Comment[ArrayExpressAccession]	E-GEOD-42490
MAGE-TAB Version	1.1						
Public Release Date	2013-12-01
Investigation Title	DNA methyltransferase inhibitor zebularine inhibits human hepatic carcinoma cells proliferation and induces apoptosis.						
Comment[Submitted Name]	DNA methyltransferase inhibitor zebularine inhibits human hepatic carcinoma cells proliferation and induces apoptosis.
Experiment Description	Hepatocellular carcinoma is one of the most common cancers in world wide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine [1-(β-ᴅ-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. Here, we demonstrated that zebularine exhibited antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis. Zebularine treatment down-regulated CDK2 and phosphorylation of retinoblastoma protein (RB), and up-regulated p21WAF/CIP1 and p53. We also found that zebularine treatment up-regulated phosphorylation of p44/42 MAPK. These results suggest that p44/42 MAPK pathway play a role in zebularine induced cell cycle arrest by regulating activity of p21WAF/CIP1 and Rb. Furthermore, we found that zebularine induced apoptosis. Although proapoptotic protein Bax levels were not affected, antiapoptotic protein Bcl-2 level was down-regulated with zebularine treatment. The data in the present study suggest that the action of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results provide a novel mechanism of zebularine-induced cell growth arrest and apoptosis in hepatocellular carcinoma. Three each independent batches of zebuluarine-treated and control HepG2 cells were subjected to illumina Human methylation 450K Beadchip analysis.						
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl					
Person Last Name	Nakabayashi	Nakabayashi					
Person First Name	Kazuhiko	Kazuhiko					
Person Email	nakabaya-k@ncchd.go.jp						
Person Affiliation	National Research Institute for Child Health and Development						
Person Address	Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo, Japan						
Person Roles	submitter						
Protocol Name	P-GSE42490-1	P-GSE42490-5	P-GSE42490-6	P-GSE42490-2	P-GSE42490-3	P-GSE42490-4	P-GSE42490-7
Protocol Description	GenomeStudio V2011.1 was used to determine the methylation level at each CpG site as beta value. Normalization to internal controls and background subtraction were applied to determine normalized average beta values. ID_REF =  VALUE = Normalized average beta value Detection Pval = 	Standard Illumina protocol	Bisulfite converted DNA was amplified, fragmented and hybridized to illumina Infinium Human Methylation450 Beadchip using standard Illumina protocol.	HepG2 cells were cultured under the presence (1000 μM) or absence of zebularine for 72 h.	HepG2 cells were maintained at 37oC under an atmosphere of 95% air and 5% CO2 in DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin.	Genomic DNA was extracted using the AllPrep DNA/RNA mini kit (Qiagen).	Arrays were imaged using iScan array scanner (illumina) using standard setting.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	BATCH						
Experimental Factor Type	batch						
Comment[SecondaryAccession]	GSE42490						
Comment[GEOReleaseDate]	2013-12-01						
Comment[ArrayExpressSubmissionDate]	2012-11-26						
Comment[GEOLastUpdateDate]	2013-12-01						
Comment[AEExperimentType]	methylation profiling by array						
Comment[AdditionalFile:Data1]	GSE42490_non_normalized.txt						
SDRF File	E-GEOD-42490.sdrf.txt