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 butyrate‐induced apoptosis. © 2002 Wiley‐Liss, Inc.