CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Avaliou-se a expressÃo das proteÃnas bax, bcl-2 e P27 no carcinoma espinocelular invasivo da cavidade bucal atravÃs da tÃcnica da imunohistoquÃmica para conhecer-se o perfil apoptÃtico destes tumores. Buscou-se detectar os fatores epidemiolÃgicos e o comportamento clÃnico dos pacientes acometidos por esta neoplasia. Procurou-se empregar parÃmetros estatÃsticos diferentes do odds ratio para comparaÃÃes posteriores. Analisando-se quarenta e oito pacientes da Santa Casa da MisericÃrdia de Fortaleza, Cearà foi possÃvel conhecer os fatores sexo, escolaridade, renda familiar, tabagismo (forma e tempo de consumo,tempo de abandono, quantidade consumida), etilismo(tipo de bebida, tempo de consumo, quantidade consumida, freqÃÃncia). Com a anÃlise do laudo histopatolÃgico da peÃa cirÃrgica soube-se das informaÃÃes referentes ao tumor em si (localizaÃÃo, gradaÃÃo histolÃgica, tamanho, linfonodos envolvidos). Obteve-se fragmentos da peÃas cirÃrgicas e foram confeccionadas lÃminas para a verificaÃÃo da invasividade atravÃs da coloraÃÃo em hematoxilina e eosina. Em seguida foi realizada a reaÃÃo de imunohistoquÃmica foi pelo mÃtodo da estrepto-avidina-biotina-peroxidase objetivando-se avaliar a expressÃo das proteÃnas supracitadas. As lÃminas com marcaÃÃo positiva foram submetidas a contagem de no mÃnimo mil cÃlulas e este resultado foi empregado nos mÃtodos LI (Labelling Index) e HS(HScore). A avaliaÃÃo dos resultados foi atravÃs de mÃtodos descritivos, sendo os testes realizados o qui-quadrado e o teste exato de Fisher com nÃvel de significÃncia a dez por cento.Os resultados apontaram uma mÃdia de idade de cinqÃenta e sete anos e o maior nÃmero de indivÃduos do sexo masculino, analfabetos, com renda familiar de um salÃrio-mÃnimo na sua maioria. A forma de consumo de tabaco, em maior nÃmero encontrada, foi o cigarro industrializado com vinte anos de consumo. A ingestÃo de bebidas destiladas superou a de fermentadas com mÃdia de vinte anos. O soalho bucal foi a localizaÃÃo com maior nÃmero de casos, com uma mÃdia de tamanho de trÃs e meio centÃmetros, o estadiamento patolÃgico predominante foi o pT2, com gradaÃÃo histolÃgica moderadamente diferenciada em maior quantidade. Dentre os cruzamentos realizados, houve correlaÃÃo estatÃstica entre o tamanho de tumores com as faixas etÃrias(P=0,084 para α=10%); tamanho dos tumores com envolvimento de linfonodos(P=0,085 para α=10%); sexo dos pacientes com envolvimento dos linfonodos (P=0,03 para α=10%). Os resultados das reaÃÃes de imunohistoquÃmica mostraram um maior percentual de casos positivos para bax (77,1%) seguido de P27(45,9%) e bcl-2 (16,6%). As mÃdias dos LI encontradas apontaram bax com 67, 766; seguida de bcl-2 com 10,804; e P27 com 7,989. Cruzando-se os H-scores dos marcadores entre si encontrou-se correlaÃÃo positiva entre bax e P27 (0,245 na correlaÃÃo de Pearson). Os parÃmetros estatÃsticos avaliados foram: mÃdia e seu erro padrÃo; mediana; moda; desvio padrÃo; curtose e seu erro padrÃo; mÃnimo; mÃximo e quartis. Os resultados mostram uma tendÃncia a apoptose nos carcinomas espinocelulares bucais / The evaluation of the expression of bax, bcl-2 and p27 proteins at invasive squamous cell carcinoma of oral cavity was done using the immunohistochemistry technique to know about apoptotic profile of these neoplasms. The epidemiological factors and the clinical behavior of the patients were detected, too. Statistical parameters different from odds ratio was employed for future comparisons. Forty-eight patients from Santa Casa da MisericÃrdia de Fortaleza, CearÃ, Brazil was analyzed and with this analysis was possible to know the prognostic factors: sex of patients, instruction grade, familiar gains, tobacco consumption (form of consumption, duration of consumption, period of forsaking and quantity consumed), alcohol consumption (nature of drinking, duration of consumption, period of forsaking, quantity consumed, periodicity). In a posterior moment, with the histopathological report from the surgical specimen was possible to know about localization of neoplasm, size of neoplasm, histological grade, nodes involvement and invasiveness. Pieces of the neoplasm were achieved from surgical specimen and glass slides were done to analyze the invasiveness by hematoxilin-eosin coloration. After the immunohistochemistry reaction by streptoavidin biotin technique was done to evaluate the expression of proteins above. The glass slides with positive reaction were submitted under a counting and, at least, one