O papel dos transportadores de monocarboxilatos em tumores sólidos

Tese de doutoramento em Ciências da SaúdeMost solid tumours rely on glycolysis for energy production, even in conditions of high oxygen tension (‘‘aerobic glycolysis’’, also known as the Warburg effect), giving rise to enhanced lactate production. This lactate production is responsible for extracellular acidification, which, conditioning the tumour environment, favours tumour invasion and suppresses anticancer immune response. Transport of lactate across the plasma membrane is mediated by a family of protoncoupled monocarboxylate transporters (MCTs), which comprises 14 members. The isoforms MCT1-MCT4 are proton symporters that exhibit different affinities for lactate, leading to different levels of tissue expression. Since some MCT isoforms (especially MCT1 and MCT4) play a role in the intracellular pH homeostasis, by exporting the accumulating lactic acid, they are upregulated in glycolytic tumours, where high levels of lactate are produced, such as high grade gliomas, colorectal carcinomas and lung cancer. However, the role of MCTs in tumours is far from being well understood and their potential as therapeutic targets is poorly explored, being the main aim of this work to further elucidate the significance of MCT expression in solid tumours. Thus, MCTs expression and their clinic-pathological value were evaluated in human series of colorectal, cervical, gastric and breast carcinomas. Also, MCT regulation by chaperones was further investigated by analysing CD147 in cervical, gastric and breast carcinomas series, as well as performing a screening in colorectal, breast, lung and ovary tumour samples, where CD44 expression was evaluated, as a putative MCT chaperone, in addition to CD147. Finally, some in vitro studies were performed, to determine the contribution of MCTs to cancer cell metabolic profile and viability. Importantly, up-regulation of MCTs, in particular MCT1, was found in colorectal, cervical and breast carcinomas, but not in gastric carcinomas, which, in fact, showed a decrease of MCT4 along towards malignancy. Also, MCT1 was associated with poor prognosis, especially in breast carcinomas, as well as in gastric carcinoma where MCT1/CD147 co-expression was associated with poorer patient prognostic. Also, as anticipated, CD147 was found to be co-expressed with both MCT1 and MCT4 in the large series studied (cervical, gastric and breast carcinomas). In the tumour screening, CD44 was only associated with MCT1 in lung cancer, however, series were very small and some results warrant further attention. Finally, the in vitro studies showed a CHCinduced inhibition of cell proliferation in human breast cancer cell lines that was, in some cases, accompanied by metabolic alterations. In conclusion, the results presented in this thesis have an important impact on the comprehension of MCT contribution to malignant phenotype and pave the way for further studies aiming to the development of cancer therapies directed to MCTs.A maioria dos tumores sólidos depende da glicólise para produção de energia, mesmo em condições de alta pressão parcial de oxigénio (“glicólise aeróbia”, também conhecida como o efeito de Warburg), dando origem a uma maior produção de lactato. Esta produção de lactato é responsável pela acidificação extracelular, o que condiciona o ambiente do tumor, favorecendo a invasão tumoral e a supressão da resposta imune contra o tumor. O transporte de lactato através da membrana plasmática é mediado pelos transportadores de monocarboxilatos (MCTs), que pertencem a uma família composta actualmente por 14 membros. O transporte mediado pelas isoformas MCT1-MCT4 é um simporte com protões e cada isoforma possui afinidade distinta para o lactato, apresentando uma distribuição diferente nos vários tecidos. Uma vez que algumas isoformas (especialmente o MCT1 e o MCT4) desempenham um papel na homeostasia intracelular, ao exportar o ácido láctico acumulado, está descrito um aumento destas isoformas em alguns tumores glicolíticos, onde são produzidos níveis elevados de lactato, como seja em gliomas de alto grau, carcinomas colorectais e cancro do pulmão. No entanto, o papel dos MCTs em tumores está longe de ser completamento elucidado e o seu potencial como alvo terapêutico ainda se encontra pouco explorado, sendo o objectivo principal deste trabalho caracterizar o papel dos MCTs em tumores sólidos. Assim, a expressão e valor clínico-patológico dos MCTs foram avaliados em séries humanas de carcinoma colorectal, do colo do útero, do estômago e da mama. Para além disso, a regulação dos MCTs por proteínas chaperonas foi investigada, analisando a expressão da CD147 nas séries de carcinoma do colo do útero, do estômago e da mama, assim como a realização de um estudopiloto onde a expressão da CD44, outra possível chaperone dos MCTs, foi avaliada em tumores colorectais, da mama, do pulmão e do ovário, para além da CD147. Finalmente, alguns estudos in vitro foram realizados de modo a elucidar a contribuição dos MCTs para o perfil metabólico e viabilidade das células tumorais. De notar que um aumento na expressão dos MCTs, em particular do MCT1, foi encontrado nos carcinomas colorectal, do colo do útero e de mama, mas não nos carcinomas gástricos, que, na realidade, mostraram uma diminuição do MCT4 com o aumento da malignidade. Além disso, a expressão do MCT1 foi associada a um pior prognóstico, em especial nos carcinomas da mama, assim como no carcinoma gástrico, onde a co-expressão do MCT1 com a CD147 foi associada a um pior prognóstico do paciente. Como antecipado, a CD147 estava co-expressa quer com o MCT1 quer com o MCT4, quando avaliado nas séries de maior dimensão (carcinomas do colo uterino, gástrico e da mama). No estudo-piloto, a CD44 estava associada apenas com o MCT1 no cancro do pulmão, no entanto, o tamanho das séries era muito reduzido e alguns dos resultados obtidos merecem ser explorados no futuro. Finalmente, os estudos in vitro mostraram que o tratamento com CHC induz uma inibição da proliferação celular em linhas celulares de tumor da mama, a qual foi, em alguns casos, acompanhada por alterações metabólicas. Em conclusão, os resultados apresentados nesta tese têm um grande impacto na compreensão da contribuição dos MCTs para o fenótipo maligno e abrem caminho para estudos posteriores que visem ao desenvolvimento de terapias anti-tumorais direccionadas aos MCTs.Fundação para a Ciência e Tecnologia (FCT) - bolsa de doutoramento SFRH/BD/27465/2006, projecto “Monocarboxylate transporters as potential therapeutic targets in cancer: inhibition studies in solid tumor models”, com a referência PTDC/SAU-FCF/104347/2008

