Expression profiling of sodium butyrate (NaB)-treated cells: identification of regulation of genes related to cytokine signaling and cancer metastasis by NaB

Oncogene (2004) 23, 6304–6315 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc Expression profiling of sodium butyrate (NaB)-treated cells: identification of regulation of genes related to cytokine signaling and cancer metastasis by NaB Jeena Joseph1,2, Giridhar Mudduluru1,2, Sini Antony1, Surabhi Vashistha1, Parthasarathi Ajitkumar1 and Kumaravel Somasundaram*,1 ONCOGENOMICS 1 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India Histone deacetylase (HDAC) inhibitors induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (NaB), a short chain fatty acid, is a HDAC inhibitor and is produced in the colonic lumen as a consequence of microbial degradation of dietary fibers. In order to dissect out the mechanism of NaB-induced growth inhibition of cancer cells, we carried out expression profiling of a human lung carcinoma cell line (H460) treated with NaB using a cDNA microarray. Of the total 1728 genes analysed, there were 32 genes with a mean expression value of 2.0-fold and higher and 66 genes with a mean expression value 3.0-fold and lower in NaBtreated cells. For a few selected genes, we demonstrate that their expression pattern by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis is matching with the results obtained by microarray analysis. Closer view at the expression profile of NaBtreated cells revealed the downregulation of a total of 16 genes associated with cytokine signaling, in particular, interferon c (IFNc) pathway. In good correlation, NaBpretreated cells failed to induce interferon regulatory factor 1, an INFc target gene, efficiently upon IFNc addition. These results suggest that NaB inhibits proinflammatory cytokine signaling pathway, thus providing proof of mechanism for its anti-inflammatory activity. We also found that NaB induced three genes, which are known metastatic suppressors, and downregulated 11 genes, which have been shown to promote metastasis. Upregulation of metastatic suppressor Kangai 1 (KAI1) by NaB in a time-dependent manner was confirmed by RT–PCR analysis. The differential regulation of metastasis-associated genes by NaB provides explanation for the antiinvasive properties of NaB. Therefore, our study presents new evidence for pathways regulated by NaB, thus providing evidence for the mechanism behind antiinflammatory and antimetastatic activities of NaB. Oncogene (2004) 23, 6304–6315. doi:10.1038/sj.onc.1207852 Published online 12 July 2004 *Correspondence: K Somasundaram; E-mail: 2 These authors contributed equally to this work Received 16 December 2003; revised 26 April 2004; accepted 26 April 2004; published online 12 July 2004 Keywords: sodium butyrate; histone deacetyalse inhibitor; chromatin remodeling; microarray; expression profiling; cytokine signaling; metastasis Introduction The acetylation and deacetylation of histones of the core proteins of the nucleosomes in chromatin play an important role in the regulation of gene expression. (Gray and Ekstrom, 2001; Khochbin et al., 2001; Urnov, 2003). The acetylation status of the histones is mainly controlled by two classes of enzymes: histone acetyl transferases (HATs) and histone deacetylases (HDAC). A number of HDAC inhibitors (HDIs) have been identified that induce growth arrest, differentiation and apoptosis in cancer cells (Gore and Carducci, 2000; Marks et al., 2000; Weidle and Grossmann, 2000). HDIs belong to a heterogeneous class of compounds that includes derivatives of short chain fatty acids, hydroxamic acids, cyclic tetrapeptides and benzamides. HDI sodium butyrate (NaB), a short chain fatty acid, occurs naturally in the body from the acetyl-CoAdependent catabolic oxidation of long chain saturated fatty acids (Lehninger et al., 1993; Widmer et al., 1996). NaB is produced in the colon of mammals as a result of anaerobic bacterial fermentation of dietary fiber, undigested starch and proteins (Cummings, 1981; Bugaut and Bentejac, 1993; McIntyre et al., 1993; Mcintosh et al., 1996; Whiteley et al., 1996). NaB serves as an energy source of colonic epithelium. In addition, NaB also has growth inhibitory effect on cancer cells, which has been attributed to its ability to induce cell cycle arrest, differentiation and apoptosis (Medina et al., 1997; Richon et al., 1998; Wang et al., 1999; Butler et al., 2000). NaB has been shown to induce p21WAF1/CIP1 in a p53-independent manner (Xiao et al., 1997; Chai et al., 2000; Siavoshian et al., 2000; Iacomino et al., 2001; Pellizzaro et al., 2001), modulate cyclin D1 (Lallemand et al., 1996; Siavoshian et al., 2000), cdc2 (Charollais et al., 1990), cdk2 (Siavoshian et al., 2000; Clarke et al., 2001) and proliferating cell nuclear antigen (Ranganna et al., 2000). In addition to the above functions, an antiinflammatory role for NaB in certain stages of mucosal Sodium butyrate inhibits cytokine signaling and metastasis J Joseph et al 6305 A B NaB 300 AP activity µmol PNP/mg/hr a - 250 200 150 100 50 0 b 0 10 20 30 40 50 60 Time (hrs) 5 mM C 120 80 1 mM 2 mM 5 mM 60 10 mM c % Live cells 100 40 20 10 mM 0 0 24 48 72 96 Time (hrs) Figure 1 NaB induces differentiation and inhibits the growth of H460 cells. (A) H460 cells were either untreated (panel a) or treated with 5 mM (panel b) and 10 mM (panel c) concentrations of NaB for 24 h. The cells were photographed under bright field. (B) H460 cells were treated with NaB (2 mM) for different periods of time as indicated in the figure. At the end of the indicated time points, AP activity was quantified as described in Materials and methods. (C) H460 cells were treated with increasing concentrations (1, 2, 5 and 10 mM) of NaB for different periods of time as indicated in the figure. At the end of indicated time points, proportion of live cells was quantified by MTT assay as described in Materials and methods. The absorbance of control cells was considered as 100% inflammation has also been proposed. Modulation of certain proinflammatory cytokines by NaB has been found to be the mechanism behind anti-inflammatory functions of this short chain fatty acid (Saemann et al., 2000). In the present study, we carried out expression profiling of the NaB-treated human non-small-cell lung cancer line H460 using cDNA microarrays. We have identified several previously known genes and some novel genes, which are differentially expressed upon NaB treatment. We demonstrate that NaB downregulates the expression of a total of 16 genes belonging to cytokine signaling pathways, particularly that of IFNg, suggesting that NaB interferes with cytokine signaling, which explains its anti-inflammatory functions. In addition, we also show that a total of 14 genes associated with metastasis are differentially regulated by NaB, thus providing evidence for the mechanism behind antimetastatic properties of NaB. Results Effect of NaB on H460 cells NaB has been (...truncated)