thousand cells were counted. The results from counting were submitted under the LI(Labelling Index) and HS(H Score) methods. The evaluation of the results was made using descriptive methods and the statistical tests were qui-square and Fisherâs exact test with 10% significance. The results showed the mean age was 57 years old, more males than females, analphabets and one minimum wage the mean familiar gain. The main form of tobacco consumption was industrialized cigarette with 20 years of consumption. The swallow of distillers drinking was bigger than fermentation ones with 20 years of drinking at mean. The floor of the mouth was the anatomic site with more number of cases and the mean size of neoplasm was 1.4 inches. The preponderant pathological staging detected was pT2 and the preponderant histological grade was moderately differentiated. Among the cross tabs realized, there were statistical correlation between size of tumors and age (P=0.084; α=10%), between size of tumors and nodes involvement (P=0,085; α=10%), between sex of patients and nodes involvement (P=0.03; α=10%). The results from immunohistochemical reactions were more positive to bax (77.1% of positive cases), P27(45.9% of positive cases) and bcl-2 (16.6% of positive cases). The mean of LI showed bax at first position (67.766); second bcl-2 (10.804) and P27(7.989). The cross tabs among HS showed statistical positive correlation between bax and P27 (0.245 at Pearsonâs correlation). The statistical parameters were: mean and its standard error, median, mode, standard deviation, kurtosis and its standard error, minimum, maximum and percentiles. The conclusions showed apoptosis propensity at oral squamous cell carcinoma

INTRODUÇÃO: O carcinoma de pulmão de pequenas células (CPPC) e o carcinoma neuroendócrino (CNE) extra-pulmonar apresentam características histopatológicas e tratamentos similares, porém os desfechos encontrados nos dois grupos podem ser diferentes. Avaliamos a expressão de alguns biomarcadores e a associação destes com taxa de resposta (TR) à quimioterapia baseada em platina e sobrevida global (SG) nos dois grupos. METODOS: Realizamos estudo retrospectivo de pacientes com CPPC e CNE extra-pulmonares tratados com quimioterapia baseada em platina. Todas as amostras tumorais foram revisadas pelo mesmo patologista (R.S.S.M.) e analisadas quanto a expressão imunoistoquímica de Ki-67, ERCC1, Bcl-2 e Lin28a, a qual foi determinada através do H-escore (calculado multiplicando o produto da intensidade da coloração - 0 a 3 - com a porcentagem de células positivas - 0 a 100 -, podendo variar de 0 a 300 - positivo quando >= 200). Os biomarcadores foram analisados tanto como variáveis contínuas quanto categóricas e a TR foi determinada por RECIST 1.1. A associação entre a expressão de cada biomarcador e a TR foi avaliada através do teste de qui-quadrado ou teste exato de Fisher para variáveis categóricas e regressão logística simples para variáveis contínuas. Sobrevida global foi estimada por Kaplan-Meier e as curvas foram comparadas por log-rank. O modelo de regressão de cox foi utilizado para avaliar associação entre SG e a expressão de biomarcadores como variável contínua. RESULTADOS: Entre Julho de 2006 e Julho de 2014, 142 pacientes foram identificados: N=82 (57,7%) com CPPC e N=60 (42,3%) com CNE extra-pulmonar. As características clínicas eram semelhantes em ambos os grupos. Mediana de ki67 foi de 60% (7-100) no CPPC e de 50% (20-95%) no segundo grupo (p=0,858). Com uma mediana de 5 ciclos por paciente (N=123 elegíveis para análise de TR), a TR foi de 86,8% no CPPC, enquanto nos com CNE extra-pulmonar, foi de 44,6% (p < 0.001). A mediana de SG (N=132 elegíveis para análise da SG) foi similar entre os grupos (10,3 meses em CPPC e 11,1 meses em CNE extra-pulmonar; p=0,069). Não houve diferença no padrão de expressão do ERCC1 (p=0,277) e do Lin28a (p=0,051) entre os grupos. Bcl2 foi expresso em 38 pacientes (46,3%) com CPPC e em 17 pacientes (28,3%) com CNE extra-pulmonar (p=0,030). Apenas no grupo com CNE extra-pulmonar, a alta expressão do Bcl2 foi associada com pior prognóstico (8,0 meses vs 14,7 meses; p=0,025). A expressão dos demais marcadores em CNE extra-pulmonar e dos quatro em CPPC não apresentou influência sobre a SG, não havendo também associação entre estes e a taxa de resposta à quimioterapia. Dentre os pacientes com CNE extra-pulmonar, não houve diferença na SG ou na TR entre os pacientes com carcinoma bem diferenciado (N=13;) e com carcinoma pouco diferenciado (N=47). CONCLUSÃO: Apesar do CPPC e