Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX

Estudio nutrigenómico de compuestos polifenólicos del cacao y del café en células tumorales humanas

[spa] La investigación en genómica nutricional combina las áreas de conocimiento de biología molecular, genética y nutrición. Los compuestos bioactivos de la dieta se podrían definir como aquellos componentes con potencial para modificar un elevado número de procesos biológicos asociados al equilibrio entre salud y enfermedad. En la presente tesis se han desarrollado tres estudios nutrigenómicos con compuestos polifenólicos del cacao y del café, con el objetivo de profundizar en el conocimiento de los mecanismos de acción por los que ejercen su actividad. En el estudio de los efectos de un extracto de polifenoles de cacao (PCE) en líneas celulares de cáncer de mama, las conclusiones obtenidas son las siguientes: 1. El tratamiento con PCE induce la expresión de CYP1A1, así como sus niveles de proteína y la actividad enzimática en dos líneas celulares de cáncer de mama, MCF-7 y SKBR3. 2. La inducción de CYP1A1 mediada por PCE tiene lugar a través de la vía de señalización de AhR. PCE induce la transcripción de CYP1A1 a través de la unión de AhR a las cajas XRE de su promotor en ambas líneas celulares. 3. PCE induce los niveles de ERα en la línea celular SKBR3 aumentando la síntesis de la proteína, pero su vida media se reduce aproximadamente 10 horas en comparación con la proteína basal. 4. La incubación con PCE y tamoxifeno (TAM) a concentraciones no citotóxicas inducen un efecto sinérgico provocando un descenso en la viabilidad de dos líneas de cáncer de mama, MCF-7 y SKBR3, pero no en la línea no tumoral HEK293T. En el análisis de los efectos de distintos metabolitos del cacao sobre la expresión de la apolipoproteína A1 en la línea celular HepG2 de hepatoma, establecimos las siguientes conclusiones: 1. La epicatequina (EPI) y los metabolitos del cacao inducen la expresión de ApoAI en la línea celular HepG2. 2. El tratamiento con EPI en células HepG2 induce la unión de factores de transcripción a dos regiones del “liver specific enhancer”, Site A y Site B. En la unión a Site A participan los factores de transcripción HNF-4, RXRα y ERα, además de HNF3β de manera indirecta. En la unión a Site B participa el factor de HNF-3β. EPI y los metabolitos del cacao estudiados inducen los niveles de mRNA de HNF-3β. 3. La transcripción de ApoAI se induce por EPI y los metabolitos del cacao estudiados a través del Site B, siendo 3-M-EPI el metabolito que provoca una mayor activación transcripcional. 4. Los factores de transcripción NFY y Sp1, además de HNF-3β, participan en la unión al Site B inducida por la incubación con 3-M-EPI. La sobreexpresión de NFY y Sp1 induce la transcripción de ApoAI a través del Site B. Finalmente en la aproximación transcriptómica en células de cáncer de colon tratadas con ácido cafeico y café soluble, podemos concluir que: 1. El tratamiento con ácido cafeico (CA) y con café (ICC) genera un cambio en el perfil de expresión de la línea tumoral HT29. 2. Se han identificado dos genes nodo, STAT5B y ATF-2, tras la generación de una red de asociación biológica a partir de los genes diferencialmente expresados comunes a ambos tratamientos, CA e ICC. 3. La validación de los cambios en la expresión de ambos genes confirma la sobreexpresión de STAT5B e infraexpresión de ATF-2 inducida por la exposición a CA e ICC. También se ha confirmado el aumento en la proteína STAT5B inducida por el tratamiento con CA y con ICC y la disminución de los niveles de ATF-2 debidos al tratamiento con CA. 4. El tratamiento con CA e ICC modula los niveles de ciclina D1, proteína regulada por STAT5B y ATF-2 en dos líneas celulares de cáncer. En HT29, la exposición a CA reduce los niveles de ciclina D1, mientras que ICC induce los niveles de ciclina D1 al igual que los de ATF-2. En MCF-7 ambos tratamientos, CA e ICC, consiguen una disminución drástica de los niveles de proteína ciclina D1, a pesar de no acompañarles la disminución de ATF-2.[eng] NUTRIGENOMIC STUDIES OF COCOA AND COFFEE POLYPHENOLS IN HUMAN TUMOR CELL LINES Nutritional genomics research combines the knowledge areas of molecular biology, genetics and nutrition. Nutrigenomics studies how diet, or compounds from diet are able to modulate gene expression. Diet compounds such as polyphenols are known to produce beneficial effects in human homeostasis. Cocoa and coffee are two polyphenol food sources well studied in nutritional research. The aim of this work was to get further insight in the understanding of the mechanism of action through which cocoa and coffee polyphenols exert their beneficial effect, using three different nutrigenomics approaches. The main conclusions of these nutrigenomic studies are: 1) The interaction between ERα and AhR upon incubation with a polyphenolic cocoa extract leads to CYP1A1 induction in breast cancer cells. The synergy between PCE and non-cytotoxic tamoxifen concentrations opens the possibility for a combination therapy based on polyphenols from cocoa that increased tamoxifen efficacy. 2) The activation of Apolipoprotein AI transcription through Site B in its promoter by cocoa flavanol metabolites is mainly mediated by an increase in HNF-3β, with a significant contribution of Sp1 and NFY, as a mechanism for the protective role of these compounds in cardiovascular diseases. 3) Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2. The results of this thesis led to three publications in international journals; i) “CYP1A1 is overexpressed upon incubation of breast cancer cells with a polyphenolic cocoa extract” in European Journal of Nutrition ii) “Cocoa flavanol metabolites activate HNF-3β, Sp1 and NFY mediated transcription of Apolipoprotein AI in human cells” in Molecular Nutrition and Food Research iii) “Coffee Polyphenols Change the Expression of STAT5B and ATF-2 Modifying Cyclin D1 Levels in Cancer Cells” in Oxidative Medicine and Cellular Longevity

