Tan Hwee Tong, Tan Sandra, Lin Qingsong, Lim Teck Kwang, Hew Choy Leong, Chung Maxey C M
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 117597, Singapore.
Mol Cell Proteomics. 2008 Jun;7(6):1174-85. doi: 10.1074/mcp.M700483-MCP200. Epub 2008 Mar 14.
Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.
结直肠癌是发达国家最常见的癌症之一，其发病率与高膳食纤维摄入量呈负相关。丁酸是纤维短链脂肪酸发酵的副产物，可诱导细胞成熟，促进癌细胞生长停滞、分化和/或凋亡。丁酸对结肠癌细胞成熟的刺激作用呈现出从生长停滞早期到凋亡级联激活的时间进程。此前我们进行了二维差异凝胶电泳（DIGE），以鉴定经24小时丁酸处理的HCT-116结肠癌细胞中差异表达的蛋白质。在此，我们使用相对和绝对定量等压标签（iTRAQ）这种稳定同位素标记方法进行定量蛋白质组学研究，该方法能够对四个样本进行多重分析，以对经丁酸处理的HCT-116细胞进行时间进程研究。此外，还使用了可切割的同位素标记亲和标签（ICAT），它能选择性标记含半胱氨酸的蛋白质，其结果补充了从iTRAQ策略获得的结果。通过实时聚合酶链反应（PCR）和蛋白质免疫印迹法对选定的蛋白质靶点进行了验证。我们提出了一个模型，以说明我们对丁酸反应性蛋白质组进行时间分析的结果，该分析揭示了几个与生长停滞、凋亡和转移相关的整合细胞过程和信号通路。这些丁酸调节通路的特征性簇是用于结直肠癌治疗的新型化学预防和治疗药物的潜在靶点。