How to decipher a microbial puzzle on microbial control: hands on

How to decipher a microbial puzzle on microbial control: hands onAntibiotics are among the most commonly prescribed drugs, but they are often misused, contributing to bacterial resistance, being an important public health problem, with clinical/economic impacts. Aiming to contribute to convey information to students on Microbiology/antibiotic use, we developed different teaching activities, with 6th and 9th grade students. To assess the impact of these activities and student learning, we applied a pre-validated questionnaire before and after completion of the activities (pre- and post-test). We observed that most students had preconceived ideas that antibiotics were effective against viruses, however, after the implemented activities, the performance of students in the post-test improved significantly for both student grades. Regarding the use of antibiotics in specific diseases, students showed lack of knowledge concerning the treatment of cold, flu and tuberculosis, with some improvement in the post-test. Concerning antibiotic use, the initial knowledge of the 9th grade students was better than the 6th grade, nevertheless the highest improvement in knowledge was observed for the 6th grade students. Our results support the use of teaching activities on Microbiology/antibiotic use that promote the development of critical thinking, analyze and solve problems. Our results support the need to educate students/communities on the proper use of antibiotics

Portuguese students' knowledge of antibiotics: a cross-sectional study of secondary school and university students in Braga

<p>Abstract</p> <p>Background</p> <p>Recent surveys show that the knowledge of the general public about the correct use of antibiotics is limited. This contributes to the problem of inappropriate antibiotic use, leading to a progressive loss of bacterial sensitivity to these drugs and the spreading of resistant strains of bacteria.</p> <p>Methods</p> <p>In this cross-sectional study, a questionnaire about antibiotic use was given to a sample of students in the 9^thand 12^thgrades of secondary school and in the first year of university in the north of Portugal.</p> <p>Results</p> <p>349 students returned completed questionnaires. Deficits were found in the students' knowledge of antibiotics and their correct use. Only 4% of 9^thgrade students were aware that antibiotics are used to treat bacteria only, while 14% of 12^thgrade students and 29% of first-year university students were aware of this. Fewer students were aware that antibiotics are used to treat tuberculosis. There were deficiencies in the knowledge of timing and duration of therapy. However close to 70% of these students are aware that inappropriate use of antibiotics can contribute to resistance to these drugs.</p> <p>Conclusion</p> <p>This study has observed a lack of general knowledge on correct antibiotic use in Portugal, as has been found in other countries. Since this may be due to a lack of formal education on this subject, we believe that a teaching unit on infectious diseases should be included in the 9^thand 12^thgrades, in all curricular areas, with emphasis on bacterial and viral pathogens and correct antibiotic use. In addition, education on the correct use of medications may need to begin at much earlier ages.</p

