Expression profiling of CC531 colon carcinoma cells reveals similar regulation of β‐catenin target genes by both butyrate and aspirin

Abstract

The CC531 cell line has been widely used to study different aspects of tumor growth and metastasis and provides an excellent experimental platform to develop novel antitumor strategies. To characterize the CC531 model at the molecular level, we screened for mutations in genes covering important signal-transduction pathways that are known to play major roles during colon carcinogenesis, the wnt and the ki-ras signaling pathways. We found both a prototypic ␤-catenin (Ctnnb1) mutation (Thr 41 Ile) and a ki-ras (G12D) mutation, providing unambiguous evidence for the constitutive activation of these pathways in CC531 cells. We further established comprehensive gene expression profiles of CC531 cells and investigated the molecular response to 2 antitumor drugs, butyrate and aspirin. Using oligonucleotide microarrays, we screened the expression levels of 7,700 genes and identified a total of 398 genes whose expression was significantly changed upon treatment with butyrate. When using aspirin, 121 genes were significantly altered. Interestingly, 36 genes were regulated by both butyrate and aspirin and 35 of them were regulated in the same direction. We found 7 differentially expressed genes, cyclin D1, cyclin E, c-myc, Fosl1, c-fos, Cd44 and follistatin, which are known targets of the ␤-catenin and/or the ras pathway. In all cases, butyrate and aspirin reversed the changes in expression normally found in response to active signaling of these oncogenic pathways. Colon cancer is the third most common malignant tumor in the Western world and the second most frequent cause of cancer death. 1 According to the concept of Fearon and Vogelstein, 2 colorectal tumors arise via a multistep process as the result of accumulating genetic and epigenetic alterations that affect different growth-regulating genes. The neoplastic process is thought to be initiated by constitutive activation of the wnt signaling pathway brought by mutations in regulatory genes, e.g., APC, Axin and CTNNB1, all resulting in the activation of gene transcription by ␤-catenin. 3 Mutations in the Ki-Ras (Kirsten-ras) gene have been found in 30 -50% of colon tumors 4 and can be observed already in small adenomas, whereas p53 gene mutations occur during carcinogenesis only at the adenoma/carcinoma transition. 5 ki-ras and Ctnnb1 mutations have been reported to occur also at high frequency in experimentally induced rodent colon tumors. 6,7 In addition, oncogenic CTNNB1 mutations have been observed in human embryonic kidney-derived tumors. 8 Several epidemiologic and pharmacologic studies indicate that NSAIDs can reduce the incidence of colorectal cancers in humans and experimental animals and reduce the number and size of polyps in patients with FAP cancer. 9 In particular, aspirin revealed chemopreventive effects on carcinogen-induced colon tumors 10,11 in rodents. The effects have been generally attributed to inhibition of the enzyme activity of COX-1 and COX-2, which are highly expressed in colorectal cancers; but additional mechanisms likely contribute to the a...

Genetic networks responsive to sodium butyrate in colonic epithelial cells

“…Based on the present results together with those of previous studies [5,6,[9][10][11][12][13][14], we suggest that a decrease in cell cycle-related genes in cluster IV (Table S2) may be closely associated with the cell growth arrest induced by SB. Of particular interest is our identification of the cell cycle-associated genetic network whose core contains CCND1, PCNA and BRCA1 (Fig.…”

Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

“…We have also shown that low levels of aspirin are more toxic to colorectal cancer cells than other cancer types such as glioma (U373MG) and breast cancer (MCF-7, MDA-231-MB) cells (data not shown). We were particularly interested in the effect of aspirin at relatively low levels (~1 mM) in comparison to other studies where microarray analysis has been utilised [see for example (30,31)], because we wished to ensure that the results obtained were as physiologically ----------------------------------------------- Table I. Continued.…”

“…Microarray analysis has been extensively utilised to examine alterations in gene transcription in response to NSAID exposure in a number of colon cancer cell lines with a subsequent large list of alterations in gene expression reported (24,(28)(29)(30)(31). This study aimed to investigate the relationship between aspirin and DNA repair genes, utilising a commercially available PCR array to analyse the expression of 84 different genes involved in DNA damage signalling pathways in the colorectal cancer cell line SW480 upon aspirin treatment.…”

Is resistant starch protective against colorectal cancer via modulation of the WNT signalling pathway?

“…Germann et al observed that 4·5 mM butyrate treatment of CC531 rat colon carcinoma cells resulted in positive modulation of expression of four WNT pathway target genes (CCND1, c-MYC, FOSL1 and FST), which have been reported to be up-regulated in CRC (40)(41)(42) . However, the effects of butyrate on c-MYC expression are complex.…”