do CNE extra-pulmonar serem tratados de forma semelhante, nesta coorte a taxa de resposta entre os grupos foi significativamente diferente. Quando comparado com CPPC, os pacientes com CNE extra-pulmonar apresentam uma menor responsividade à quimioterapia baseada em platina, mas com tendência a maior SG. Dentre os CNE extra-pulmonares, a alta expressão de Bcl-2 foi associada a pior prognóstico. Os demais biomarcadores não apresentaram papel preditor de resposta ou prognóstico / INTRODUCTION: Small cell lung cancer (SCLC) and high-grade extrapulmonary neuroendocrine carcinomas (EPNEC) share similar histopathological features and treatment, but outcomes may differ. We evaluated the expression of biomarkers and their association with response rate (RR) to platin-based chemotherapy and overall survival (OS) in these entities. METHODS: We conducted a retrospective analysis of patients with advanced EPNEC and SCLC treated with platinum-based chemotherapy. A single pathologist (R.S.S.M.) revised all samples. Paraffin-embedded tumor samples were tested for Ki-67, ERCC1, Bcl-2 and Lin28a expression by immunohistochemistry (IHC). Final IHC score (H-score) was calculated multiplying the intensity of staining by grading (0-300, with >= 200 considered positive). Biomarkers were analyzed as both categorical and continuous variables. RR was determined by RECIST 1.1. Associations between each biomarkers expression and RR were assessed using Chi-square or Fisher\'s exact test for categorical variables and univariate logistic regression for continuous variables. OS was estimated by the Kaplan-Meier method and curves were compared by log-rank. Cox regression analysis was used to evaluate any association between biomarkers expression (continuous variables) and OS. RESULTS: From July 2006 to July 2014, 142 patients were identified: N=82 (57,7%) with SCLC and N=60 (42,3%) with EPNEC. Baseline clinical characteristics were similar. Median Ki67 was 60% (7-100) among SCLC patients and 50% (20-95%) in EPNEC (p=0,858). With a median of 5 cycles per patient in both groups (N=123 evaluable patients), the RR was significantly higher in the SCLC group (86,8% vs 44.6%; p < 0.001). Median OS (N=132 evaluable patients) was similar between the groups (10.3 months in SCLC and 11.1 months in EPNEC; p=0,069). In the EPNEC group, there wasn\'t any difference in OS or RR between the patients with welldifferentiated (N=13) and poorly differentiated carcinoma (N=47). ERCC1 (p=0.277) and Lin28a (p=0.051) were similarly expressed between the groups. Bcl2 was expressed in 38 SCLC patients (46.3%) and in 17 EPNEC patients (28.3%; p=0.030). Only in the EPNEC group, Bcl2 high expression was associated with worse survival (8.0 months vs 14.7 months; p = 0.025). RR to chemotherapy was not influenced by the expression of the ERCC1, Lin28a, Bcl-2, Ki-67 in either EPNEC or SCLC groups. CONCLUSION: Even though SCLC and EPNEC are treated similarly, in this cohort, the rate response differed significantly. When compared with SCLC, patients with EPNEC apparently had tumors less responsive to platin-based chemotherapy, but tended to live longer. In EPNEC treated with platin, high expression of Bcl2 was associated with poor prognosis. We could not identify additional predictive or prognostic biomarkers

L’amélioration du développement du médicament est un véritable défi et ceci encore plus dans le domaine de l’oncologie dans lequel le besoin d’avoir de nouvelles alternatives thérapeutiques est primordial. De plus, on note que le taux d’approbation des nouveaux anticancéreux après leur entrée en phase 1 fait partie des plus bas taux de toutes les aires thérapeutiques. De ce fait, ce processus doit être amélioré et l’utilisation de nouvelles approches faisant le lien entre développement préclinique et clinique par anticipation des propriétés pharmacocinétiques et d’efficacité pourrait être une perspective intéressante.L’objectif de ce travail est l’élaboration de stratégies basées sur la modélisation mathématique de données précliniques in vivo et in vitro afin d’anticiper le comportement chez l’homme d’un nouvel inhibiteur de bcl-2 développé par les Laboratoires Servier pour soutenir le développement clinique. Ce projet a été mené suivant différentes étapes :Premièrement, un modèle semi-mécanistique décrivant le mode d’action de la molécule a été établi chez la souris.Une stratégie d’extrapolation inter-espèces des caractéristiques PK utilisant la modélisation PBPK a été effectuée afin d’anticiper les profils temps-concentration chez l’homme.Des