Lactate-induced il-8 pathway in endothelial cells-letter

Vegran and colleagues proposed a model in which the lactate released from tumor cells through MCT4 would be taken up by endothelial cells via the MCT1 transporter and stimulate angiogenesis, using human umbilical vein endothelial cells (HUVECs) as model of tumor endothelial cells. By analyzing a total of 505 cases of human tumor samples immunostained for MCT1, we do not confirm plasma membrane expression of MCT1 in the endothelial cells of tumor-associated vessels

Expression of Monocarboxylate Transporters 1, 2, and 4 in Human Tumours and Their Association with CD147 and CD44

Monocarboxylate transporters (MCTs) are important cellular pH regulators in cancer cells; however, the value of MCT expression in cancer is still poorly understood. In the present study, we analysed MCT1, MCT2, and MCT4 protein expression in breast, colon, lung, and ovary neoplasms, as well as CD147 and CD44. MCT expression frequency was high and heterogeneous among the different tumours. Comparing with normal tissues, there was an increase in MCT1 and MCT4 expressions in breast carcinoma and a decrease in MCT4 plasma membrane expression in lung cancer. There were associations between CD147 and MCT1 expressions in ovarian cancer as well as between CD147 and MCT4 in both breast and lung cancers. CD44 was only associated with MCT1 plasma membrane expression in lung cancer. An important number of MCT1 positive cases are negative for both chaperones, suggesting that MCT plasma membrane expression in tumours may depend on a yet nonidentified regulatory protein

Expression of Monocarboxylate Transporters 1, 2, and 4 in Human Tumours and Their Association with CD147 and CD44

Monocarboxylate transporters (MCTs) are important cellular pH regulators in cancer cells; however, the value of MCT expression in cancer is still poorly understood. In the present study, we analysed MCT1, MCT2, and MCT4 protein expression in breast, colon, lung, and ovary neoplasms, as well as CD147 and CD44. MCT expression frequency was high and heterogeneous among the different tumours. Comparing with normal tissues, there was an increase in MCT1 and MCT4 expressions in breast carcinoma and a decrease in MCT4 plasma membrane expression in lung cancer. There were associations between CD147 and MCT1 expressions in ovarian cancer as well as between CD147 and MCT4 in both breast and lung cancers. CD44 was only associated with MCT1 plasma membrane expression in lung cancer. An important number of MCT1 positive cases are negative for both chaperones, suggesting that MCT plasma membrane expression in tumours may depend on a yet nonidentified regulatory protein

Expression of monocarboxylate transporters 1, 2, and 4 in human tumours and their association with CD147 and CD44

Expression of monocarboxylate transporters 1, 2, and 4 in human tumours and their association with CD147 and CD44.Monocarboxylate transporters (MCTs) are important cellular pH regulators in cancer cells; however, the value of MCT expression in cancer is still poorly understood. In the present study, we analysed MCT1, MCT2, and MCT4 protein expression in breast, colon, lung, and ovary neoplasms, as well as CD147 and CD44. MCT expression frequency was high and heterogeneous among the different tumours. Comparing with normal tissues, there was an increase in MCT1 and MCT4 expressions in breast carcinoma and a decrease in MCT4 plasma membrane expression in lung cancer. There were associations between CD147 and MCT1 expressions in ovarian cancer as well as between CD147 and MCT4 in both breast and lung cancers. CD44 was only associated with MCT1 plasma membrane expression in lung cancer. An important number of MCT1 positive cases are negative for both chaperones, suggesting that MCT plasma membrane expression in tumours may depend on a yet nonidentified regulatory protein.Celine Pinheiro received a Ph.D. fellowship from the Portuguese Science and Technology Foundation (SFRH/BD/27465/2006). The authors acknowledge NCI (National Cancer Institute) Tumour Repository MTA, MD, USA for the multitumour tissue microarray (TARP)

