Protección del ácido clorogénico frente a lesiones inducidas por venzo(a)pireno en Saccharomyces cerevisiae / Protection of chlorogenic acid against benzo(a)pyrene-induced lesions in Saccharomyces cerevisiae / Proteção do ácido clorogênico contra lesões induzidas por benzo(a)pireno em Saccharomyces cerevisiae

El objetivo del presente estudio fue analizar el efecto del ácido clorogénico, uno de los compuestos polifenólicos con mayor concentración en la infusión de Ilex paraguariensis, sobre el daño celular y molecular inducido por el benzo(a)pireno. La infusión de Ilex paraguariensis ("mate") es bebida por la mayoría de los habitantes de Argentina, Paraguay, sur de Brasil y Uruguay. La levadura Saccharomyces cerevisiae (cepas SC7K lys2-3; SX46A y SX46Arad14() se utilizó como modelo eucariota. Las células en crecimiento exponencial se expusieron a concentraciones crecientes de benzo(a)pireno y a tratamientos combinados con una concentración de 250 ng/mL de benzo(a)pireno y ácido clorogénico a una concentración igual a la encontrada en la infusión de yerba mate. Luego de los tratamientos se determinaron fracciones de sobrevida, frecuencia mutagénica y roturas de doble cadena de ADN así como la modulación en la expresión de la proteína Rad14 a través de un análisis de Western Blot. Se observó un aumento significativo en las fracciones de sobrevida así como una disminución en la frecuencia mutagénica después de la exposición combinada con benzo(a)pireno y ácido clorogénico en comparación con los tratamientos con benzo(a)pireno como único agente. En la cepa mutante deficiente en la proteína Rad14 se observó un aumento significativo en la sensibilidad a benzo(a)pireno en comparación con la cepa SC7K lys2-3. En los tratamientos combinados de benzo(a)pireno y ácido clorogénico se observó una importante disminución de la letalidad. Con respecto a la determinación de roturas de doble cadena de ADN no se observó fraccionamiento cromosómico a la concentración de benzo(a)pireno utilizada en los experimentos. Los análisis de Western Blot mostraron un aumento en la expresión de la proteína Rad14 en las muestras tratadas con benzo(a)pireno como único agente en comparación con la muestra control. Adicionalmente se observó una disminución en la expresión de la proteína cuando en los tratamientos se utilizaron benzo(a)pireno y ácido clorogénico combinados. Los resultados indican que el ácido clorogénico disminuye significativamente la actividad mutagénica producida por el benzo(a)pireno, la cual no se encuentra relacionada con un incremento en la remoción de las lesiones inducidas por el sistema de reparación por escisión de nucleótidos.

The aim of this study was to analyze the effect of chlorogenic acid, a polyphenolic compound found at high concentrations in Ilex paraguariensis infusions, on cellular and molecular damage induced by benzo(a)pyrene. Ilex paraguariensis infusions ("mate") are consumed by most of the population in Argentina, Paraguay, southern Brazil and Uruguay. Saccharomyces cerevisiae yeast (SC7K lys2-3; SX46A and SX46Arad14( strains) were used as eukaryotic model organisms. Cells in an exponential growth phase were exposed to increasing concentrations of benzo(a)pyrene, as well as combined treatments of benzo(a)pyrene at a concentration of 250 ng/mL and chlorogenic acid at a concentration matching that which is commonly found in mate. Determinations of surviving fraction, mutagenic frequency and double strand DNA breaks induction were performed, along with the assessment of the modulation of the expression of protein Rad14 by Western Blot. A significant increase of surviving fractions and a decrease in mutagenic frequency were observed after exposure to benzo(a)pyrene plus chlorogenic acid, contrary to benzo(a)pyrene alone. A substantial increase in sensitivity to benzo(a)pyrene was observed for the Rad14 protein-deficient mutating strain when compared to the SC7K lys2-3 strain. An important decrease in lethality was observed when combined benzo(a)pyrene and chlorogenic acid treatments were applied. As for the determination of DSBs, no chromosomic fractionation was observed at the benzo(a)pyrene concentration tested in the experiments. Western Blot analysis showed an increase in the expression of protein Rad14 for samples treated with benzo(a)pyrene as a single agent when compared against the control sample. Additionally, the expression of this protein was observed to diminish when combined treatments with benzo(a)pyrene and chlorogenic acid were used. Results obtained indicate that chlorogenic acid significantly decreases the mutagenic activity of benzo(a)pyrene, which is not related to an increase in the removal of lesions induced by nucleotide excision repair system.