stratégies d’extrapolation de la partie PD basées sur différentes hypothèses ont été proposées pour prédire une efficacité chez l’homme et des doses à tester lors de l’étude clinique.Les prédictions obtenues ont ainsi été comparées aux résultats cliniques issus de la première étude réalisée chez l’homme confirmant le caractère utile de telles approches et la supériorité des stratégies bâties à l’aide de concepts semi-mécanistiques par rapport aux approches plus empiriques.Ce projet souligne donc le grand intérêt d’élaborer des approches translationnelles inter-espèces durant le développement du médicament et pourrait favoriser leur utilisation afin d’accélérer le développement de nouvelles entités, diminuant ainsi les risques d’échecs ainsi que les coûts financiers / Improvement of drug development is a very challenging question and even more in the field of oncology wherein the need for new medicines is crucial. In addition, the rate of approval for anticancer drugs after entry in phase I clinical trial was reported as one of the lowest of all therapeutic areas. Thereby, this process has to be improved, and the use of new approaches fulfilling the gap between preclinical and clinical settings by anticipating human pharmacokinetics and efficacy could be an interesting solution.The work is focused on the building of strategies based on mathematical modeling of in vivo and in vitro preclinical data to anticipate the behavior of a new bcl-2 inhibitor developed by Servier laboratories in human to support clinical development. This project was elaborated following different steps:Firstly, a semi-mechanistic relationship was established in mice to describe the mechanism of action of the compound.PK extrapolation strategy using PBPK modeling was performed to anticipate human concentration-time profiles.PD extrapolation strategies based on different assumptions were proposed to predict human efficacy and doses to be tested in clinical trial.Predictions obtained were consequently compared to clinical results from a First in Human study confirming the usefulness of such approaches and the superiority of mechanism-based strategies compared to more empirical approaches.Therefore, this project highlights the large interest of elaborating interspecies translational approaches during drug development and could promote their use to accelerate new entities development, decreasing the risks of failure and financial costs.

Les cancers des voies aéro-digestives supérieures (VADS) sont des cancers de mauvais pronostic avec 50% de rechute des patients dans les deux ans qui suivent leur traitement. La surexpression des protéines anti-apoptotiques de la famille Bcl-2 est un des marqueurs corrélés avec la résistance aux divers traitements. L’objectif de ce travail a été d’évaluer l’efficacité d’une approche thérapeutique associant l’irradiation à un inhibiteur des protéines anti-apoptotiques Bcl-2 et Bcl-XL, l’ABT-737 (Laboratoires Abbott), ceci à la fois sur des lignées issues de cancers de VADS, mais également sur une sous-population de cellules souches cancéreuses (CSC).La première partie de ce travail a démontré que cette association thérapeutique provoque une radio-sensibilisation des lignées cellulaires étudiées in vitro, avec augmentation de la mort cellulaire par apoptose. Cet effet synergique fait intervenir la mitochondrie, module le métabolisme des céramides et modifie l’expression de certaines protéines de la famille Bcl-2 dont l’équilibre est déterminant dans la destinée de la cellule cancéreuse. De plus, le traitement par l’ABT-737 permet de ralentir très sensiblement la croissance tumorale après irradiation sur un modèle in vivo.La deuxième partie de ce travail a permis de révéler une efficacité cytotoxique encore plus importante de l’ABT-737 sur une sous-population de CSC, résistante à l’apoptose, proliférante et tumorigène, dont le rôle dans la résistance aux divers traitements fait l’objet de nombreux travaux. A cette efficacité cytotoxique s’ajoute une diminution de la migration et de l’invasion des CSC, effet très prometteur dans la lutte contre la récidive locale et la dissémination métastatique, caractéristiques des cancers de VADS de haut grade / Head and neck squamous cell carcinomas (HNSCC) are frequently characterized by chemotherapy and radiation resistance and 5-year survival rates have lingered around 50% for several decades. The frequent resistance of HNSCC is due, in large part, to aberrant inhibition of apoptosis and overexpression of antiapoptotic members of the Bcl-2 