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Title: 2D-DIGE analysis of butyrate-treated HepG2 cells
Keywords: 2D-DIGE,HCC,Butyrate,HPLC
Issue Date: 30-Nov-2011
Citation: LAU SIANG LIN (2011-11-30). 2D-DIGE analysis of butyrate-treated HepG2 cells. ScholarBank@NUS Repository.
Abstract: Hepatocellular carcinoma (HCC) is one of the top causes of cancer deaths in Asia Pacific, and its incidence is predicted to increase in the next decade. The lethality of this disease can be seen from the high mortality rates and poor prognosis with a 5-year survival rate of only 5%. For patients with extrahepatic metastasis, the life expectancy decreases to a mere 3 months. This is due to lack of reliable biomarkers, poor understanding of HCC tumorigenesis and limited clinical therapies. Butyrate, a physiological saccharolytic fermentative by-product of the colonic microflora, is an attractive chemotherapeutic agent with curative potentials due to its cancer-specific cytostatic and apoptotic effects. As the liver is the second site of butyrate delivery via hepatic portal transport and butyrate is well tolerated by hepatocytes, butyrate treatment could be an avenue to lengthen the lifespan of HCC patients awaiting liver transplants. Despite treatment feasibility, butyrate¿s mode of actions against neoplasm, such as cell arrest induction and metastasis abrogation, remains oblique in liver cancer cells. In this study, we employed a novel proteomics workflow using heparin affinity chromatography and 2-Dimension Difference Gel Electrophoresis (2D-DIGE) to identify butyrate-induced changes in proteins in the HepG2 liver cancer cell line. We focused on cytoskeletal-related proteins in cell migration and invasion pathways which are enriched via heparin interaction. Our results revealed that the three HCC cell lines HepG2, HCC-M and Hep3B exhibited reduced cell viability significantly after butyrate treatment. The treatment however resulted in significant increased cell motility in all the 3 cell lines. From this proteomics screening of butyrate-treated HepG2 cells, a total of 52 proteins¿ expressions were detected to be dysregulated. These butyrate-induced proteins are mainly involved in glucose catabolism, urea cycle and nucleic acid synthesis. This study also yielded a list of cancer-associated proteins that were regulated after treatment thus suggesting the reversal of the malignant phenotype of HCC cells by butyrate. A group of proteins involved in cell migration and cytoskeletal reorganization was identified in this study which included LRRC15, NME1, and GSN. These proteins may help us to understand the mechanisms behind the changes in cell mobility after butyrate treatment. Gelsolin (GSN) is one of the targets up-regulated by butyrate treatment in HepG2 cells indicating its possible involvement in butyrate¿s effect on cell motility or invasion. HepG2 with siRNA-mediated GSN knockdown resulted in increased basement membrane invasion. This suggested that up-regulation of GSN by butyrate is crucial for butyrate-mediated suppression of metastasis.
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