O objetivo deste estudo foi analisar o efeito do ácido clorogênico, um dos compostos polifenólicos com maior concentração na infusão de Ilex paraguariensis, sobre o dano celular e molecular induzido pelo benzopireno. A infusão de Ilex paraguariensis ("mate") é consumida pela maioria dos habitantes da Argentina, Paraguai, sul do Brasil e Uruguai. A levedura Saccharomyces cerevisiae (cepas SC7K lys2-3; SX46A e SX46Arad14() foi utilizada como modelo eucariótico. Células em crescimento exponencial foram expostas a concentrações crescentes de benzopireno e tratamentos combinados foram realizados com uma concentração de 250 ng/mL de benzo(a)pireno e ácido clorogênico, igual à encontrada na infusão de erva-mate. Após os tratamentos, foram determinadas as frações de sobrevivência, frequência mutagênica e quebras de fita dupla do DNA, bem como a modulação na expressão da proteína Rad14 por meio de análise de Western Blot. Um aumento significativo nas frações de sobrevivência, bem como uma diminuição na frequência mutagênica foram observados após a exposição combinada de benzo(a)pireno e ácido clorogênico em comparação com tratamentos de agente único de benzo(a)pireno. Um aumento significativo na sensibilidade ao benzo(a)pireno foi observado na cepa mutante deficiente em proteína Rad14 em comparação com a cepa SC7K lys2-3. Nos tratamentos combinados de benzo(a)pireno e ácido clorogênico, observou-se uma diminuição significativa na letalidade. Com relação à determinação das quebras de fita dupla de DNA, não foi observado fracionamento cromossômico na concentração de benzo(a)pireno utilizada nos experimentos. A partir da análise de Western Blot, observou-se um aumento na expressão da proteína Rad14 nas amostras tratadas com benzo(a)pireno como agente único em relação à amostra controle. Além disso, uma diminuição na expressão da proteína foi observada quando combinados de benzo(a)pireno e ácido clorogênico foram usados ​​nos tratamentos. Os resultados obtidos neste trabalho indicam que o ácido clorogênico diminui significativamente a atividade mutagênica produzida pelo benzo(a)pireno, a qual não está relacionada a um aumento na remoção de lesões induzidas pelo sistema de reparo por excisão de nucleotídeos.

Functional and transcriptomic characterization of carboplatin-resistant A2780 ovarian cancer cell line

BACKGROUND: Ovarian cancer is a significant cancer-related cause of death in women worldwide. The most used chemotherapeutic regimen is based on carboplatin (CBDCA). However, CBDCA resistance is the main obstacle to a better prognosis. An in vitro drug-resistant cell model would help in the understanding of molecular mechanisms underlying this drug-resistance phenomenon. The aim of this study was to characterize cellular and molecular changes of induced CBDCA-resistant ovarian cancer cell line A2780. METHODS: The cell selection strategy used in this study was a dose-per-pulse method using a concentration of 100 µM for 2 h. Once 20 cycles of exposure to the drug were completed, the cell cultures showed a resistant phenotype. Then, the ovarian cancer cell line A2780 was grown with 100 µM of CBDCA (CBDCA-resistant cells) or without CBDCA (parental cells). After, a drug sensitivity assay, morphological analyses, cell death assays and a RNA-seq analysis were performed in CBDCA-resistant A2780 cells. RESULTS: Microscopy on both parental and CBDCA-resistant A2780 cells showed similar characteristics in morphology and F-actin distribution within cells. In cell-death assays, parental A2780 cells showed a significant increase in phosphatidylserine translocation and caspase-3/7 cleavage compared to CBDCA-resistant A2780 cells (P < 0.05 and P < 0.005, respectively). Cell viability in parental A2780 cells was significantly decreased compared to CBDCA-resistant A2780 cells (P < 0.0005). The RNA-seq analysis showed 156 differentially expressed genes (DEGs) associated mainly to molecular functions. CONCLUSION: CBDCA-resistant A2780 ovarian cancer cells is a reliable model of CBDCA resistance that shows several DEGs involved in molecular functions such as transmembrane activity, protein binding to cell surface receptor and catalytic activity. Also, we found that the Wnt/3-catenin and integrin signaling pathway are the main metabolic pathway dysregulated in CBDCA-resistant A2780 cells.