protein family. The aim of this study was to examine, in association with radiation, the impact of ABT-737, a potent small-molecule inhibitor of Bcl-XL and Bcl-2, on HNSCC cells and cancer stem cells (CSC).The first part of our work demonstrated that ABT-737 strongly synergized with radiotherapy to promote HNSCC cell death and loss of clonogenic survival. This effect involves mitochondrial damage, modulates ceramide metabolism and modify the expression of some proteins of the Bcl-2 family whose interactions with other family members determine cell fate. Moreover, we found that this combination is able to significantly slow tumor growth.The second part of our work revealed that ABT-737, even without radiation, had a preferential cytotoxic activity in vitro towards CSC. Thus, as CSC have a greater capacity for tumor relapse, increased motility and invasiveness, our data suggest that ABT-737 could effectively complement a first line of therapy with chemotherapy or radiotherapy in order to target residual quiescent HNSCC CSC

Les cellules invariantes « natural killer » T (iNKT) constituent une population particulière de LT non conventionnelle qui exprime un récepteur TCRαβ semi-invariant composé de la chaine Vα14-Jα18 associée aux chaines Vβ8, -7 ou -2 chez la souris et dont le développement a lieu dans le thymus. Ainsi, les cellules iNKT sont capables de reconnaitre via leur TCR des antigènes de type glycolipidique présentés par une molécule de classe I non polymorphique : le CD1d. Ces cellules sont connues pour être impliquées dans diverses réponses immunes grâce à leur capacité à produire rapidement des cytokines immunorégulatrices. De la même façon que les LTc SP CD4+, il existe trois phénotypes de cellules iNKT : Th1, -2 et -17 permettant de distinguer trois sous-populations de cellules iNKT. La sous-population iNKT1 dite iNKT conventionnelle exprime des récepteurs appartenant au lignage NK. Cette sous-population est localisée préférentiellement dans le foie, le thymus et la rate et produit majoritairement de l’IFN-. La sous-population iNKT2, qui reste jusqu’à aujourd’hui insuffisamment décrite, se localise préférentiellement dans les poumons et produit majoritairement de l’IL-4 et de l’IL-13. La sous-population iNKT17 a été caractérisée et mise en évidence au sein de notre laboratoire comme une sous-population de cellules iNKT exprimant le facteur de transcription RORt et capable de sécréter de l’IL-17 en réponse à l’IL-1 et l’IL-23 et se localisant principalement dans les ganglions périphériques et la peau. A ce jour, seul le développement des cellules iNKT conventionnelles est bien connu tandis que celui des cellules iNKT17 demeurent ignorés. Ainsi, ayant remarqué une faible proportion des cellules iNKT17 dans le thymus de la souris C57BL/6 comparées aux autres sous-populations de cellules iNKT, nous nous sommes intéressés dans un premier temps à expliquer les causes de cette faible distribution de cette sous-population, ainsi qu’à définir la séquence d’acquisition de ses marqueurs lors de son développement thymique et de sa migration en périphérie. Les résultats montrent que ces cellules n’ont aucun défaut de prolifération, ni de réponse aux cytokines de l’homéostasie permettant d’expliquer leur plus faible nombre. En revanche, nous avons constaté une absence d’accumulation thymique de ces cellules qui ont la capacité de migrer en périphérie, accompagnée d’une sensibilité plus accrue à la mort par apoptose et une diminution de l’expression des facteurs de survie comme le Bcl-2 pouvant ainsi expliquer leur nombre réduit. Les analyses de leur développement aux stades précoces ont montré un biais préétabli de leur faible nombre observable dès le stade CD44-. L’étude de leur ontogénique a permis de montrer une cinétique d’acquisition séquentielle des marqueurs CCR6 et CD138 permettant d’établir pour la première fois un modèle de maturation thymique de cette sous-population iNKT17 qui était encore inconnue. Ainsi, au stade précoce HSAhigh, les cellules iNKT RORt+ observé correspondent à des précurseurs communs des cellules iNKT et non pas à des précurseurs des cellules iNKT17 montrant que la différenciation de ces cellules ne se fait pas au stade de sélection positive et que leur faible nombre dépend des signaux que ces cellules reçoivent lors de leur engagement vers le lignage “Th17 like“. / Invariant natural killer cells T (iNKT) constitute a particular population of unconventional LT which expresses a semi-invariant TCRαβ receptor composed of the Vα14-Jα18 chain associated with the