Unknown,Transcriptomics,Genomics,Proteomics

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Diethyl maleate inhibits MCA+TPA transformed cell growth via modulation of GSH, MAPK, and cancer pathways

ABSTRACT: Murine or human cancer cells have high glutathione levels. Depletion of the elevated GSH inhibits proliferation of cancer cells. Molecular basis for this observation is little understood. In an attempt to find out the underlying mechanism, we reproduced these effects in transformed C3H10T1/2 and BALB/c 3T3 cells using diethyl maleate and studied cytogenomic changes in the whole mouse genome using spotted 8 M-CM-^W 60K arrays. Transformed cells revealed an increase in GSH levels. GSH depletion by DEM inhibited the growth of transformed cells. The non-cytotoxic dose of DEM (0.25 mM) resulted in GSH depletion, ROS generation, cell cycle arrest, apoptosis, decrease in anchorage independent growth, gene expression changes and activation of all three members of the MAPK family. Increase in intracellular GSH levels by GSHe countered the effect of DEM. These results support the physiological importance of GSH in regulation of gene expression for transformed cell growth restraint. This study is of interest in not only understanding the molecular biology of the transformed cells, but also in identifying new targets for development of gene therapy together with the chemotherapy. Agilent one-color experiment; Organism: Mus musculus; Agilent Custom Mouse Whole Genome Mouse 8x60k gene expression designed by Genotypic Technology Private Limited; Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442).

ORGANISM(S): Mus musculus

SUBMITTER: Dr Sushil Kumar

PROVIDER: E-GEOD-44650 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Diethyl maleate inhibits MCA+TPA transformed cell growth via modulation of GSH, MAPK, and cancer pathways.

Priya Shivam S Nigam Akanksha A Bajpai Preeti P Kumar Sushil S

Chemico-biological interactions 20140509

Murine or human cancer cells have high glutathione levels. Depletion of the elevated GSH inhibits proliferation of cancer cells. Molecular basis for this observation is little understood. In an attempt to find out the underlying mechanism, we reproduced these effects in transformed C3H10T1/2 and BALB/c 3T3 cells using diethyl maleate and studied cytogenomic changes in the whole mouse genome using spotted 8 × 60 K arrays. Transformed cells revealed an increase in GSH levels. GSH depletion by DEM ...[more]

PMID: 24814887

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Project description:The two-stage cell transformation assay is an in vitro model cell culture system to identify the ability of chemicals to act as initiators or promoters of cell transforma- tion and also to study the cellular and molecular mechanisms of chemically induced morphological and neoplastic cell transformation. The global gene expression profiles of 3- methylcholanthrene (MCA)+12-O-tetradecanoylphorbol-13- acetate (TPA)-transformed C3H/10T1/2 cells are not known. Therefore, we have investigated the global transcriptional profile of MCA+TPA-transformed C3H10T1/2 cells using an 8M-CM-^W60 k probe microarray. The study revealed a differential regulation of pathways and gene expressions. Multifold dysregulation was seen in pathways of cancer, phagosomal activity, and tumor cell microenvironment information pro- cessing systems, notably the neuroactive ligandM-bM-^@M-^Sreceptor in- teraction, actin cytoskeleton regulation, tight junction, axon guidance, and cell adhesion molecules. The genes FGF1, EIF4E1B, MAGI1,and GRIA3 showed upregulation; these encoded the pluripotent fibroblast growth factor, the transla- tion initiation factor, the tight junction scaffolding protein, and the antiapoptotic as well as the enhancer of proliferation and migration, respectively. The genes CXCL7/CXCL5/CXCL12, H2DMB1,and HSPA1A showed downregulation; these encoded the chemotactic agent protein, the protein involved Total mRNA was isolated from the control as well as the MCA+TPA-transformed C3H/10T1/2 cells and complemen- tary RNA (cRNA) was prepared from mRNA (1 M-NM-<g). One- color microarray processing was performed. Acceptable qual- ity of the total RNA sample was ascertained by its electropho- resis trace and integrity assay using a Bioanalyzer which profiled RNA by RIN interpretation. The T7 promoter-based linear amplification was used to generate labeled complemen- tary RNA to amplify target material and incorporate cyanine 3-labeled CTP using AgilentM-bM-^@M-^Ys low-input RNA linear amplifi- cation kit one color (cat no. 5188-5339). The fluorescence- labeled cRNA samples were hybridized onto a Genotypic- designed Custom Whole Genome Mouse 8x60k slide (AMADID no. 26986) in duplicate using AgilentM-bM-^@M-^Ysinsitu hybridization kit (no. 5184-3568). Hybridization was carried out in AgilentM-bM-^@M-^Ys Surehyb Chambers at 65M-BM-0C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers (part no. 5188-5327). Fluorescence data were collected using an Agilent Microarray Scanner (G2567AA) and analyzed with program Gene Spring GX, version 11.5 (Agilent Technologies, Bangalore, India). Significantly dysregulated genes were identified. Statistical t test p value was calculated based on volcano plot using Gene spring GX Software.

Project description:Objectives: Eosinophils cultured with all-trans RA (ATRA) and 9-cis RA showed dramatically induced cell survival, and the efficacy of RAs (10-6 M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Using a gene microarray, we studied mRNA profile regulated by RAs. Methods: Peripheral venous blood was obtained from subjects with mild eosinophilia. Eosinophils were isolated by sedimentation with 6% dextran followed by centrifugation on 1.088 Percoll (Pharmacia, Uppsala, Sweden) density gradients. The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified eosinophils were resuspended in RPMI 1640 medium (Gibco, Grand Island, NY) with 10% FCS, and incubated with vehicle, 10-6 M of 9-cis RA, or ATRA in 1% human serum albumin-coated plates, each 4 Ã 106 cells, for 4 h. Then cells were lysed, and total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) as per the manufacturerâs instructions. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis. Results: There was little variation between the samples treated by 9-cisRA and ATRA. The comparison between vehicle control and 9-cisRA, or vehicle control and ATRA, identified an increase (greater than twofold change over control) in 19 and 11 transcripts, respectively. The number of decreased transcripts (less than 0.5-fold) resulting fromby 9-cisRA or ATRA exposure wasere 19 and 8 transcripts, respectively. In these transcripts, the expression changes relative to vehicle control were smaller in withthe ATRA compared with 9-cis RA, suggesting that 9-cis RA induced gene transcription more effectively rather than ATRA. Of note, among the ten genes with the biggestmost decrease relative tofrom vehicle control, we found an apoptosis- related gene, caspase-3. Conclusions: We screened several genes involved in RA-induced prolonged eosinophil survival. Purified peripheral blood eosinophils were incubated with vehicle, 10-6 M of 9-cis RA, or ATRA in 1% human serum albumin-coated plates, each 4 Ã 106 cells, for 4 h. Then cells were lysed, and total RNA was isolated. RNA was repurified with phenol-chloroform extraction and ethanol precipitation. For each cell culture condition, the total RNA from three donors was pooled, enabling us to acquire adequate amounts of RNA for microarray analysis.

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Diethyl maleate inhibits MCA+TPA transformed cell growth via modulation of GSH, MAPK, and cancer pathways

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Diethyl maleate inhibits MCA+TPA transformed cell growth via modulation of GSH, MAPK, and cancer pathways.

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