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Evaluación de la calidad de vida de pacientes bipolares chilenos: propiedades psicométricas y utilidad diagnóstica de la versión chilena del cuestionario Quality of Life Bipolar Disorder (QoL.BD-CL) / Assessment of a version adapted and translated into Spanish of the Quality of Life Bipolar Disorder Questionnaire

Background: The Quality of life Bipolar Disorder (QoL.BD) Questionnaire specifically measures quality of life in patients with bipolar disorder. Aim: To adapt a version translated into Spanish of the questionnaire and assess its validity in Chilean patients. Material and Methods: The QoL. BD was adapted to the Chilean population through the back-translation method and then administered to 32 adult patients with a bipolar disorder and 31 subjects without the disease, both groups with similar socioeconomic status. To confirm the diagnosis, the International Neuropsychiatric Interview (MINI), Young (YMRS) and Hamilton (HAM-D) scales were applied. Quality of life was assessed using the SF-36v.2 survey. We determined internal consistency, reliability, convergent validity, the cut-off point, and the sensibility and specificity of the scale. Results: The Chilean version of the Questionnaire [QoL. BD-CL] had a high reliability (α = 0.95) and a high validity in reference to external criteria (correlation coefficients with SF-36 ranging from 0.453 and 0.819; p < 0.01). A cut-off point of 170, with sensitivity of 87.9% and specificity of 80% was determined. Conclusions: QoL.BD-CL has adequate psychometric properties, as well as an adequate sensitivity and specificity to distinguish between negative and positive perceptions of life quality in Chilean patients with bipolar disorders.

Efeitos da luz UV-A e visível em células da pele e no cabelo / Effects UV-A and visible light on skin cells and hair

A luz solar apresenta ondas eletromagnéticas em ampla faixa espectral, incluindo as regiões do ultravioleta (UV-C, UV-B, UV-A), visível e infravermelho. Cada região interage com a pele de forma dependente da fotofísica e da fotoquímica dos seus respectivos compostos absorvedores. A luz UV-A causa a geração de espécies reativas de oxigênio e de nitrogênio (EROs e ERNs) através da fotossensibilização de moléculas endógenas (co-enzimas de flavina, porfirinas, melaninas). Quando fotossensibilizadores produzem quantidades de EROs e ERNs maiores do que a capacidade celular de supressão destas espécies, caracteriza-se um quadro de desbalanço redox, que causa lesão em biomoléculas como os ácidos nucleicos, lipídeos e as proteínas. Essas lesões podem levar à morte celular ou a outras transformações fenotípicas e genotípicas e também estimulam a liberação de citocinas pró-inflamatórias. Com a finalidade de melhor compreender a dinâmica dos mecanismos de resposta celular após exposição ao UV-A e ao visível, nós caracterizamos inicialmente as propriedades fotofísicas da melanina e detectamos a produção de oxigênio singlete (1O2) pela fotossensibilização no visível e a supressão desta espécie excitada pela reação do oxigênio singlete com a dupla ligação reativa dos grupos indóis presentes na melanina. Estes processos também foram observados no cabelo e levaram-nos a propor um modelo que explica o efeito da luz visível na estrutura e cor dos cabelos. Demonstramos também que a feomelanina produz mais (30%) 1O2 do que a eumelanina, que sofre maior modificação na sua estrutura por fotodegradação. O efeito destes processos na pele foi estudado a nível celular. Demonstramos que células epiteliais com maior teor de melanina apresentaram maior geração de 1O2 que causa lesão no DNA e morte necro-apoptótica após irradiação com luz visível. A foto-oxidação da melanina pela luz visível nos motivou a estudar um pigmento que fosse foto-protetor não somente contra luz UV-B mas também contra luz visível. A pigmentação com Acetil-Tirosina se mostrou atóxica e protetora contra luz UV-B e visível ao contrário do pigmento com tirosina, que se mostrou protetor do UV-B mas tóxico no visível. Este efeito foi relacionado com a localização celular do polímero e não com a estrutura do mesmo. A luz UV-A, por sua vez, promove o acúmulo de lipofuscina dentro dos vacúolos autofágicos de queratinócitos da pele e que também ativa a fototoxicidade pela luz visível. A lipofuscina dentro dos vacúolos autofágicos é foto-oxidada pela luz visível, causando lesão no DNA e morte celular programada tipo II. Doses UV-A que desencadeiam a liberação de citocinas também foram caracterizados

