Tan Hwee Tong, Zubaidah Ramdzan M, Tan Sandra, Hooi Shing Chuan, Chung Maxey C M
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 117597.
J Proteome Res. 2006 May;5(5):1098-106. doi: 10.1021/pr050435r.
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 38 kDa were down-regulated while two with Mr approximately 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.
丁酸是一种4碳短链脂肪酸，在预防结直肠癌中，纤维的保护作用归因于丁酸。为了更好地理解丁酸在这种疾病化学预防作用的“蓝图”，我们在使用肝素亲和色谱进行预分级分离后，对经丁酸处理的HCT - 116结肠癌细胞进行了二维差异凝胶电泳（2 - D DIGE）。在二维差异凝胶电泳中，将这种富集步骤与重叠的窄范围固定化pH梯度胶条（pH 4 - 7和pH 6 - 11）相结合，检测到46个差异表达斑点。其中24个通过质谱分析得到鉴定，发现5个斑点为不均一核核糖核蛋白A1（hnRNP A1）。38 kDa的三种同工型下调，而两种约26 kDa的上调。经抗磷酸酪氨酸和抗磷酸丝氨酸抗体免疫印迹验证，这些代表hnRNP A1的磷酸化同工型。通过二维电泳、亚细胞分级分离和蛋白质印迹分析，我们进一步表明，全长hnRNP A1在丁酸处理后发生下调、裂解和细胞质滞留。这些表明，hnRNP A1的调节可能在丁酸介导的生长停滞和细胞凋亡中起重要作用。
