Journal
JOURNAL OF PROTEOME RESEARCH
Volume 5, Issue 5, Pages 1098-1106Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr050435r
Keywords
HCT-116 colorectal cancer cells; butyrate; apoptosis; heparin affinity chromatography; 2-D DIGE; narrow range IPG; hnRNP A1; subcellular fractionation
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Butyrate, a 4-carbon short chain fatty acid, is responsible for the protective effects of fiber in colorectal cancer prevention. To better understand the 'blueprint' of butyrate's chemopreventive role in this disease, we performed 2-dimensional difference gel electrophoresis (2-D DIGE) of butyrate-treated HCT-116 colorectal cancer cells after pre-fractionation using heparin affinity chromatography. A combination of this enrichment step with overlapping narrow range IPGs (pH 4-7 and pH 6-11) in 2-D DIGE resulted in the detection of 46 differentially expressed spots. Twenty-four of these were identified by MS analyses, and 5 spots were found to be heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Three isoforms of 38kDa were down-regulated while two with M-r approximate to 26 kDa were up-regulated. These represent phosphorylated isoforms of hnRNP A1 as verified by immunoblotting with anti-phosphotyrosine and anti-phosphoserine antibodies. Using 2-DE, subcellular fractionation and western blot analysis, we further showed that full-length hnRNP A1 underwent down-regulation, cleavage and cytoplasmic retention upon butyrate treatment. These indicate that modulations of hnRNP A1 may play a significant role in the mediation of growth arrest and apoptosis by butyrate.
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