Sunlight presents electromagnetic radiation over a wide spectral range, including the regions of ultraviolet (UV-C, UV-B, UV-A), visible and infrared. Each region interacts with skin dependending on the photophysics and photochemistry of the respective absorbing compounds. UV-A light causes the generation of reactive oxygen and nitrogen species (ROS and RNS) by photosensitization of endogenous molecules (flavin coenzymes, porphyrins, melanins). When photosensitizers produce amounts of ROS and RNS larger than the cell capacity to suppress these species, a set of redox imbalance, which damages biomolecules such as nucleic acids, lipids and proteins. This damage cause cell death and to other phenotypic and genotypic changes and also stimulates the release of proinflammatory cytokines. In order to better understand the dynamics of the mechanisms of cellular responses after exposure to UV-A and visible light, we initially characterized the photophysical properties of melanin and detected the production of singlet oxygen (1O2) by photosensitization in the visible, as well as the suppression of these excited species by reaction of singlet oxygen with the double bonds of the reactive groups presented in the melanin indols. These processes were also observed in hair and led us to propose a model that explains the effects of visible light on the structure and color of hair. We also demonstrated that pheomelanin produces more (30%) 1O2 than eumelanin, which undergoes a quick change on its structure by photodegradation. The effect of these processes in the skin was studied at the cellular level. We demonstrated that epithelial cells with larger melanin content have stronger generation of 1O2, which causes DNA damage and necro-apoptotic death after irradiation with visible light. The photo-oxidation of melanin by visible light has motivated us to study a pigment that was not only able to protect against UV-B but also against visible. Pigmentation with Acetyl-Tyrosine proved nontoxic and protective against UV-B and visible light instead of pigmentation with Tyrosine, which shielded against UV-B but showed toxicity in the visible. This effect was associated with the polymer, cell location and not with its structure. UV-A light, in turn, promotes the accumulation of lipofuscin, within autophagic vacuoles of keratinocytes also enabling phototoxicity in the visible light. The lipofuscin within the autophagic vacuoles is fotooxidized by visible light, causing DNA damage and programmed cell death type II. Linear dose of UV-A that trigger the release of cytokines were also characterized

Effect of Heat Shock Protein 72 Expression on Etoposide-induced Cell Death of Rat Retinal Ganglion Cells

PURPOSE: To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. METHODS: Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. RESULTS: Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. CONCLUSIONS: Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.

Regulação da morte celular pelo fator de transcrição NFAT1 / Regulation of cell death by the transcription factor NFAT1

Os fatores de transcrição da família NFAT (nuclear factor of activated T cells) foram inicialmente descritos por desempenhar um papel central na resposta imune. Esta família de fatores de transcrição é composta por cinco diferentes genes, NFAT1-5, que codificam diferentes isoformas protéicas. Os membros NFAT1-4 possuem dois importantes domínios de ativação da transcrição (TAD – transcription activation domain) localizados nas regiões N- e C-terminais (TAD-N; TAD-C) da proteína. Estes domínios possuem relativamente pouca conservação de sequência e são, possivelmente, as principais regiões responsáveis pelas diferenças na regulação gênica entre os diferentes membros da família NFAT. Diversos estudos já demonstraram que as proteínas NFAT desempenham papéis não redundantes no processo de tumorigênese. Recentemente, nosso grupo demonstrou que o NFAT1/C reverte a transformação celular induzida pelo oncogene H-RasV12 em fibroblastos NIH3T3 pela ativação do mecanismo de morte celular. O NFAT1 já foi descrito por regular genes envolvidos na apoptose, tais como FasL, TNF-alfa e Nur77. O NFAT1 regula forma direta a expressão de FasL e TNF-alfa e de forma indireta o Nur77 agindo como um coativador de MEF2D. Nesta dissertação, nós demonstramos que as isoformas predominantes do NFAT1, B e C (CANFAT1/ B; CA-NFAT1/C), são capazes de induzir apoptose em fibroblastos NIH3T3. Além disso, demonstramos que a retirada dos resíduos 699 a 888 do TAD-C abole por completo a capacidade de induzir apoptose do NFAT1/C e induz a hiperproliferação de fibroblastos NIH3T3. No entanto, apesar da apoptose induzida pelo NFAT1/C ser dependente de resíduos de aminoácidos presentes no seu TAD-C, demonstramos que o TAD-C não é capaz de induzir apoptose em fibroblastos NIH3T3 por si só. O TAD-C também não age como um dominante negativo do NFAT1/C indicando que esta região da proteína não interage com nenhuma proteína parceira para induzir apoptose. Além disso, demonstramos que a apoptose induzida pelo NFAT1/C é dependente da sua ligação ao DNA. Em conjunto, estes dados sugerem que o gene induzido para ativar a apoptose no nosso modelo é diretamente regulado pelo NFAT1/C e depende do seu TAD-C para ser ativado.