Coffee polyphenols change the expression of STAT5b and ATF-2 modifying cyclin D1 levels in cancer cells

Background: Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective: To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole genome microarrays. Results: ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were dowregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions: Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2

Nutritional genomics. A new approach in nutrition research

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32393There is an increasing evidence that nutritional genomics represents a promise to improve public health. This goal will be reached by highlighting the mechanisms through which diet can reduce the risk of common polygenic diseases. Nutritional genomics applies high throughput functional genomic technologies and molecular tools in nutrition research, allowing a more precise and accurate knowledge of nutrient-genome interactions in both health and disease. Understanding the inter-relationships among genes, genes products, and dietary habits is fundamental to identify those who will benefit the most or be placed at risk by nutritional interventions. This chapter provides an overview of this novel nutritional approach, including the most relevant results of our recent research on the nutrigenomic effects of food polyphenols on cancer cells. Those studies would highlight the molecular mechanisms underlying the chemopreventive effects of those bioactive food compounds

Coffee polyphenols change the expression of STAT5b and ATF-2 modifying cyclin D1 levels in cancer cells

Background: Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective: To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole genome microarrays. Results: ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were dowregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions: Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2

The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells

Generation of biological association networks: A novel strategy to detect new targets in cancer therapy

Podeu consultar el llibre complet a: http://www.trnres.com/ebookcontents.php?id=149The aim of this work was to design a novel strategy to detect new targets for anticancer treatments. The rationale was to build Biological Association Networks from differentially expressed genes in drug-resistant cells to identify important nodes within the Networks. These nodes may represent putative targets to attack in cancer therapy, as a way to destabilize the gene network developed by the resistant cells to escape from the drug pressure. As a model we used cells resistant to methotrexate (MTX), an inhibitor of DHFR. Selected node-genes were analyzed at the transcriptional level and from a genotypic point of view. In colon cancer cells, DHFR, the AKR1 family, PKC¿, S100A4, DKK1, and CAV1 were overexpressed while E-cadherin was lost. In breast cancer cells, the UGT1A family was overexpressed, whereas EEF1A1 was overexpressed in pancreatic cells. Interference RNAs directed against these targets sensitized cells towards MTX