Dataset Information

Identification of genes associated with methotrexate resistance in methotrexate-resistant osteosarcoma cell lines.

ABSTRACT: This study aimed to better understand the mechanisms underlying methotrexate (MTX)-resistance in osteosarcoma.The raw transcription microarray data GSE16089 collected from three MTX-sensitive osteosarcoma (Saos-2) cell samples and three MTX-resistant osteosarcoma (Saos-2) cell samples were downloaded from Gene Expression Omnibus. After data processing, the differentially expressed genes (DEGs) were identified. Next, DEGs were submitted to DAVID for functional annotation based on the GO (Gene Ontology) database, as well as pathway enrichment analysis based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Transcription factors (TFs) and tumor-associated genes (TAGs) were identified with reference to TRANSFAC and TAG, and TSGene databases, respectively. The protein-protein interaction (PPI) network of the gene-encoded products was constructed, and the subnetwork with the highest score was also detected using Search Tool for the Retrieval of Interacting Genes and BioNet package.A total of 690 up-regulated genes and down-regulated 626 genes were identified. Up-regulated DEGs (including AARS and PARS2) were associated to transfer RNA (tRNA) aminoacylation while down-regulated DEGs (including AURKA, CCNB1, CCNE2, CDK1, and CENPA) were correlated with mitotic cell cycle. Totally, 13 TFs (including HMGB2), 13 oncogenes (including CCNA2 and AURKA), and 19 tumor suppressor genes (TSGs) (including CDKN2C) were identified from the down-regulated DEGs. Ten DEGs, including nine down-regulated genes (such as AURKA, CDK1, CCNE2, and CENPA) and one up-regulated gene (GADD45A), were involved in the highest score subnetwork.AARS, AURKA, AURKB, CENPA, CCNB1, CCNE2, and CDK may contribute to MTX resistance via aminoacyl-tRNA biosynthesis pathway, cell cycle pathway, or p53 signaling pathway.

SUBMITTER: Yang XR

PROVIDER: S-EPMC4558632 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of genes associated with methotrexate resistance in methotrexate-resistant osteosarcoma cell lines.

Yang Xiao-Rong XR Xiong Yan Y Duan Hong H Gong Ren-Rong RR

Journal of orthopaedic surgery and research 20150904

<h4>Background</h4>This study aimed to better understand the mechanisms underlying methotrexate (MTX)-resistance in osteosarcoma.<h4>Methods</h4>The raw transcription microarray data GSE16089 collected from three MTX-sensitive osteosarcoma (Saos-2) cell samples and three MTX-resistant osteosarcoma (Saos-2) cell samples were downloaded from Gene Expression Omnibus. After data processing, the differentially expressed genes (DEGs) were identified. Next, DEGs were submitted to DAVID for functional a ...[more]

PMID: 26337976

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Project description:IntroductionMethotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use.MethodsTo better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX.ResultsMTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested.ConclusionAltogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.

Project description:A summary of the work associated to these microarrays is the following: The need for an integrated view of all data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize phenomena in terms of 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). Seven cell lines representative of different types of cancer including colon cancer (HT29 and Caco2), breast cancer (MCF7 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 microarrays covering the whole human genome and analyzed with the GeneSpring GX software package, v.7.3.1. Genes deregulated in common in the two colon cancer cell lines studied, were subject of Biological Association Networks construction. Dikkopf homolog-1 (DKK1) was a clear node of this network, and functional validations of this target using a siRNA showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of differentially expressed genes in the two breast cancer cell lines studied. siRNA treatment against UGT1A showed also an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was a gene overexpressed in common among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Biological Association Networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using iRNA technology against these three genes show chemosensitization toward MTX. Two cell lines are compared, which are Saos-2 osteosarcoma cells sensitive to methotrexate and Saos-2 cells resistant to 10e-6M methotrexate. Six samples are provided which correspond to triplicates of each cell line. The samples provided were analyzed using the specific software GeneSpring GX.

Dataset Information

Identification of genes associated with methotrexate resistance in methotrexate-resistant osteosarcoma cell lines.

Publications

Identification of genes associated with methotrexate resistance in methotrexate-resistant osteosarcoma cell lines.

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