Proteome analysis of butyrate‐treated human colon cancer cells (HT‐29)

Abstract

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.

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

“…Nine fractions were obtained using step gradients of mobile phase B: unbound, 0 -5, 5-10, 10 -15, 15-20, 20 -30, 30 -40, 40 -50, and 50 -100%. The eluting fractions were captured alternatively onto two 0.3 ϫ 1-mm trap columns (3-m C 18 PepMap TM , 100 Å) (Dionex-LC Packings) and washed with 0.05% TFA followed by gradient elution in a 0.2 ϫ 50-mm reverse phase column (monolithic polystyrene-divinylbenzene) (Dionex-LC Packings). The mobile phases used for this second dimensional separation were 2% ACN with 0.05% TFA (A) and 80% ACN with 0.04% TFA (B).…”

“…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). Therefore, the stimulation of cell maturation by butyrate implicated a temporal orchestration of various cellular processes.…”

Nutrition and nutrigenomics: an overview

“…For this reason, it was thought that there was a causal relationship between the major serum beta-carotene concentrations and the reduction of risk. Evidently this has not been proven to be true and it is presumed that the reason for this is that, while excessive intake of vitamin A or beta-carotene may have toxic effects, they did not appear in nutrition benefit as in this case these nutrients were part of the nutrient complex made by a given diet, but it is not possible to distinguish at this level from which nutrients depend on the observed effects [57][58][59][60][61][62][63][64][65][66][67][68][69][70].…”

“…If this type of experimental evidence has highlighted the difficulty of transferring bioactive nutrient-derived observations in foodprocessing applications, they clearly highlighted the concept of different nutritionally-suited foods in the body. These concentrations can then be subjected to diagnostic analysis: if the simple serum concentration of beta-carotene is associated with a significant reduction in the risk of pathology in the populations, simultaneous analysis of a large number of nutrients in the single subject will be able to produce even more detailed information on the one hand in terms of objective assessment of the nutrition regime adopted, on the other in terms of the resulting risk entity configured [57][58][59][60][61][62][63][64][65][66][67][68][69][70].…”

Proteomics in pathology research

“…11 Despite these limitations, due to highly automated and roboticized spot picking technologies, numerous reports have successfully used 2D-GE followed by MALDI-TOF analysis to determine differential expression of protein profiles in normal vs tumor tissues including breast, colon, 59 lung, esophageal tissues 63 and cellular responses to stimulating and differentiating agents such as LPS 64 and response to Fas, 65 cytotoxic agents such as butyrate. 66 The average number of differentially expressed proteins identified by 2D-GE followed by MS analysis is in the range of 30-70 proteins.…”