Down-Modulation of Expression, or Dephosphorylation, of IG20/MADD in Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Resistant Thyroid Cancer Cells Makes Them Susceptible to Treatment with This Ligand
Down-Modulation of Expression, or Dephosphorylation, of IG20/MADD in Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Resistant Thyroid Cancer Cells Makes Them Susceptible to Treatment with This Ligand
复制标题
DOI：
10.1089/thy.2012.0155
复制
发表时间：
2013-01-01
期刊：
影响因子：
6.6
通讯作者：
Prabhakar, Bellur S.
中科院分区：
文献类型：
--
作者：
Li, Liang-Cheng;Jayarama, Shankara;Prabhakar, Bellur S.
背景：IG20/MADD基因在甲状腺癌组织和细胞系中过表达，与肿瘤坏死因子相关的凋亡诱导配体(TRAIL)耐药有关。MADD蛋白抵抗TRAIL诱导的细胞凋亡的能力依赖于其被Akt的磷酸化。有趣的是，尽管TRAIL诱导TRAIL敏感细胞中的磷酸化Akt(PACK)和磷酸化MADD(PMADD)水平显著降低，但在TRAIL耐药细胞中却不能做到这一点。在本研究中，我们探讨了Akt对MADD的磷酸化是否参与了甲状腺癌细胞对TRAIL的耐药。方法：通过荧光激活细胞分类(FACS)分析不同的甲状腺癌细胞株对TRAIL诱导的细胞凋亡的敏感性。我们用流式细胞仪检测各种TRAIL耐药因子，用定量逆转录聚合酶链式反应检测IG20/MADD的表达。用免疫印迹法检测TRAIL处理后甲状腺癌细胞中PACT和pMADD的表达水平。结果：bCPAP和TPC1细胞对TRAIL诱导的细胞凋亡敏感，而KTc1和FTC133细胞对TRAIL诱导的细胞凋亡具有抵抗力。TRAIL的不同易感性与死亡受体、诱骗受体或TRAIL的表达水平无关。与bCPAP和TPC1相比，KTC1和FTC133细胞中IG20/MADD的表达水平较高，并且在IG20/MADD被敲除后对TRAIL敏感。有趣的是，经TRAIL处理后，TRAIL敏感的bCPAP和TPC1细胞的PACT和pMADD水平降低，而耐药的KTC1和FTC133细胞的PACT和pMADD水平保持不变。虽然在bCPAP和TPC1细胞中表达具有结构性活性的Akt使它们对TRAIL产生抗性，但用LY294002预处理KTC1和FTC133细胞使它们对TRAIL敏感。结论：pMADD在某些甲状腺癌细胞中是一个重要的TRAIL耐药因子，提示下调IG20/MADD的表达或磷酸化均可使TRAIL耐药的甲状腺癌细胞对TRAIL敏感。
Background: The IG20/MADD gene is overexpressed in thyroid cancer tissues and cell lines, and can contribute to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance. The ability of the MADD protein to resist TRAIL-induced apoptosis is dependent upon its phosphorylation by Akt. Interestingly, while TRAIL induces a significant reduction in the levels of phospho-Akt (pAkt) and phospho-MADD (pMADD) in TRAIL-sensitive cells, it fails to do so in TRAIL-resistant cells. In this study, we investigated if MADD phosphorylation by Akt was contributing to TRAIL resistance in thyroid cancer cells.Methods: We determined the susceptibility of different thyroid cancer cell lines to TRAIL-induced apoptosis by fluorescence-activated cell sorting (FACS) analysis. We tested for various TRAIL resistance factors by FACS analyses or for IG20/MADD expression by quantitative reverse transcription-polymerase chain reaction. We determined the levels of pAkt and pMADD upon TRAIL treatment in thyroid cancer cells by Western blotting. We tested if down-modulation of IG20/MADD gene expression using shRNA or phosphorylation using a dominant negative Akt (DN-Akt) or pretreatment with LY294002, a PI3 kinase inhibitor, could help overcome TRAIL resistance.Result: BCPAP and TPC1 cells were susceptible, while KTC1 and FTC133 cells were resistant, to TRAIL-induced apoptosis. The differential susceptibility to TRAIL was not related to the levels of expression of death receptors, decoy receptors, or TRAIL. KTC1 and FTC133 cells showed higher levels of IG20/MADD expression relative to BCPAP and TPC1, and were rendered susceptible to TRAIL treatment upon IG20/MADD knockdown. Interestingly, upon TRAIL treatment, the pAkt and pMADD levels were reduced in TRAIL-sensitive BCPAP and TPC1 cells, while they remained unchanged in the resistant KTC1 and FTC133 cells. While expression of a constitutively active Akt in BCPAP and TPC1 cells rendered them resistant to TRAIL, pretreating KTC1 and FTC133 cells with LY294002 rendered them TRAIL-sensitive. Moreover, expression of a DN-Akt in KTC1 and FTC133 cells reduced the levels of pAkt and pMADD and sensitized them to TRAIL-induced apoptosis.Conclusion: Our results show that pMADD is an important TRAIL resistance factor in certain thyroid cancer cells and suggest that down-modulation of either IG20/MADD expression or phosphorylation can render TRAIL-resistant thyroid cancer cells sensitive to TRAIL.