Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Microarray analysis of HK-2 cells treated with cisplatin

ABSTRACT: Chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. HK-2 cells were cultured to confluence in 100mm dishes. Total RNA was extracted (QIAGEN, Valencia, CA, USA), and the concentration in the samples was measured using a Micro UV-Vis fluorescence spectrophotometer (Malcom, Tokyo, JAPAN). Sample of 10ɥg of Total RNA from HK-2 cells were labeled with biotin (3'IVT Labeling Kit, Affymetrix, USA) and hybridized (GeneAtlas Hybridization, Wash, and Stain Kit for 3' IVT Arrays, Affymetrix). The Microarray platform used was Affymetrix HG U219 Array Strip. The arrays were scanned in a GeneAtlas scanner controlled by GeneAtlas Software (Affymetrix, Sta. Clara, USA). Genes were later filtered according to expression fold change (Affymetrix Expression Console software, Affymetrix) Using Affymetrix GeneAtlas System, we determined the gene expression profiles of HK-2 cells treated with cisplatin. Two replicates were made for each timepoints (control, 6h and 24h).

ORGANISM(S): Homo sapiens

SUBMITTER: Sangdun Choi

PROVIDER: E-GEOD-69644 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aim： Use microarray analysis to understand the molecular mechanism underlying the effect of aristolochic acid (AA), a major active component of plants from the Aristolochiaceae family, in normal human kidney (HK-2) cells. Methods： HK-2 cells were treated with AA for 24 hours and cell viability was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Complementary DNA microarrays were used to investigate the gene expression pattern of HK-2 cells exposed to AA and the results of this study were in triplicate. Quantitative real-time RT-PCR assay was used to verify the microarray data for selected nuclear factor kappa B (NF-κB)-regulated genes. Furthermore, subcellular localization of NF-κB p65 was visualized by immunofluorescence confocal microscopy in HK-2 cells. NF-κB activity was examined by luciferase reporter assay in HK-2/NF-κB transgenic cells. Results： AA exhibited a dose-dependent cytotoxic effect in HK-2 cells and induced alterations in gene expression profiles related to DNA damage response, stress response, etc. In addition, 9 biological pathways associated with immunomodulatory functions were down-regulated in AA-treated HK-2 cells. Network analysis revealed that NF-κB played a central role in the network topology. Among NF-kB-regulated genes, 8 differentially expressed genes were verified by real-time RT-PCR. The inhibition of NF-κB activity by AA was further confirmed by immunofluorescence confocal microscopy and by NF-κB luciferase reporter assay. Conclusion： Our data revealed that AA could suppress NF-κB activity in normal human cells, perhaps partially accounting for the reported anti-inflammatory effects of some plants from the genus Aristolochia. HK-2 cells were grown in keratinocyte serum-free basal medium (Gibco) supplemented with 5 ng/ml of recombinant epidermal growth factor and 50 μg/ml of bovine pituitary extract without antibiotics in 5 % CO2 at 370C. HK-2 cells were seeded in 25-T flasks and incubated for 24 h before aristolochic acid treatment. Aristolochic acid (10 90 μM) were added to HK-2 cells for 24 h. The control cells received equal amounts of water only.

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Microarray analysis of HK-2 cells treated with cisplatin

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