 23,96 mL/min/1,73m²) e de TFG (79,56 ± 20,68 para 69,94 ± 21,40 versus 84,38 ± 19,96 para 62,87 ± 20,72 mL/min/1,73m²) do que os portadores do gene. Já os pacientes com o alelo Val do polimorfismo GSTP1 Ile105Val estiveram sob risco 6,32 vezes maior de ocorrência de vômitos acentuados (IC 95%: 2,05-19,51), 3,35 vezes maior de ototoxicidade acentuada (IC 95%: 1,03-10,96) e maior redução de TFG (80,87 ± 21,73 para 66,97 ± 24,96 versus 85,35 ± 18,76 para 62,19 ± 17,40 mL/min/1,73m²) do que os portadores do genótipo Ile/Ile. Maior concentração urinária de CDDP foi observada em pacientes com a deleção homozigótica do gene GSTT1, quando comparados aos pacientes com a presença do gene (429,58 ± 116,24 versus 253,42 ± 95,20 ug CDDP/mg creatinina). Concluímos que estes polimorfismos desempenham papéis importantes na ocorrência de eventos adversos da terapêutica e excreção urinária do CDDP. Acreditamos que estes resultados possam constituir a base preliminar para o tratamento personalizado futuro de pacientes com CCECP / Abstract: Cisplatin (CDDP) associated with radiotherapy (RT) is used in treatment of patients with head and neck squamous cell carcinoma (HNSCC). The response to treatment as well as its side effects vary among individuals, and this fact may be explaned by the genetic variability in metabolism of CDDP. The aim of this study was to access if inherited ability to cellular CDDP detoxification, mediated by GSTM1, GSTT1 and GSTP1 enzymes alters the therapeutic and side effects of CDDP and RT and urinary concentration of CDDP in HNSCC patients. We evaluated, prospectively, 90 consecutive HNSCC patients, who received CDDP associated RT as treatment. Genotypes of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms were analyzed by multiplex polymerase chain reaction (PCR) and PCR followed by restriction enzyme digestion, respectively, in peripheral blood DNA. Treatment response was assessed by clinical examination and computed tomography of neck. Treatment side effects were ranked through questionnaire and laboratory tests. Renal and hearing toxicities were assessed using, respectively, estimated creatinine clearance and glomerular filtration 51Cr-EDTA and pure tone threshold audiometry. Urinary doses of CDDP were performed by high performance liquid chromatography (HPLC). Patients with the GSTT1-null genotype had a 0.09 times (95% CI:0.02-0.41) decreased risk for vomiting, 0.22 times (95% CI:0.05-0.89) decreased risk for ototoxicity, less creatinine clearance decreases (81.69 ± 21.40 to 84.13 ± 25.69 versus 93.16 ± 28.94 to 77.52 ± 23.96 mL/min/1.73m²) and glomerular filtration 51Cr-EDTA (79.56 ± 20.68 to 69.94 ± 21.40 versus 84.38 ± 19.96 to 62.87 ± 20.72 mL/min/1.73m²) than GSTT1-present genotype. Patients with Val allele of GSTP1 Ile105Val polymorphism had 6.32 higher risk presenting high grade vomiting (95% CI: 2.05-19.51), 3.35 higher risk to high grade ototoxicity (95% CI: 1.03-10.96) and higher glomerular filtration 51Cr-EDTA reduction (80.87 ± 21.73 to 66.97 ± 24.96 versus 85.35 ± 18.76 to 62.19 ± 17.40 mL/min/1.73m²) when compared to wild genotype. Higher CDDP urinary level was observed in patients with the GSTT1-null genotype, compared to patients with the gene (429.58 ± 116.24 versus 253.42 ± 95.20 ug CDDP/mg creatinine). We concluded that these genetic polymorphisms have important roles in complete response rate, in occurrence of side effects, and in urinary CDDP excretion. We believe that this data may constitute preliminary basis of future personalized treatment of HNSCC patients / Doutorado / Clinica Medica / Doutor em Clínica Médica

Les dérivés de platine (cisplatine, oxaliplatine et carboplatine) et les taxanes (paclitaxel et docétaxel) font partie des grandes familles d’agents anticancéreux couramment utilisés en chimiothérapie. Ils permettent de traiter les tumeurs solides telles que les cancers de l’ovaire, du poumon, de la prostate, du sein, tête et cou. Cependant, ils sont à l’origine d’importantes neuropathies périphériques qui peuvent devenir irréversibles et laisser les patients avec des séquelles importantes. Il s'agit de vertiges, acouphènes, engourdissements, pertes de sensibilité, allodynies au toucher ou aux variations de température. Ces effets secondaires sont fortement contraignants et réduisent considérablement la qualité de vie de 30 à 50% des patients. L'importance clinique est considérable, puisque cela conduit environ 1/3 des patients atteints à l’arrêt et/ou au changement des traitements, bien que ceux-ci soient efficaces sur les tumeurs.Les dérivés de platine sont des agents pontants : leurs interactions avec les bases purines de l’ADN forment des adduits, qui vont être reconnus par des protéines de dommage à l’ADN, conduisant à la mort cellulaire. Les taxanes bloquent la dépolymérisation des microtubules, ce qui induit la mort des cellules en divisions. Le fait que deux mécanismes d’action antitumorale très différents provoquent, à court ou à moyen terme, les mêmes neuropathies périphériques, est paradoxal et n’a pas d’explication.Le but de mon projet de thèse était d’étudier le ou les mécanismes à l’origine de ces neuropathies et de développer une formulation visant à les réduire. Pour cela, j’ai étudié les effets des taxanes et dérivés de platine sur les canaux ioniques, impliqués dans la nociception. Ces canaux ioniques, appelés