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Homo sapiens

ABSTRACT: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCa on resistance to methotrexate in human HT29 colon cancer cells

OTHER RELATED OMICS DATASETS IN: E-GEOD-16066E-GEOD-16085E-GEOD-16070E-GEOD-16089E-GEOD-16080E-GEOD-16082

PROVIDER: PRJNA122643 | ENA |

REPOSITORIES: ENA

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Project description:A summary of the work associated to these microarrays is the following:; Methotrexate (MTX) is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we have performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the DHFR gene or as a result of a transcriptome screening using microarrays. Genes adjacent to dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCa genes in treated cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. We provide functional evidences indicating that caveolin 1 and E-cadherin may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy. Experiment Overall Design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M MTX. Six samples are provided which correspond to triplicated of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX v7.3.1.

Project description:A summary of the work associated to these microarrays is the following: MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatment. MiRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed microRNAs in resistant cells were identified and miR-224, which was greatly underexpressed and displayed robust raw signal values, was selected for further studies. Putative targets were predicted using TargetScan 5.1 software and intersected with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them, ARL3, CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells toward MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells. KEYWORDS Methotrexate, miRNAs, drug resistance, DHFR Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M MTX. Six samples are provided which correspond to 3 samples for the control condition and 3 samples for the resistant condition. Data files from miRNA and mRNA (previously submitted to GEO as GSE11440) microarrays were analyzed with GeneSpring GX11.5 software (Agilent Technologies) to find differentially expressed miRNAs and their cellular target genes in the resistant cell lines compared to their sensitive counterparts.

Project description:The abstract of the associated publication (Selga E, NoÃ© V, Ciudad CJ. Biochemical Pharmacology 2007 Sep 8; Article in press) is the following:; While studying differentially expressed genes between sensitive and 10-5 M Methotrexate (MTX) resistant HT29 human colon cancer cells, we identified some members of the aldo-keto reductase (AKR) superfamily. The study was followed with the member AKR1C1 (EC 1.1.1.213), validating its increase in mRNA and protein levels in MTX resistant cells. The genomic content for AKR1C1 remained unchanged between sensitive and resistant cells, thereby excluding a mechanism of AKR1C1 gene amplification. Thus, we cloned the AKR1C1 human promoter and performed luciferase experiments that revealed a transcriptional regulation of the gene in the resistant cells. Computational studies showed a putative binding site for the transcription factor Sp1. The co-transfection of Sp1 or Sp3 with different constructs of AKR1C1 promoter deletions, including and excluding the proximal GC-box, demonstrated a key role for these factors in regulating AKR1C1 transcriptional activity. Gel-shift assays revealed an increase in Sp1 and Sp3 binding in resistant compared to sensitive cells, without differences in Sp1 protein levels. Dephosphorylation of the extracts coincided with a decrease in Sp1 binding, which is consistent with a process of regulation of Sp1 by phosphorylation. We also investigated the possible relationship between AKR1C1 expression and MTX action. Overexpression of AKR1C1 counteracted the S-phase accumulation of cells and apoptosis caused by MTX treatment. This suggests a role of AKR1C1 in cell proliferation. Finally, overexpression of AKR1C1 in MTX sensitive HT29 cells conferred resistance to the chemotherapeutic agent and silencing of AKR1C1 by means of iRNA technology sensitized the cells to MTX. Experiment Overall Design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M methotrexate. Six samples are provided, which correspond to triplicates of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX 7.3.1

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Homo sapiens

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