Bottone Jr FG, Moon Y, Kim JS, Alston-Mills B, Ishibashi M, Eling TE.. The anti-invasive activity of cyclooxygenase inhibitors is regulated by the transcription factor ATF3 (activating transcription factor 3). Mol Cancer Ther 4: 693-703

Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, P.O. Box 12233, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
Molecular Cancer Therapeutics (Impact Factor: 5.68). 06/2005; 4(5):693-703. DOI: 10.1158/1535-7163.MCT-04-0337
Source: PubMed


We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds.

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Available from: Frank Bottone Jr, Jun 06, 2014
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    • "The following protocol was used for each PCR: 94°C for 1 min (1 cycle), followed by 9°C for 1 min, 57°C for 1 min, and 72°C for 2 min (26 cycles), and a final extension phase at 72°C for 5 min. Primer sequences for human ATF-3 were 5′-CGGTTTCCGTCTGGGCTTCT-3′ (forward) and 5′-GCACCTCAAAGCTGTTCCGTCCC-3′ (reverse) [16]; for human p53 were 5′-CGGTTTCCGTCTGGGCTTCT-3′ (forward) and 5′-GCACCTCAAAGCTGTTCCGTCCC-3′ (reverse); for human GAPDH mRNA were 5′- TGAAGGTCGGTGTGAACGGATTTGG-3′ (forward) and 5′-ACGACATACTCAGCACCGGCCTCAC-3′ (reverse). PCR products were run on a 2% agarose gel containing 0.5 g/mL ethidium bromide. "
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    ABSTRACT: Background and Purpose Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Urotensin II (U-II), a potent vasoactive peptide, participates in vascular and myocardial remodeling after injury. We investigated the protective effect of U-II on doxorubicin (DOX)-induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved in this process. Experimental Approach Cultured HUVECs were treated with vehicle, DOX (1 µM), U-II, or U-II plus DOX. Apoptosis was evaluated by DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining. Western blot analysis was employed to determine the related protein expression and flow cytometry assay was used to determine the TUNEL positive cells. Key Results U-II reduced the quantity of cleaved caspase-3 and cytosol cytochrome c and increased Bcl-2 expression, which results in protecting HUVECs from DOX-induced apoptosis. U-II induced Activating transcription factor 3 (ATF3) at both mRNA and protein levels in U-II-treated cells. Knockdown of ATF3 with ATF3 siRNA significantly reduced ATF3 protein levels and U-II protective effect under DOX-treated condition. U-II downregulated p53 expression in DOX-induced HUVECs apoptosis, and it rapidly activated extracellular signal-regulated protein kinase (ERK) and Akt. The DOX induced change of p53 was not affected by U-II antagonist (urantide) under ATF-3 knockdown. The inhibitory effect of U-II on DOX-increased apoptosis was attenuated by inhibitors of ERK (U0126) and PI3K/Akt (LY294002). Conclusion and Implications Our observations provide evidence that U-II protects HUVECs from DOX-induced apoptosis. ERK-Akt phosphorylation, ATF3 activation, and p53 downregulation may play a signal-transduction role in this process.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "In this context, we are aware of the complexity of sulindac actions and of the Wnt/β-catenin signaling pathway. For instance, the anti-invasive activity of sulindac was also regulated by ATF3 in colon cancer cells [47]. Therefore, we tested the effect of sulindac with respect to the inhibition of S100A4 expression and cell migration in additional colon cancer cell lines. "
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    ABSTRACT: Colon cancer metastasis is often associated with activation of the Wnt/β-catenin signaling pathway and high expression of the metastasis mediator S100A4. We previously demonstrated the transcriptional regulation of S100A4 by β-catenin and the importance of the interconnection of these cellular programs for metastasis. Here we probe the hypothesis that the nonsteroidal anti-inflammatory drug sulindac sulfide can inhibit colon cancer metastasis by intervening in β-catenin signaling and thereby interdicting S100A4. We treated colon cancer cell lines heterozygous for gain-of-function and wild-type β-catenin with sulindac. We analyzed sulindac's effects on β-catenin expression and subcellular localization, β-catenin binding to the T-cell factor (TCF)/S100A4 promoter complex, S100A4 promoter activity, S100A4 expression, cell motility, and proliferation. Mice intrasplenically transplanted with S100A4-overexpressing colon cancer cells were treated with sulindac. Tumor growth and metastasis, and their β-catenin and S100A4 expressions, were determined. We report the expression knockdown of β-catenin by sulindac, leading to its reduced nuclear accumulation. The binding of β-catenin to TCF was clearly lowered, resulting in reduced S100A4 promoter activity and expression. This correlated well with the inhibition of cell migration and invasion, which could be rescued by ectopic S100A4 expression. In mice, sulindac treatment resulted in reduced tumor growth in the spleen (P = .014) and decreased liver metastasis in a human colon cancer xenograft model (P = .025). Splenic tumors and liver metastases of sulindac-treated mice showed lowered β-catenin and S100A4 levels. These results suggest that modulators of β-catenin signaling such as sulindac offer potential as antimetastatic agents by interdicting S100A4 expression.
    Full-text · Article · Feb 2011 · Neoplasia (New York, N.Y.)
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    • "Colon cancer cells Reduced tumorigenicity Bottone et al. (2005) Phenotypes indicate the consequences of ATF3 deficiency in the corresponding stress models or the consequences of ectopically expressing ATF3 (transgenic or injection models). a Gain-of-function approach. "
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    ABSTRACT: Activating transcription factor 3 (ATF3) gene encodes a member of the ATF family of transcription factors and is induced by various stress signals, including many of those that induce the unfolded protein response (UPR). Emerging evidence suggests that ATF3 is a hub of the cellular adaptive-response network and studies using various mouse models indicate that ATF3 plays a role in the pathogenesis of various diseases. One way to investigate the potential relevance of ATF3 to human diseases is to determine its expression in patient samples and test whether it correlates with disease progression or clinical outcomes. Due to the scarcity and preciousness of patient samples, methods that can detect ATF3 on archival tissue sections would greatly facilitate this research. In this chapter, we briefly review the roles of ATF3 in cellular adaptive-response and UPR, and then describe the detailed steps and tips that we developed based on general immunohistochemistry (IHC) protocols to detect ATF3 on paraffin embedded sections.
    Full-text · Article · Jan 2011 · Methods in enzymology
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