2-D DIGE Analysis of Butyrate-Treated HCT-116 Cells after Enrichment with Heparin Affinity Chromatography

Tan, Hwee Hoon; Zubaidah, Ramdzan M.; Tan, Sandra; Hooi, Shing Chuan; Chung, Maxey C. M.

doi:10.1021/pr050435r

Cited by 19 publications

(40 citation statements)

References 59 publications

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“…Interestingly a subset of proteins found in this study had also been identified in our previous work using 2-D DIGE (17), and they showed regulation in a similar manner by butyrate treatment. Such proteins include cytoskeletal 8, ornithine aminotransferase, cytochrome c oxidase polypeptide VIb, and Tu elongation factor.…”

Section: Protein Identification From Itraq-and Icat-labeled Peptidessupporting

confidence: 84%

“…Cell Culture-HCT-116 colorectal cancer cells were cultured and treated with 5 mM sodium butyrate as reported previously except that three treatment time points (24,36, and 48 h) were used (17).…”

Section: Methodsmentioning

confidence: 99%

“…Two-dimensional Gel Electrophoresis (2-DE)-2-DE was performed as described previously (17). Briefly harvested cells were lysed in the extraction buffer and clarified with centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…In our earlier work, a functional proteomics approach using a prefractionation strategy coupled with two-dimensional (2-D) 1 DIGE analysis was undertaken to identify candidate proteins regulated by 24-h butyrate treatment in HCT-116 cells (17). We have also demonstrated the high sensitivity of the cell line to butyrate-induced growth inhibition and apoptosis in a time-and dose-dependent manner (18).…”

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confidence: 99%

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Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Tan¹,

Tan

Lin

et al. 2008

Molecular & Cellular Proteomics

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Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a shortchain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrateresponsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer. Molecular & Cellular Proteomics 7:1174 -1185, 2008.In developed countries, colorectal cancer is a prevalent disease with high mortality and morbidity rates (1). This disease has emerged as the top malignancy in Singapore. Environmental factors are responsible for about 80% of the cases, whereas genetic predisposition accounts for the minority 20% of cases. Epidemiological evidence suggests that high intake of dietary fiber reduces the incidence and risk of this neoplasm (2, 3). A wealth of studies has shown that butyrate produced from anaerobic fermentation of indigestible carbohydrate is the molecule responsible for the chemopreventive properties of a fiber-rich diet (4 -6).Although butyrate serves as an energy source for normal colonocytes, in vivo and in vitro studies have shown that at physiological concentrations this natural short-chain fatty acid mediates cell maturation with the promotion of growth arrest followed by differentiation and/or apoptosis of cancer cells (7-11). These biological effects are crucial in colorectal cancer therapy as colonic transformation is characterized by multistage alterations of tissue homeostasis resulting in aberrant cell division and/or cell death (12, 13). Butyrate has been purported as a potential anticancer agent. This initiated notable research in identifying proteins that contribute to its biological effects (14, 15). However, most of these investigations focused on one target at any one time and were thus unable to systematica...

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Section: Protein Identification From Itraq-and Icat-labeled Peptidessupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

“…Two-dimensional Gel Electrophoresis (2-DE)-2-DE was performed as described previously (17). Briefly harvested cells were lysed in the extraction buffer and clarified with centrifugation.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Tan¹,

Tan

Lin

et al. 2008

Molecular & Cellular Proteomics

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“…The subcellular localization of hnRNP A1 is dictated by the phosphorylation status of its C terminus, which affects the Trn1-dependent nuclear import of hnRNP A1. 20 The phosphorylation of hnRNP A1 occurs under conditions of diverse stresses such as osmotic shock, 19 UV irradiation, 19 heat shock, 35 or treatment of cells with sodium butyrate 36 and arsenite. 35 Cytoplasmic hnRNP A1 accumulates in stress granules, the sites of mRNA triage from which target mRNAs are exported to processing bodies for degradation.…”

