Involvement of p21Waf1/Cip1 and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate
Involvement of p21Waf1/Cip1 and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate
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DOI：
10.1093/carcin/21.1.7
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发表时间：
2000-01-01
期刊：
影响因子：
4.7
通讯作者：
Zalewski, PD
中科院分区：
文献类型：
--
作者：
Chai, F;Evdokiou, A;Zalewski, PD
丁酸盐是一种在结肠中产生的短链脂肪酸，通过抑制组蛋白去乙酰化酶、从头合成蛋白质和激活DEVD-caspase(凋亡DNA断裂和膜起泡的主要影响因子)等一系列过程诱导癌细胞凋亡，我们现在在LIM 1215结直肠癌细胞中表明，丁酸盐除了激活DEVD-caspase和诱导凋亡外，还增加通用的细胞周期蛋白依赖性激酶抑制物p21(Waf1/Cip1)的表达和裂解，导致视网膜母细胞瘤蛋白的低磷酸化。伴随这些分子变化的是G(0)/G(1)和S期细胞的进行性丧失。P21的表达与DEVD-caspase激活所需的必需蛋白的表达动力学相似，表明丁酸在抗凋亡和促凋亡机制中的平行作用。对丁酸诱导的LIM 1215细胞进行3个周期的筛选，筛选出耐受丁酸诱导的LIM 1215细胞。流式细胞仪检测显示这些细胞p21表达明显增强，并处于细胞周期停滞状态。另一方面，在没有丁酸盐的情况下，这些细胞培养2-3天后，p21基因表达下调，丁酸诱导的细胞对凋亡的敏感性恢复，经丁酸处理的细胞在凋亡过程中的Western blotting始终显示出对照细胞所没有的15 kDa条带。这条带在DEVD-caspase激活开始后立即变得明显，与贴壁的非凋亡细胞相比，在浮动的凋亡细胞群中丰富，在缺乏DEVD-caspase活性的丁酸抗性细胞中缺失。多肽caspase抑制剂部分阻断了p15的出现，在这里，我们首次表明p21是结直肠癌细胞中效应半胱氨酸酶的靶点，并且对丁酸诱导的细胞凋亡的抵抗是p21裂解失败的特征。
Butyrate, a short chain fatty acid produced in the colon, induces apoptosis in cancer cell lines by a sequential process involving inhibition of histone deacetylase, de novo protein synthesis and activation of DEVD-caspase, a major effector of apoptotic DNA fragmentation and membrane blebbing, We now show in LIM 1215 colorectal cancer cells, that butyrate, in addition to activating DEVD-caspase and inducing apoptosis, also increases expression and 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 cells. Expression of p21 had similar kinetics to that of the essential protein required for DEVD-caspase activation, indicating parallel effects of butyrate 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 cells showed markedly enhanced p21 expression and were in cell cycle arrest as determined by flow cytometry. On the other hand, subsequent culture of these cells for 2-3 days in the absence of butyrate resulted in downregulation of p21 and restoration of sensitivity to apoptosis by butyrate, Western blots of butyrate-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band (p15) that was not present in control cultures. This band became apparent immediately after the onset of DEVD-caspase activation, was enriched in the floating apoptotic cell population when compared with the adherent, non-apoptotic cells and was absent in butyrate-resistant cells lacking DEVD-caspase activity. Peptide caspase inhibitors partially blocked appearance 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 butyrate-induced apoptosis is characterized by failure of p21 cleavage.