Vβ8, -7 or -2 chains in mice and which develops in the thymus. Thus, iNKT cells are able to recognize glycolipid antigens via their TCR presented by a non-polymorphic class I molecule: CD1d. These cells are known to be involved in various immune responses because of their ability to rapidly produce cytokines. However, like conventional SP T CD4+ lymphocytes, iNKT cells can differentiate into three phenotypes: Th1, -2 and -17. The iNKT1 subset also named conventional iNKT cells expresses receptors belonging to the NK lineage, is mainly located in the liver, thymus and spleen and produces mainly IFN-. The iNKT2 subset which until now remains insufficiently described, is localized preferentially in the lungs and produces mainly IL-4 and IL-13. The iNKT17 subset has been characterized in our laboratory as a subset of iNKT cells expressing the RORt transcription factor and capable of secreting IL-17 in response to IL-1 and IL-23 and located mainly in the peripheral lymph nodes and the skin. To date, only the development of conventional iNKT cells is well known while that of iNKT17 cells remains unknown. Thus, having noticed the low distribution of the iNKT17 cells present in the thymus of the C57BL/6 mouse compared to other iNKT cell subset, we were initially interested in explaining the causes of this poor distribution of this subset, as well as to define the acquisition sequence of its markers during its thymic development and peripheral migration. The results show that these cells have no defect of proliferation or response to cytokines of homeostasis that can explain their lower number in the thymus. In contrast, we found a lack of thymic accumulation of these cells that have the ability to migrate peripherally, accompanied by increased sensitivity to death by apoptosis and decreased expression of survival factors such as Bcl-2 which can explain their reduced number. Analyzes of their development at early stages showed a pre-established bias of their low number from the CD44- stage. The study of their ontogeny has shown a sequential acquisition kinetics of CCR6 and CD138 markers to establish for the first time a model of thymic maturation of this iNKT subset which was still unknown.

No description available.

MicroARN (miARN) ont récemment émergé comme un acteur central du gène réseau de régulation impliqués dans la prise du destin cellulaire. L'apoptose, un actif processus, par lequel des cellules déclenchent leur auto-destruction en réponse à un signal, peut être contrôlé par les miARN. Il a également été impliqué dans une variété de maladies humaines, comme les maladies du cœur, et a été pensé comme une cible pour le traitement de la maladie. Tanshinone IIA (TIIA), un monomère de phenanthrenequinones utilisé pour traiter maladies cardiovasculaires, est connu pour exercer des effets cardioprotecteurs de l'infarctus du myocarde en ciblant l'apoptose par le renforcement de Bcl-2 expression. Pour explorer les liens potentiels entre le miARN et l'action anti-apoptotique de TIIA, nous étudié l'implication possible des miARN. Nous avons constaté que l'expression de tous les trois membres de la famille miR-34, miR-34a, miR-34b et miR-34c ont été fortement régulée à la hausse après l'exposition soit à la doxorubicine, un agent endommageant l'ADN ou de pro-oxydant H2O2 pendant 24 heures. Cette régulation à la hausse causé significativement la mort cellulaire par apoptose, comme déterminé par fragmentation de l'ADN, et les effets ont été renversés par les ARNs antisens de ces miARN. Le prétraitement des cellules avec TIIA avant l'incubation avec la doxorubicine ou H2O2 a empêché surexpression de miR-34 et a réduit des apoptose. Nous avons ensuite établi BCL2L2, API5 et TCL1, en plus de BCL2, comme les gènes nouveaux cibles pour miR-34. Nous avons également élucidé que la répression des ces gènes par MiR-34 explique l'effet proapoptotique dans les cardiomyocytes. Ce que la régulation positive de ces gènes par TIIA realisée par la répression de l'expression de miR-34 est probable le mécanisme moléculaire de son effet bénéfique contre ischémique lésions cardiaques. / MiRNAs (miRNAs) have recently emerged as a central player of gene regulatory network involved in decision of cell fate. Apoptosis, an active process that leads to cell death, has been shown to be controlled by miRNAs. It has also been implicated in a variety of human disease, such as heart disease, and established as a target process for disease therapy. Tanshinone IIA (TIIA), a monomer of phenanthrenequinones used to treat