Paclitaxel effect on the IRS/PI3-kinase/Akt/mTOR pathway in breast cancer adenocarcinoma and lung cancer carcinoma

Orientador: José Barreto Campello CarvalheiraDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A elucidação das vias de crescimento celular e a observação de que essas vias estão alteradas no câncer humano incentivou a procura de inibidores específicos. Proteínas adaptadoras, que se ligam a múltiplos elementos de uma cascata de sinalização e coordenam a sinalização celular, bem como quinases intracelulares podem ser candidatos ideais a alvos para o bloqueio da sinalização celular. Nesse sentido, a via de sinalização IRS/PI3K/Akt/mTOR e a AMPK aparecem como alvos para o bloqueio de crescimento e indução de apoptose de células tumorais. Por outro lado, a descoberta dos taxanos, ésteres de alcalóides complexos, compostos por um sistema de anéis e com diversas ramificações laterais que são essenciais para a ação única contra os microtúbulos, é provavelmente a mais importante adição ao arsenal quimioterápico do final do século vinte. Entretanto, os efeitos da associação entre taxanos e moduladores da atividade da AMPK e de bloqueadores da mTOR em linhagens de câncer de mama e de pulmão são apenas parcialmente conhecidos. Assim, o objetivo do projeto foi avaliar o efeito do tratamento concomitante entre taxanos e moduladores da atividade da AMPK em diferentes linhagens de câncer de mama e de pulmão. As células de adenocarcinoma de mama (MCF-7) e de carcinoma de pulmão (A549) foram tratadas com metformina, ativador da AMPK, e com paclitaxel; e animais SCID foram inoculados com células de câncer de pulmão A549 e tratados com metformina ou paclitaxel ou então uma combinação das duas drogas. Os resultados obtidos demonstram que AMPK é ativada por metformina e que mTOR, p70S6K e 4EBP-1 são inibidas pelo tratamento com metformina de maneira dependente do tempo e da dose a que foram submetidos nas células MCF-7 e A549. Além disso, observamos que o tratamento com paclitaxel ativou não apenas a AMPK, mas também p53 e sestrina 2, e inibiu as proteínas mTOR, p70S6K e 4EBP-1 de maneira dependente do tempo e da dose nas duas linhagens de células utilizadas. Assim, na associação de 2-DG com paclitaxel e de metformina com paclitaxel, verificamos que as proteína ativadas pela associação de drogas eram p53, sestrina 2 e AMPK, enquanto mTOR, p70S6K e 4EBP-1 encontravam-se inibidas nas células MCF-7 e A549. Também observamos que o tratamento de metformina com paclitaxel resulta em aumento no número de células com parada na fase G2/M do ciclo celular e diminui o crescimento tumoral em animais com diminuição da proliferação e aumento da apoptose, em relação aos tratamentos isolados e ao grupo controle. Assim, podemos sugerir que a associação de um ativador da AMPK, que leva a uma diminuição da atividade de vias de crescimento, proliferação e diferenciação celular pode ser uma alternativa mais eficiente que o tratamento com paclitaxel isoladoAbstract: Metformin is a widely-used antidiabetic drug whose anti-cancer effects, mediated by the activation of AMPK and reduction of mTOR signaling, have become noteworthy. Chemotherapy produces genotoxic stress and induces p53 activity, which can cross-talk with AMPK/mTOR pathway. Herein, we investigate whether the combination of metformin and paclitaxel has an effect in cancer cell lines. Human tumors were xenografted into SCID mice and the cancer cell lines were treated only with paclitaxel or metformin, or a combination of both drugs. Western Blotting, flow cytometry and immunohistochemistry were then used to characterize the effects of the different treatments. The results presented herein, demonstrate that the addition of metformin to paclitaxel leads to quantitative potentialization of molecular signaling through AMPK and a subsequent potent inhibition of the mTOR signaling pathway. Treatment with metformin and paclitaxel resulted in an increase in the number of cells arrested in the G2/M phase of the cell cycle, decreased tumor growth and increased apoptosis in tumor-bearing mice, when compared to individual drug treatments. We have provided evidence for a convergence of metformin and paclitaxel induced signaling at the level of AMPK. This mechanism illustrates how different drugs may cooperate to augment anti-growth signals, and suggests that target activation of AMPK by metformin may be a compelling ally in cancer treatmentMestradoMedicina ExperimentalMestre em Ciência

