Zeng W, Zhang J, Qi M, Peng C, Su J, Chen X, Yuan Z
Department of Dermatology, Xiangya Hospital, Central South University, Changsha, People's Republic of China.
Department of Plastic Surgery, Xiangya Hospital, Central South University, Changsha, People's Republic of China.
Cell Death Dis. 2014 Jun 5;5(6):e1282. doi: 10.1038/cddis.2014.192.
Nascent polypeptide-associated complex α (αNAC) is reportedly overexpressed in several types of cancers and regulates cell apoptosis under hypoxic conditions in HeLa cells. The aim of our study was to investigate the apoptotic function of αNAC in cancer progression. First, we observed the cellular effects of αNAC depletion. Mouse αNAC was used to restore the protein level and verify the effect. An Annexin V assay, a caspase activity reporter assay, an apoptotic molecular marker, and a colony formation assay were used as markers to investigate the mechanisms of cell death caused by αNAC depletion. The Cancer 10-pathway reporter assay was used to screen downstream pathways. PCR site-directed deletion based on the functional domains of αNAC was used to construct deletion mutants. Those functional domain deletion mutants were used to recover the apoptotic phenotype caused by αNAC depletion. Finally, the role of αNAC in TNF-related apoptosis-inducing ligand (TRAIL) treatment was investigated in vitro. We found that depletion of αNAC in multiple types of cancer cells induce typical apoptotic cell death. This anti-apoptotic function is mediated by the FADD/c-Jun N-terminal kinase pathway. Intact αNAC is required for the direct binding of FADD as well as its anti-apoptosis function. Either αNAC depletion or the deletion of the ubiquitin-associated domain of αNAC sensitizes L929 cancer cells to mTRAIL treatment. Our study revealed a αNAC anti-apoptotic function in multiple types of cancer cells and suggested its potential in cancer therapy.
据报道，新生多肽相关复合物α（αNAC）在多种癌症中过表达，并在缺氧条件下调节HeLa细胞的细胞凋亡。我们研究的目的是探讨αNAC在癌症进展中的凋亡功能。首先，我们观察了αNAC缺失的细胞效应。使用小鼠αNAC来恢复蛋白水平并验证其效果。采用膜联蛋白V检测、半胱天冬酶活性报告检测、凋亡分子标志物和集落形成检测作为标志物，以研究αNAC缺失导致细胞死亡的机制。使用癌症10通路报告检测来筛选下游通路。基于αNAC的功能域进行PCR定点缺失，构建缺失突变体。使用这些功能域缺失突变体来恢复由αNAC缺失引起的凋亡表型。最后，在体外研究了αNAC在肿瘤坏死因子相关凋亡诱导配体（TRAIL）治疗中的作用。我们发现，多种癌细胞中αNAC的缺失会诱导典型的凋亡细胞死亡。这种抗凋亡功能由FADD/c-Jun氨基末端激酶途径介导。完整的αNAC是FADD直接结合及其抗凋亡功能所必需的。αNAC的缺失或αNAC泛素相关结构域的缺失都会使L929癌细胞对mTRAIL治疗敏感。我们的研究揭示了αNAC在多种癌细胞中的抗凋亡功能，并表明了其在癌症治疗中的潜力。