nocicepteurs, sont transcrits dans les corps cellulaires des ganglions spinaux (DRG), avant d’être principalement adressés à l’extrémité des nerfs périphériques. Ils détectent les stimuli mécaniques, thermiques et chimiques et génèrent ou transmettent les potentiels d’action correspondants. Le fait de perturber l’expression génique et/ou l’activité de ces nocicepteurs, aura donc pour conséquence de modifier les seuils de sensibilité et la transmission de différents stimuli.Mes résultats ont permis de quantifier dans des cultures primaires de DRG et in vivo chez la souris, le remodelage génique de nocicepteurs induit par les traitements de chimiothérapie, en corrélation avec l’apparition de neuropathies périphériques que j'ai mesurées par des tests comportementaux. Cela nous a permis d'identifier une famille de molécules candidates, qui pourraient potentiellement contrer le mécanisme identifié dans ces travaux. L'une de ces molécules permet de rétablir l’expression génique de nocicepteurs et aussi de supprimer les neuropathies périphériques chez la souris. Ce travail devrait se poursuivre dans le cadre d'un processus de valorisation, ayant pour objectif d'aboutir à un traitement préventif et/ou curatif, de ces neuropathies chez les patients. / Platinum-based drugs (cisplatin, oxaliplatin and carboplatin) and taxanes (paclitaxel and docetaxel) are among the most common drugs families used in chemotherapy. They are used for treatment of numerous human cancers including bladder, breast, head and neck, lung, ovarian, prostate and testicular cancers. However, these anticancer drugs cause significant peripheral neuropathies, that can become irreversible and leave serious clinical sequelae in patients. These include tinnitus, dizziness, tingling, numbness, loss of sensitivity, allodynia to touch or temperature changes and hyperalgesia. These side effects are highly restrictive and significantly reduce the quality of life of 30-50% of patients. The clinical significance is considerable, since it leads to about one-third of patients with stopping and/or changing treatments, although these are effective on tumors.Platinum-based drugs are DNA binding agents: they generate DNA lesions on the purine bases of DNA, that will be recognized by DNA damage response proteins, leading to cell death. Taxanes block the cell cycle in mitosis, by stabilizing the microtubule cytoskeleton against depolymerization. The fact that these two completely different antitumor mechanisms of action, induce the same peripheral neuropathies in the short to medium term, is paradoxical and has no explanation.The aim of my PhD research project was to study the mechanism(s) behind these neuropathies and to develop a new formulation to prevent and/or reduce them. Therefore, I studied the effects of taxanes and platinum-based drugs on several ion channels, involved in nociception. These ion channels, called nociceptors, are transcribed into the cell bodies of dorsal root ganglia (DRGs) and located to the peripheral nerve endings. They detect mechanical, thermal and chemical stimuli and generate or transmit the corresponding action potentials. Changes in gene expression and/or activity of nociceptors, will therefore modify the nociceptive thresholds and the transmission of different stimuli.My results allowed me to quantify, in primary cell culture of mouse DRGs and in vivo, the remodeling of ion channel expression induced by chemotherapy, in correlation with the development of peripheral neuropathies, that I measured by behavioral assessment. These results allowed us to identify a family of candidate molecules, that could potentially counteract the mechanism identified in this work. I showed that one of these molecules, can restore the gene expression of nociceptors and suppress peripheral neuropathies in mice. This work should continue as part of a valorization process, aiming to lead to a preventive and/or curative treatment, of these neuropathies in patients.

In the 1960s, Rosenberg and his colleagues confirmed the anti-cancer activity of cisplatin. Although cisplatin was capable of killing testicular cancer cells there were also serious side effects. It was necessary to find alternate ways of overcoming side effects, and soon many researchers have discovered novel platinum compounds that show similar reactivity. Recently, replacing one chloride group to a heterocyclic amine group showed significant cytotoxicity with a different binding activity than cisplatin. Previously in our lab, [Pt(Me5dien)(NO3)]+ and [Pt(Et2dien)Cl]+ have been synthesized and reacted with NAcetyl- L-methionine (N-AcMet) and Guanosine 5’-monophosphate (5’-GMP) showed unusual reactivity. Unlike most previously studied platinum triamine compounds, Me5dien compound was reacting faster with 5’-GMP than N-AcMet, due to the bulkiness of the triamine ligand. When both N-AcMet and 5’-GMP were reacted with Et2dien, 5’- GMP displaced one amine group of the triamine ligand and replaced that spot to form a bis-adducts, when the pH was kept below 4. Here a new novel platinum compound has been synthesized with a seven-membered chelate ring triamine ligand, Chloro[2-(4- methyl-1,4-diazepan-1-yl)ethanamine]platinum(II) chloride ([Pt(L)Cl]+). The unusual binding activity of [Pt(L)Cl]+ showed a unique pair of products under 1H NMR, 195Pt NMR and LC/MS spectrometry.