Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.We would like to thank Dr. Estela M. A. F. Bevilacqua and Dr. Ruy Jaeger for allowing us to use their ELISA plate readers, MSc. Roberto Cabado for the assistance in the performance of the confocal microscope and MSc. Adam A. Martens for the assistance with the western blotting. We also thank Dr. Gilberto A. Paula, Daniel D. Barreto, Paula C. G. Melo and Thiago F. Costa for helping with statistical analysis and FAPESP, CNPq and CAPES for financial support

Multipolar mitosis and aneuploidy after chrysotile treatment: a consequence of abscission failure and cytokinesis regression

Chrysotile, like other types of asbestos, has been associated with mesothelioma, lung cancer and asbestosis. However, the cellular abnormalities induced by these fibers involved in cancer development have not been elucidated yet. Previous works show that chrysotile fibers induce features of cancer cells, such as aneuploidy, multinucleation and multipolar mitosis. In the present study, normal and cancer derived human cell lines were treated with chrysotile and the cellular and molecular mechanisms related to generation of aneuploid cells was elucidated. The first alteration observed was cytokinesis regression, the main cause of multinucleated cells formation and centrosome amplification. The multinucleated cells formed after cytokinesis regression were able to progress through cell cycle and generated aneuploid cells after abnormal mitosis. To understand the process of cytokinesis regression, localization of cytokinetic proteins was investigated. It was observed mislocalization of Anillin, Aurora B, Septin 9 and Alix in the intercellular bridge, and no determination of secondary constriction and abscission sites. Fiber treatment also led to overexpression of genes related to cancer, cytokinesis and cell cycle. The results show that chrysotile fibers induce cellular and molecular alterations in normal and tumor cells that have been related to cancer initiation and progression, and that tetraploidization and aneuploid cell formation are striking events after fiber internalization, which could generate a favorable context to cancer development

Molecular characterization of a mariner-like element in the Atta sexdens rubropilosa genome

Mobile elements are widely present in eukaryotic genomes. They are repeated DNA segments that are able to move from one locus to another within the genome. They are divided into two main categories, depending on their mechanism of transposition, involving RNA (class I) or DNA (class II) molecules. The mariner-like elements are class II transposons. They encode their own transposase, which is necessary and sufficient for transposition in the absence of host factors. They are flanked by a short inverted terminal repeat and a TA dinucleotide target site, which is duplicated upon insertion. The transposase consists of two domains, an N-terminal inverted terminal repeat binding domain and a C-terminal catalytic domain. We identified a transposable element with molecular characteristics of a mariner-like element in Atta sexdens rubropilosa genome. Identification started from a PCR with degenerate primers and queen genomic DNA templates, with which it was possible to amplify a fragment with mariner transposable-element homology. Phylogenetic analysis demonstrated that this element belongs to the mauritiana subfamily of mariner-like elements and it was named Asmar1. We found that Asmar1 is homologous to a transposon described from another ant, Messor bouvieri. The predicted transposase sequence demonstrated that Asmar1 has a truncated transposase ORF. This study is part of a molecular characterization of mobile elements in the Atta spp genome. Our finding of mariner-like elements in all castes of this ant could be useful to help understand the dynamics of mariner-like element distribution in the Hymenoptera.FAPESPFAPESPCNPqCNP

Molecular characterization of a mariner-like element in the Atta sexdens rubropilosa genome

Mobile elements are widely present in eukaryotic genomes. They are repeated DNA segments that are able to move from one locus to another within the genome. They are divided into two main categories, depending on their mechanism of transposition, involving RNA (class I) or DNA (class II) molecules. The mariner-like elements are class II transposons. They encode their own transposase, which is necessary and sufficient for transposition in the absence of host factors. They are flanked by a short inverted terminal repeat and a TA dinucleotide target site, which is duplicated upon insertion. The transposase consists of two domains, an N-terminal inverted terminal repeat binding domain and a C-terminal catalytic domain. We identified a transposable element with molecular characteristics of a mariner-like element in Atta sexdens rubropilosa genome. Identification started from a PCR with degenerate primers and queen genomic DNA templates, with which it was possible to amplify a fragment with mariner transposable-element homology. Phylogenetic analysis demonstrated that this element belongs to the mauritiana subfamily of mariner-like elements and it was named Asmar1. We found that Asmar1 is homologous to a transposon described from another ant, Messor bouvieri. The predicted transposase sequence demonstrated that Asmar1 has a truncated transposase ORF. This study is part of a molecular characterization of mobile elements in the Atta spp genome. Our finding of mariner-like elements in all castes of this ant could be useful to help understand the dynamics of mariner-like element distribution in the Hymenoptera.FAPESPFAPESPCNPqCNP

Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells

Olhares suíços sobre o Portugal de Salazar. 2 : as guerras (1936-1945)

Entre 1936 e 1945 quase todas as fontes consultadas, quer diplomáticas, quer jornalísticas, dão uma imagem positiva da política de Salazar, a qual defendem sem que praticamente sobre ela exerçam qualquer análise crítica. Durante a guerra civil espanhola, se excluirmos a imprensa comunista, que, no entanto, fala muito pouco de Portugal, os representantes suíços em Lisboa e os editorialistas dos principais quotidianos helvéticos justificam a atitude filofranquista do governo português. No que diz respeito à política externa portuguesa e à neutralidade durante a Segunda Guerra Mundial, as mesmas fontes, obnubiladas, sem dúvida, pelo mito da ditadura «branda e moderada» de Salazar, manifestam uma grande compreensão e simpatia pelas diferentes tomadas de posição do ditador português, por razões ideológicas, pelo facto de Portugal ser um pequeno país como a Suíça e também pelo papel de Portugal no abastecimento da Confederação durante o conflito.ABSTRACT: Between 1936 and 1945, an overwhelming majority of the documents analyzed in this article give a very positive image of Salazar’s regime. During the Spanish civil war, with the exception of a very few left-leaning newspapers, the Swiss journalists and the diplomats in charge at the Swiss Legation in Lisbon defended and justified the support given to the nationalist cause in Spain by the Portuguese government. Concerning the Portuguese neutrality during the Second World War, the same sources adopted the Salazar’s point of view and analyzed his politics with a lot of sympathy and understanding. This was mainly for four reasons: they sympathized partially with the ideology of the Estado Novo; Portugal is a small country, like Switzerland; they were blinded by the myth of the so-called «soft» and «moderate» dictatorship of Salazar; and, finally, because Portugal was an important trade partner for the Confederation during the conflict

Decreased expression of ADAMTS-1 in human breast tumors stimulates migration and invasion

Abstract\ud \ud \ud \ud Background\ud \ud ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines.\ud \ud \ud \ud Results\ud \ud In a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels.\ud \ud \ud \ud Conclusions\ud \ud ADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.This investigation was supported by The State of São Paulo Research Foundation (FAPESP grants 2006/54963-0, 2006/01026-0, 2008/57103-8, 2010/07699-1), and Brazilian National Council for Scientific and Technological Development (CNPq grant 470779/2007-1). The authors also want to thank Dr. Stanley Zucker (Stony Brook University; Stony Brook, NY, USA) and Dr. Rama Khokha (University of Western Ontario, Toronto, Canada) for suggestions made on data analysis

The multiple facets of drug resistance: one history, different approaches

Abstract\ud Some cancers like melanoma and pancreatic and ovarian cancers, for example, commonly display resistance to chemotherapy, and this is the major obstacle to a better prognosis of patients. Frequently, literature presents studies in monolayer cell cultures, 3D cell cultures or in vivo studies, but rarely the same work compares results of drug resistance in different models. Several of these works are presented in this review and show that usually cells in 3D culture are more resistant to drugs than monolayer cultured cells due to different mechanisms. Searching for new strategies to sensitize different tumors to chemotherapy, many methods have been studied to understand the mechanisms whereby cancer cells acquire drug resistance. These methods have been strongly advanced along the years and therapies using different drugs have been increasingly proposed to induce cell death in resistant cells of different cancers. Recently, cancer stem cells (CSCs) have been extensively studied because they would be the only cells capable of sustaining tumorigenesis. It is believed that the resistance of CSCs to currently used chemotherapeutics is a major contributing factor in cancer recurrence and later metastasis development. This review aims to appraise the experimental progress in the study of acquired drug resistance of cancer cells in different models as well as to understand the role of CSCs as the major contributing factor in cancer recurrence and metastasis development, describing how CSCs can be identified and isolated.We thank Roberdo Cabado for helping with the Figure 1 final art. The works\ud of our laboratory are supported by FAPESP, CNPq and CAPES

The Fate of Chrysotile-Induced Multipolar Mitosis and Aneuploid Population in Cultured Lung Cancer Cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes