Reconstitution of caspase-3 confers low glucose-enhanced tumor necrosis factor-related apoptosis-inducing ligand cytotoxicity and akt cleavage
Reconstitution of caspase-3 confers low glucose-enhanced tumor necrosis factor-related apoptosis-inducing ligand cytotoxicity and akt cleavage
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DOI：
10.1158/1078-0432.ccr-03-0136
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发表时间：
2004-03-15
影响因子：
11.5
通讯作者：
Kim, JH
中科院分区：
文献类型：
--
作者：
Lee, YJ;Froelich, CJ;Kim, JH
目的和实验设计：我们之前观察到，葡萄糖剥夺会增强人前列腺腺癌细胞 DU-145 细胞中肿瘤坏死因子相关凋亡诱导配体 (TRAIL) 诱导的细胞凋亡以及 caspase 激活（caspase-3、-9 和 -8）。在本研究中，我们使用 caspase-3 缺陷的 MCF-7 乳腺癌细胞来检测 caspase-3 在葡萄糖剥夺增强的 TRAIL 细胞毒性中的可能作用。结果：葡萄糖剥夺和 200 ng/ml TRAIL 联合处理在 caspase-3 cDNA 转染细胞 (MCF-7/casp-3) 中显着诱导细胞毒性，但在对照载体转染细胞中则不然（MCF-7/载体）。我们还观察到，在 MCF-7/casp-3 细胞中，用 TRAIL 处理后，Akt（一种抗凋亡蛋白）的水平降低，但在 MCF-7/载体细胞中却没有降低。在 MCF-7/casp-3 细胞中缺乏葡萄糖的情况下，TRAIL 会促进 Akt 的减少。然而，用 20 muM Z-LEHD-FMK（一种 caspase-9 抑制剂）进行预处理，可以保护 MCF-7/casp-3 细胞免受 TRAIL 和葡萄糖剥夺诱导的细胞毒性的组合处理。该化合物还可以防止联合治疗期间 Akt 水平的降低。此外，这种 Akt 减少并未受到蛋白酶体抑制剂 MG-132 的抑制。定点诱变数据显示，在 TRAIL 和葡萄糖剥夺治疗期间，Akt 在氨基酸 108 处被切割，但不是 119。结论：我们的结果表明 caspase-3 参与了 Akt 水平的降低，并且其参与是通过 caspase-9 激活介导的。 Akt 水平的降低也是由于 Akt 的裂解而不是 Akt 的降解。
Purpose and Experimental Design: We have previously observed that glucose deprivation enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death as well as caspase activation (caspase-3, -9, and -8) in human prostate adenocarcinoma DU-145 cells. In this study, we used caspase-3-deficient MCF-7 breast cancer cells to examine the possible role of caspase-3 in glucose deprivation-enhanced TRAIL cytotoxicity.Results: Combined glucose deprivation and 200 ng/ml TRAIL treatment markedly induced cytotoxicity in caspase-3 cDNA transfected cells (MCF-7/casp-3) but not in control vector transfected cells (MCF-7/vector). We also observed that the level of Akt, an antiapoptotic protein, was reduced by treatment with TRAIL in MCF-7/casp-3 cells but not in MCF-7/vector cells. The reduction of Akt by TRAIL was promoted in the absence of glucose in MCF-7/casp-3 cells. However, pretreatment with 20 muM Z-LEHD-FMK, a caspase-9 inhibitor, protected MCF-7/casp-3 cells from the combinatorial treatment of TRAIL and glucose deprivation-induced cytotoxicity. This compound also prevented the reduction of Akt level during the combinatorial treatment. Moreover, this Akt reduction was not inhibited by treatment with MG-132, a proteosome inhibitor. Data from site-directed mutagenesis show that Akt was cleaved at amino acid 108, but not 119, during treatment with TRAIL and glucose deprivation.Conclusions: Our results suggest that caspase-3 is involved in the reduction of Akt level, and its involvement is mediated through caspase-9 activation. The reduction of Akt level is also due to cleavage of Akt rather than degradation of Akt.