Glucose addiction in cancer therapy : advances and drawbacks

Glucose addiction in cancer therapy: advances and drawbacks.In contrast to differentiated normal cells, which primarily use mitochondrial respiration to generate the energy needed for cellular processes, most cancer cells rely on glycolysis, even in sufficient oxygen conditions, a phenomenon known as the “Warburg effect” or aerobic glycolysis. In the last years, much attention to the metabolic reprogramming of cancer cells has been paid by many research groups and, as a result, this altered energy metabolism was recognized in 2011 as one of the “hallmarks of cancer”. Aerobic glycolysis allows a rapid growth of tumor cells, with high rates of glucose consumption and lactic acid production, leading to cellular acidosis. Metabolic reprogramming renders cancer cells dependent on specific metabolic enzymes or pathways that could be exploited in cancer therapy. The development of treatments that target tumor glucose metabolism is receiving renewed attention, with several drugs targeting metabolic pathways currently in clinical trials. However, the search for suitable targets may be limited by the high plasticity of the metabolic network that can induce compensatory routes. Moreover, deregulated glucose metabolism has been also shown as a prominent feature associated with resistance to either classical chemotherapy or oncogene-targeted therapies, strengthening the clinical potential of combining these therapies with glycolysis inhibitors. The aim of this review is to compare the advances of the different therapeutic strategies targeting the glucose “addiction” of tumor cells, highlighting the potential of these findings to be translated into effective weapons against cancer. Further, we will also present and discuss recent evidence for the involvement of glucose metabolism as a compensatory response to the use of drugs that target different signaling pathways, in which combination with glycolysis inhibitors are potentially useful

Peritumoural, but not intratumoural, lymphatic vessel density and invasion correlate with colorectal carcinoma poor-outcome markers

To evaluate whether lymphatic vessel density (LVD) and lymphatic vessel invasion (LVI) are useful markers of worse outcome in colorectal carcinoma and if LVD and LVI correlate to the classical clinical-pathological parameters, we analysed 120 cases of colorectal carcinomas selected from the files of Division of Pathology, Hospital das Clinicas, São Paulo University, Brazil. Assessment of LVD and LVI was performed by immunohistochemical detection of lymphatic vessels, using the monoclonal antibody D2-40. Higher LVD was found in the intratumoural area, when comparing with normal and peritumoural areas (p < 0.001). However, peritumoural LVD, but not intratumoural, correlated with both colonic-wall-invasion depth (p = 0.037) and liver metastasis (p = 0.012). Remarkably, LVI was found associated with local invasion (p = 0.016), nodal metastasis (p = 0.022) and hepatic metastasis (p < 0.001). Peritumoural LVD and LVI are directly related to histopathological variables indicative of poor outcome such as lymph-node status and liver metastasis

Monocarboxylate transport inhibition potentiates the cytotoxic effect of 5-fluorouracil in colorectal cancer cells

Cancer cells rely mostly on glycolysis to meet their energetic demands, producing large amounts of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). The role of MCTs in the survival of colorectal cancer (CRC) cells is scarce and poorly understood. In this study, we aimed to better understand this issue and exploit these transporters as novel therapeutic targets alone or in combination with the CRC classical chemotherapeutic drug 5-Fluorouracil. For that purpose, we characterized the effects of MCT activity inhibition in normal and CRC derived cell lines and assessed the effect of MCT inhibition in combination with 5-FU. Here, we demonstrated that MCT inhibition using CHC (a-cyano-4-hydroxycinnamic acid), DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and quercetin decreased cell viability, disrupted the glycolytic phenotype, inhibited proliferation and enhanced cell death in CRC cells. These results were confirmed by specific inhibition of MCT1/4 by RNA interference. Notably, we showed that 5-FU cytotoxicity was potentiated by lactate transport inhibition in CRC cells, either by activity inhibition or expression silencing. These findings provide novel evidence for the pivotal role of MCTs in CRC maintenance and survival, as well as for the use of these transporters as potential new therapeutic targets in combination with CRC conventional therapy.Ricardo Amorim was recipient of the fellowships SFRH/BI/51118/ 2010 and SFRH/BD/98002/2013, from Fundação para a Ciência e Tecnologia (FCT, Portugal). This study was supported by the FCT grant ref. PTDC/SAU-FCF/104347/2008, under the scope of “Programa Operacional Temático Factores de Competitividade” (COMPETE) of “Quadro Comunitário de Apoio III” and co-financed by Fundo Comunitário Europeu FEDER. This work was also supported by FEDER through POFC – COMPETE and by FCT through project PEst-OE/BIA/UI4050/2014 and Helena Pereira’s fellowship (SFRH/BD/73139/2010)