Involvement of p21(Waf1/Cip1) and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate

Butyrate, a short chain fatty acid produced in the colon, induces apoptosis in cancer cell lines by a sequential process involving inhibition of histo ne deacetylase, de novo protein synthesis and activation of DEVD-caspase, a major effector of apoptotic DNA fragmentation and membrane blebbing, We no w show in LIM 1215 colorectal cancer cells, that butyrate, in addition to a ctivating DEVD-caspase and inducing apoptosis, also increases expression an d cleavage of the universal cyclin-dependent kinase inhibitor p21(Waf1/Cip1 ) and leads to hypo-phosphorylation of retinoblastoma protein. Accompanying these molecular changes was a progressive loss of G(0)/G(1) and S phase ce lls. Expression of p21 had similar kinetics to that of the essential protei n required for DEVD-caspase activation, indicating parallel effects of buty rate on anti-apoptotic and pro-apoptotic mechanisms. LIM 1215 cells, which were resistant to butyrate-induced apoptosis, were selected by three cycles of exposure to butyrate and removal of floating apoptotic cells. These cel ls showed markedly enhanced p21 expression and were in cell cycle arrest as determined by flow cytometry. On the other hand, subsequent culture of the se cells for 2-3 days in the absence of butyrate resulted in downregulation of p21 and restoration of sensitivity to apoptosis by butyrate, Western bl ots of butyrate-treated cells undergoing apoptosis consistently demonstrate d a 15 kDa band (p15) that was not present in control cultures. This band b ecame apparent immediately after the onset of DEVD-caspase activation, was enriched in the floating apoptotic cell population when compared with the a dherent, non-apoptotic cells and was absent in butyrate-resistant cells lac king DEVD-caspase activity. Peptide caspase inhibitors partially blocked ap pearance of p15, Here we show, for the first time, that p21 is a target of effector caspases in colorectal cancer cells and that the resistance to but yrate-induced apoptosis is characterized by failure of p21 cleavage.