Butyrate, a 4-carbon fatty acid, has been shown to cause growth arrest and apoptosis of cancer cells in vitro and in vivo. The signaling pathways leading to changes in cell growth are unclear. We used a functional proteomics approach to delineate the pathways and mediators involved in butyrate action in HT-29 cells at 24 hr posttreatment. Using 2-dimensional gel electrophoresis, we showed that butyrate treatment resulted in alterations in the proteome of HT-29 cells. MALDI-TOF mass spectrometry was used to identify butyrate-regulated spots. First, our results revealed that the expression of various components of the ubiquitin-proteasome system was altered with butyrate treatment. This suggests that, in addition to the regulation of gene expression through the histone deacetylase pathway, proteolysis could be a means by which butyrate may regulate the expression of key proteins in the control of cell cycle, apoptosis and differentiation. Second, we found that both proapoptotic proteins (capase-4 and cathepsin D) and antiapoptotic proteins (hsp27, antioxidant protein-2 and pyruvate dehydrogenase E1) were simultaneously upregulated in butyrate-treated cells. Western blotting was carried out to confirm butyrate regulation of the spots. Both cathepsin D and hsp27 showed a time-dependent increase in expression with butyrate treatment in HT-29 cells. However, in HCT-116 cells, which were 5-fold more sensitive to butyrate-induced apoptosis, the upregulation of cathepsin D with time was not accompanied by a similar increase in hsp27 levels. Thus, the simultaneous upregulation of both proapoptotic and antiapoptotic proteins in HT-29 cells may account for their relative resistance to butyrateinduced apoptosis.

Currently, one of the most popular applications of proteomics is in the area of cancer research. In Africa, Southeast Asia, and China, hepatocellular carcinoma is one of the most common cancers, occurring as one of the top five cancers in frequency. This project was initiated with the purpose of separating and identifying the proteins of a human hepatocellular carcinoma cell line, HCC-M. After two-dimensional gel electrophoresis separation, silver staining, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses, tryptic peptide masses were searched for matches in the SWISS-PROT and NCBI nonredundant databases. Approximately 400 spots were analyzed using this approach. Among the proteins identified were housekeeping proteins such as alcohol dehydrogenase, alpha-enolase, asparagine synthetase, isocitrate dehydrogenase, and glucose-6-phosphate 1-dehydrogenase. In addition, we also identified proteins with expression patterns that have been postulated to be related to the process of carcinogenesis. These include 14-3-3 protein, annexin, prohibitin, and thioredoxin peroxidase. This study of the HCC-M proteome, coupled with similar proteome analyses of normal liver tissues, tumors, and other hepatocellular carcinoma cell lines, represents the first step towards the establishment of protein databases, which are valuable resources in studies on the differential protein expressions of human hepatocellular carcinoma.

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Hepatocellular carcinoma (HCC or hepatoma) is the most common primary cancer of the liver. It is responsible for approximately one million deaths each year, mainly in underdeveloped and developing countries. The aetiological factors identified in the development of HCC included persistent infection by hepatitis B and hepatitis C viruses, and exposure to aflatoxins. Although immunization can protect individuals from being infected by the hepatitis B virus, the early detection of HCC in those who have been infected by the virus remains a challenge. Thus most HCCs present late and are not suitable for curative treatment. Hence there is a tremendous interest and urgency to identify novel HCC diagnostic marker(s) for early detection, and tumour specific disease associated proteins as potential therapeutic targets in the treatment of HCC. Screening for these HCC proteins has been facilitated by proteomics, a key technology in the global analysis of protein expression and understanding gene function. Present and earlier proteome analyses of HCC have used predominantly experimental in vitro systems. The protein expression profiles of several hepatoma cell lines such as HepG2, Huh7, SK-Hep1, and Hep3B have been compared with normal liver, and nontransformed cell lines (Chang and WRL-68), while a comprehensive proteome analysis to create a protein database was carried out for the cell line HCC-M. In the future, proteome analyses utilizing tumour tissues, which reflect the pathological state of HCC more closely, will be undertaken. This work will complement the gene expression studies of HCC which are already underway. Efforts have also been directed at the proteome analysis of hepatic stellate cells, as these cells play an important role in liver fibrosis. Since liver fibrosis is reversible but not cirrhosis, it is of considerable importance to identify therapeutic targets that can slow its progression.