cardiovascular diseases, is known to exert cardioprotective effects in myocardial infarction by targeting apoptosis through enhancing Bcl-2 expression. To explore the potential link between miRNAs and the anti-apoptotic action of TIIA, we studied the possible involvement of miRNAs. We found that expression of all three members of the miR-34 family, miR-34a, miR-34b and miR-34c that have been known to mediate the apoptotic effect of p53 in cancer cells, were robustly upregulated after exposure to either the DNA-damaging agent doxorubicin or pro-oxidant H2O2 for 24 hr in cultured neonatal rat ventricular myocytes. This upregulation caused significant apoptotic cell death, as determined by DNA fragmentation, and the effects were reversed by the antisense to these miRNAs. Pretreatment of cells with TIIA prior to incubation with doxorubicin or H2O2 prevented upregulation of miR-34 and reduced apoptosis. We then established BCL2L2, API5 and TCL1, in addition to BCL2, as the novel target genes for miR-34. We further unraveled that repression of these genes by miR-34 accounts for its proapoptotic effect in cardiomyocytes whereas upregulation of these genes by TIIA through downregulating miR-34 is likely the molecular mechanism for its beneficial effect against ischemic myocardial injuries.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

In this Master’s Thesis Research the results can be summarized from two major tasks: (1) In our first task, we utilized our two protein system (BAG-1 and HSP 70) as part of beta testing of a computational software 1 that can take three dimensional x-ray crystallography information about protein complexes and predict the strength of atom –atom interactions between amino-acid residues Open Contact predicts binding hotspots that can be used to identify short amino acid chains or peptides that mimic that particular binding segment of the larger protein. These peptides are called pepidyl-biomimetics. The peptide can potentially act as an antagonist drug by binding to the hotspot on protein A before protein B of the A-B complex can form. Two potential peptide candidates were identified. In particular, a helical peptide was discovered that demonstrated a variety of different types of atom-atom interactions. (2) Our second task is to experimentally test the helical peptide for its ability to block the binding that occurs between the 70-kilodalton Heat Shock Protein (HSP-70) and the Bcl-2 Associated Athanogene (BAG-1) Protein. As reviewed here, the binding between HSP-70 and BAG-1 elicits a cascade of cellular events that maintain high cancer growth rates and a greatly increased resistance to chemotherapy. In addition, BAG-1 has been implicated in a number of onco-signal pathways, as reviewed here, and its inhibition alone is believed to act as an agent against cancer cell growth

The primary focus of this research is the mechanisms of cell death in head and neck squamous cell carcinoma (HNSCC) treatment. These cancers typically originate in squamous cells that line the moist mucosal surfaces of head and neck. HNSCC is commonly treated with a platinum based agent, cisplatin. While the drug does offer strong antitumor effects, its prolonged use often results in tumor-acquired resistance, which limits treatment effectiveness. We have shown that cisplatin treatment induces the expression of a pro-apoptotic BCL-2 family member Noxa, which then initiates caspase- dependent apoptosis through its binding and sequestration of pro-survival protein MCL-1 for its inactivation. Without Noxa induction, cell death is significantly reduced when treating HNSCCs with cisplatin. The objectives of this study are (1) to determine the molecular mechanisms by which Noxa induces cell death in HNSCC cells; (2) to determine the molecular mechanisms of cisplatin-resistance in isogenic HNSCC cell lines. We observed an increase of apoptosis by ectopic expression of Noxa in all HNSCC cell lines tested, but not in immortalized human normal oral keratinocytes (NOK), suggesting that Noxa overexpression is sufficient to induce tumor-specific cell death. Noxa-induced cell death was mediated by BAX and BAK activation. BAK activation was mediated through Noxa binding to MCL-1, but not BCL-XL. Cisplatin- resistant cells induced less Noxa and apoptosis, supporting that Noxa induction is prerequisite for apoptosis induced by cisplatin. Taken together, Noxa induces tumor- specific cell death in HNSCC cells primarily through BAX and BAK activation, which suggests the therapeutic potential of this protein.