Downregulation of the antiapoptotic Mcl-1 protein and apoptosis in Ma-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin
Downregulation of the antiapoptotic Mcl-1 protein and apoptosis in Ma-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin
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DOI：
10.1002/ijc.20371
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发表时间：
2004-11-10
影响因子：
6.4
通讯作者：
Fodstad, O
中科院分区：
文献类型：
--
作者：
Andersson, Y;Juell, S;Fodstad, O
含有假单胞菌外毒素（PE）的免疫毒素（ITs）通过抑制蛋白质合成和促进细胞凋亡发挥作用，但其诱导细胞凋亡的机制及其与蛋白质合成抑制的关系尚不清楚。我们在用425.3PE处理的MA-II人乳腺癌细胞中研究了这些作用，425.3PE是一种与425.3抗体共价连接的未修饰PE，其靶向EGF受体。这种IT诱导蛋白质合成的有效抑制，同时诱导细胞凋亡。因此，用10 ng/ml IT处理细胞5小时，导致85%的蛋白质合成抑制，同时半胱天冬酶-3、-8和-9活化和PARP失活。即使在72小时的IT处理，预孵育的广谱半胱天冬酶抑制剂z-VAD-FMK引起细胞存活率显着增加，而不影响IT诱导的蛋白质合成抑制。有趣的是，z-VAD-FMK和组织蛋白酶B/L抑制剂z-FA-FMK的组合在24小时后完全阻止了IT诱导的MA-II细胞的细胞死亡，表明组织蛋白酶活化对于IT诱导的细胞死亡的最佳诱导可能是重要的。IT治疗后2.5小时的抗凋亡蛋白Mcl-1的水平显着下降，但不Bcl-2和Bcl-X-L。此外，Mcl-1的表达是不敏感的半胱天冬酶抑制剂，但完全阻止了lactacystin蛋白酶体抑制剂，这表明IT诱导的细胞凋亡可能是由Mcl-1水平的降低引发的。线粒体膜电位（AT mito）下降，同时与caspase激活，表明参与的Deltapsi mito作为一个调节器的IT诱导的细胞凋亡。我们的研究结果表明，425.3PE介导的细胞死亡涉及同时诱导MA-II细胞凋亡和蛋白质合成抑制，从而有助于理解IT诱导凋亡的机制。(C)2004 Wiley-Liss，Inc.
Pseudomonas exotoxin (PE)-containing immunotoxins (ITs) act by arresting protein synthesis and promoting apoptosis, but the mechanisms of the induced apoptosis and the relationship to protein synthesis inhibition is not well elucidated. We studied these effects in MA-II human breast cancer cells treated with 425.3PE, an unmodified PE covalently linked to the 425.3 antibody, which targets the EGF receptor. This IT induced efficient inhibition of protein synthesis with simultaneous induction of apoptosis. Thus, treatment of cells with 10 ng/ml of IT for 5 hr caused 85% inhibition of protein synthesis in parallel with caspase-3, -8 and -9 activation and PARP inactivation. Even after 72 hr of IT treatment, preincubation with the broad-spectrum caspase inhibitor z-VAD-FMK caused a significant increase in cell survival without affecting IT-induced protein synthesis inhibition. Interestingly, a combination of z-VAD-FMK and the cathepsin B/L inhibitor z-FA-FMK prevented completely IT-induced cell death in MA-II cells after 24 hr, indicating that cathepsin activation may be important for optimal induction of IT-induced cell death. IT treatment caused after 2.5 hr a significant decrease in the level of the antiapoptotic protein Mcl-1 but not of Bcl-2 and Bcl-X-L. Furthermore, Mcl-1 expression was not sensitive to caspase inhibitors but was totally prevented by the lactacystin proteasome inhibitor, suggesting that IT-induced apoptosis may be triggered by a reduction in the Mcl-1 level. Mitochondrial membrane potential (AT mito) decreased concurrently with caspase activation, showing the involvement of Deltapsi mito as a regulator of IT-induced apoptosis. Our results demonstrate that 425.3PE-mediated cell death involves simultaneous induction of apoptosis and protein synthesis inhibition in MA-II cells, thus contributing to an understanding of the mechanisms involved in IT-induced apoptosis. (C) 2004 Wiley-Liss, Inc.