Section: Discussionmentioning

confidence: 99%

hnRNP A1 regulates UV-induced NF-κB signalling through destabilization of cIAP1 mRNA

et al. 2008

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cIAP1 is an important member of the inhibitor of apoptosis family of proteins and is involved in the regulation of the NF-jBsignalling pathway downstream of the TNF receptor. We report here that UV irradiation leads to downregulation of cIAP1 expression because of enhanced cIAP1 mRNA destabilization. An AU-rich element located within the 3 0 untranslated region of cIAP1 mRNA is sufficient to mediate cIAP1 mRNA instability. Furthermore, we have identified hnRNP A1 as a cIAP1 3 0 UTR-binding protein. hnRNP A1 is a primarily nuclear protein, but accumulates in the cytoplasm after exposure of cells to UV irradiation. Indeed, we find that hnRNP A1 enhances the destabilization of cIAP1 mRNA during UV irradiation. Moreover, siRNAmediated knockdown of hnRNP A1 restores cIAP1 levels and prevents UV irradiation-induced activation of the NF-jB signal transduction pathway, suggesting that hnRNP A1 is an essential post-transcriptional modulator of cIAP1 expression, and thus cIAP1 activity. The inhibitor of apoptosis (IAP) family of proteins are key regulators of programmed cell death. 1 The IAP family comprises eight distinct members that participate in diverse cellular processes including cell cycle, signal transduction, ubiquitylation, and caspase inhibition. 2 Although it was initially believed that all IAPs are capable of inhibiting distinct caspases, the recent data suggest that only XIAP is a bona fide caspase inhibitor. 3 cIAP1 and cIAP2 were identified through an interaction with the TNF-receptor complex proteins TRAF1 and TRAF2, which regulate NF-kB signalling through the TNF receptor. 4 The C-terminal RING finger domain of cIAP1 possesses an ubiquitin E3 ligase activity and may sensitize cells to apoptosis by direct ubiquitylation and removal of TRAF2 following activation of the TNF receptor, resulting in the inhibition of NF-kB pathways. 5 Indeed, cIAP1 degradation induced by Smac mimetics leads to activation of both the canonical and non-canonical NF-kB pathways, strengthening the importance of IAPs in the regulation of NF-kB signalling. [6][7][8] Although cIAP1 and cIAP2 perform similar functions within the cell, their expression is regulated differently. The expression of cIAP2 is controlled primarily at the level of transcription in an NF-kB-dependent manner. 9 Also, the protein turnover of cIAP2 is regulated by the ubiquitin ligase activity of cIAP1. 10 In contrast, cIAP1 levels are controlled at the level of protein synthesis. The 5 0 untranslated region (UTR) of cIAP1 contains an upstream open reading frame that reduces the basal translation of cIAP1. 11 The 5 0 UTR of cIAP1 also harbours an internal ribosome entry site (IRES) element that mediates translational induction of cIAP1 in response to stress. 12,13 This complex regulatory network reflects the distinct spatial and temporal requirement for cIAP1 and cIAP2 proteins in response to various physiological conditions.Here, we describe an additional regulatory mechanism that governs the expression of cIAP1. We find that following UV irradiation the levels...

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Effect of CyDye minimum labeling in differential gel electrophoresis on the reliability of protein identification

et al. 2007

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Differential 2-DE (DIGE) is a widely applied tool for the quantitative analysis of differentially represented proteins. The method involves covalent minimal labeling of proteins prior to their electrophoretic separation using CyDye DIGE Fluor minimal dyes. This methodology creates two different species per protein, the labeled (approx. 1-2%) and unlabeled (approx. 98-99%) ones, which differ in their molecular masses by 434-464 Da, depending on the attached dye. DIGE followed by automated spot picking according to the CyDye coordinates misses in many instances the exact positions where the maximum amounts of the considered proteins are located. This fact leads to a loss in sensitivity of the subsequent MALDI-MS analyses and results in a reduced reliability of protein identification and sequence coverage. In this paper, the migration differences of labeled and unlabeled species are quantified together with the impact of this effect on the certainty of protein identification and sequence coverage investigating proteins up to 90 kDa.

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2-D DIGE Analysis of Butyrate-Treated HCT-116 Cells after Enrichment with Heparin Affinity Chromatography

Cited by 19 publications

References 59 publications

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

hnRNP A1 regulates UV-induced NF-κB signalling through destabilization of cIAP1 mRNA

Effect of CyDye minimum labeling in differential gel electrophoresis on the reliability of protein identification

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