Carlota Oleaga

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

BackgroundThe 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).MethodsSeven 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.ResultsDikkopf 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.ConclusionsBiological association networks identified DKK1, UGT1A s and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics.

Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the Match™ software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.

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 methotrexate-induced 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.

Cocoa flavanol metabolites activate HNF‐3β, Sp1, and NFY‐mediated transcription of apolipoprotein AI in human cells

SCOPE To identify the mechanisms by which cocoa induces HDL levels and since apolipoprotein AI (ApoAI) is the major protein in HDLs, we analyzed, upon incubation with cocoa metabolites, ApoAI mRNA levels, its transcriptional regulation, and the levels of the transcription factors involved in this process. METHODS AND RESULTS Epicatechin and cocoa metabolites caused an increase in ApoAI expression in HepG2 cells. Electrophoretic mobility shift assays revealed the involvement of Sites A and B of the ApoAI promoter in the induction of ApoAI mRNA upon incubation with cocoa metabolites. Using supershift assays, we demonstrated the binding of HNF-3β, HNF-4, ER-α, and RXR-α to Site A and the binding of HNF-3β, NFY, and Sp1 to Site B. Luciferase assays performed with a construct containing Site B confirmed its role in the upregulation of ApoAI by cocoa metabolites. Incubation with 3-methyl-epicatechin led to an increase in HNF-3β mRNA, HNF-3β, ER-α, Sp1, and NFY protein levels and the activation of ApoAI transcription mediated by NFY, Sp1, and ER-α. CONCLUSION The activation of ApoAI transcription through Site B 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.

Stability and immunogenicity properties of the gene-silencing polypurine reverse Hoogsteen hairpins.

Gene silencing by either small-interference RNAs (siRNA) or antisense oligodeoxynucleotides (aODN) is widely used in biomedical research. However, their use as therapeutic agents is hindered by two important limitations: their low stability and the activation of the innate immune response. Recently, we developed a new type of molecule to decrease gene expression named polypurine reverse Hoogsteen hairpins (PPRHs) that bind to polypyrimidine targets in the DNA. Herein, stability experiments performed in mouse, human, and fetal calf serum and in PC3 cells revealed that the half-life of PPRHs is much longer than that of siRNAs in all cases. Usage of PPRHs with a nicked-circular structure increased the binding affinity to their target sequence and their half-life in FCS when bound to the target. Regarding the innate immune response, we determined that the levels of the transcription factors IRF3 and its phosphorylated form, as well as NF-κB were increased by siRNAs and not by PPRHs; that the expression levels of several proinflammatory cytokines including IL-6, TNF-α, IFN-α, IFN-ß, IL-1ß, and IL-18 were not significantly increased by PPRHs; and that the cleavage and activation of the proteolytic enzyme caspase-1 was not triggered by PPRHs. These determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response.

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 downregulated 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.