Microbiota determines insulin sensitivity in TLR2-KO mice

Environmental factors have a key role in the control of gut microbiota and obesity. TLR2 knockout (TLR2(-/-)) mice in some housing conditions are protected from diet-induced insulin resistance. However, in our housing conditions these animals are not protected from diet-induced insulin-resistance. Aim: The aim of the present study was to investigate the influence of our animal housing conditions on the gut microbiota, glucose tolerance and insulin sensitivity in TLR2(-/-) mice. Material and methods: The microbiota was investigated by metagenomics, associated with hyperinsulinemic euglycemic clamp and GTT associated with insulin signaling through immunoblotting. Results: The results showed that TLR2(-/-) mice in our housing conditions presented a phenotype of metabolic syndrome characterized by insulin resistance, glucose intolerance and increase in body weight. This phenotype was associated with differences in microbiota in TLR2(-/-) mice that showed a decrease in the Proteobacteria and Bacteroidetes phyla and an increase in the Firmicutesphylum, associated with and in increase in the Oscillospira and Ruminococcus genera. Furthermore there is also an increase in circulating LPS and subclinical inflammation in TLR2(-/-). The molecular mechanism that account for insulin resistance was an activation of TLR4, associated with ER stress and JNK activation. The phenotype and metabolic behavior was reversed by antibiotic treatment and reproduced in WT mice by microbiota transplantation. Conclusions: Our data show, for the first time, that the intestinal microbiota can induce insulin resistance and obesity in an animal model that is genetically protected from these processes234CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação465693/2014-8We also acknowledge the financial support INCT de Obesidade e Diabetes 465693/2014-8 (FAPESP) and CAPES/CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico)

Oral Glutamine Supplementation Reduces Obesity, Pro-Inflammatory Markers, and Improves Insulin Sensitivity in DIO Wistar Rats and Reduces Waist Circumference in Overweight and Obese Humans

In the present study, we aimed to investigate whether chronic oral glutamine (Gln) supplementation may alter metabolic parameters and the inflammatory profile in overweight and obese humans as well as whether Gln may modulate molecular pathways in key tissues linked to the insulin action in rats. Thirty-nine overweight/obese volunteers received 30 g of Gln or alanine (Ala-control) for 14 days. Body weight (BW), waist circumference (WC), hormones, and pro-inflammatory markers were evaluated. To investigate molecular mechanisms, Gln or Ala was given to Wistar rats on a high-fat diet (HFD), and metabolic parameters, euglycemic hyperinsulinemic clamp with tracers, and Western blot were done. Gln reduced WC and serum lipopolysaccharide (LPS) in overweight volunteers. In the obese group, Gln diminished WC and serum insulin. There was a positive correlation between the reduction on WC and LPS. In rats on HFD, Gln reduced adiposity, improved insulin action and signaling, and reversed both defects in glucose metabolism in the liver and muscle. Gln supplementation increased muscle glucose uptake and reversed the increased hepatic glucose production, in parallel with a reduced glucose uptake in adipose tissue. This insulin resistance in AT was accompanied by enhanced IRS1 O-linked-glycosamine association in this tissue, but not in the liver and muscle. These data suggest that Gln supplementation leads to insulin resistance specifically in adipose tissue via the hexosamine pathway and reduces adipose mass, which is associated with improvement in the systemic insulin action. Thus, further investigation with Gln supplementation should be performed for longer periods in humans before prescribing as a beneficial therapeutic approach for individuals who are overweight and obese

Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells

AimTo evaluate the potential use of Cephaeline as a therapeutic strategy to manage mucoepidermoid carcinomas (MEC) of the salivary glands.Material and MethodsUM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation.ResultsA single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity.ConclusionCephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells’ viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/173145/1/jop13252_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/173145/2/jop13252.pd