Transcription factors of the NFAT family (nuclear factor of activated T cells) were initially described to play a central role in immune response. This family of transcription factors consists of five different genes, NFAT1-5, which encode different protein isoforms. Members NFAT1-4 possess two significant transcription activation domains (TAD) located in the N- and C-terminal protein regions (TAD-N and TAD-C). These domains have relatively little conservation of sequence and are, possibly, the main regions responsible for differences in gene regulation between different NFAT family members. Several studies have shown that NFAT proteins play non-redundant roles in the process of tumorigenesis. Recently, our group has shown that the NFAT1/C reverses HRasV12- induced cell transformation in NIH3T3 fibroblasts by the activation of cell death mechanism. The NFAT1 has been described to regulate genes involved in apoptosis such as FasL, TNF-alfa and Nur77. The NFAT1 directly regulates the expression of FasL and TNF-alfa and indirectly the expression of Nur77 acting as a coactivator of MEF2D. In this dissertation, we demonstrated that the predominant isoforms of NFAT1, B and C (CA-NFAT1/B, CANFAT1/ C), are able to induce apoptosis in NIH3T3 fibroblasts. Furthermore, we demonstrated that the removal of residues 699 to 888 of TAD-C completely abolishes the ability to induce apoptosis of NFAT1/C and induces the proliferation of NIH3T3 fibroblasts. However, despite the apoptosis induced by NFAT1/C to be dependent on amino acid residues present in their TAD-C, we demonstrated that the TAD-C is not able to induce apoptosis in NIH3T3 fibroblasts alone. The TAD-C also does not act as a dominant negative NFAT1/C indicating that this region of the protein does not interact with any protein partner to induce apoptosis. Furthermore, we demonstrated that apoptosis induced by NFAT1/C is dependent on its binding to DNA. Together, these results suggest that the gene induced to activate apoptosis in our model is directly regulated by NFAT1/C and depends on your TAD-C to be activated.

GITR expression on T-cell receptor-stimulated human CD8+ T cell in a JNK-dependent pathway.

Glucocorticoid-induced tumor necrosis factor receptor (TNFR) (GITR) family-related gene is a member of the TNFR super family. GITR works as one of the immunoregulatory molecule on CD4+ regulatory T cells and has an important role on cell survival or cell death in CD4+ T cells. Little is known about the expression of GITR on human CD8+ T cells on antigen-specific and non-specific activation. Here, we report that expression of GITR on human CD8+ T cells on T-cell receptor (TCR) (anti-CD3)-mediated stimulation is dependent on the JNK pathway. The activation of CD8+ T cells was measured by the expression of IL-2 receptor-α (CD25), GITR and by IFN-γ production upon re-stimulation with anti-CD3 antibody. We studied the signaling pathway of such inducible expression of GITR on CD8+ T cells. We found that a known JNK-specific inhibitor, SP600125, significantly down-regulates GITR expression on anti-CD3 antibody-mediated activated CD8+ T cells by limiting JNK phosphorylation. Subsequently, after stimulation of the CD8+ cells, we tested for the production of IFN-γ by the activated cells following restimulation with the same stimulus. It appears that the expression of GITR on activated human CD8+ T cells might also be regulated through the JNK pathway when the activation is through TCR stimulation. Therefore, GITR serves as an activation marker on activated CD8+ cells and interference with JNK phosphorylation, partially or completely, by varying the doses of SP600125 might have implications in CD8+ cytotoxic T cell response in translational research.

Apoptosis: programmed cell death

A apoptose e um processo fisiologico e determinado geneticamente que leva a morte celular. Na necrose celular ocorre lesäo da celula por tumefaçäo citoplasmatica, perda de integridade da membrana e ruptura com um tipo de morte celular "acidental". Diferentemente, a apoptose e caracterizada por alteraçöes celulares que se iniciam no nucleo com fragmentaçäo do DNA com atrofia e morte celular "programada". As celulas que sofrem apoptose säo degradadas e absorvidas pelas celulas vizinhas sem sinais inflamatórios locais. O fenomeno da apoptose foi conhecido a partir do estudo de um neumatoide primitivo, o Caenorhabidtis elegans que possui somente 1090 celulas, das quais 131 säo programadas para morrer durante seu ciclo de vida...