NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug.
NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug.
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DOI：
10.1186/1479-5876-10-252
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发表时间：
2012-12-21
影响因子：
7.4
通讯作者：
D'Atri S
中科院分区：
文献类型：
--
作者：
Caporali S;Levati L;Graziani G;Muzi A;Atzori MG;Bonmassar E;Palmieri G;Ascierto PA;D'Atri S
大多数DNA损伤性化疗药物激活转录因子核因子κB（NF-κB）。然而，NF-κB活化可以保护免受或有助于该试剂的生长抑制作用。我们先前发现DNA甲基化药物替莫唑胺（TMZ）以错配修复（MMR）系统依赖性方式激活NF-κB的正调节剂AKT。我们研究了TMZ是否激活NF-κB以及AKT是否参与了这一分子事件。我们还评估了抑制NF-κB对肿瘤细胞对TMZ的反应的功能后果。在TMZ处理的MMR缺陷（HCT 116，293 TL α-）和/或MMR熟练（HCT 116/3-6，293 TL α+，M10）细胞中研究了AKT磷酸化、NF-κB转录活性、IκB-α降解、NF-κ B2/p52产生以及RelA和NF-κB2/p52核转位。在用AKT 1靶向siRNA瞬时转染的HCT 116/3-6和M10细胞中，或使用表达野生型或激酶死亡突变体AKT 1的同基因MMR-精通细胞系pUSE 2和KD 12，研究了AKT参与TMZ诱导的NF-κB活化。使用NF-κB抑制剂NEMO结合域（NBD）肽或抗RelA siRNA在HCT 116/3-6和M10细胞中研究抑制NF-κB对TMZ敏感性的影响。TMZ可增强HCT 116/3-6和M10细胞NF-κB转录活性，激活AKT，诱导IκB-α降解和RelA核转位，但对HCT 116细胞无影响。TMZ可促进M10细胞NF-κB2/p52的生成和核转位，并促进IL-8和MCP-1的分泌。TMZ也能诱导293 TL α+细胞的RelA核转位，但不能诱导293 TL α-细胞的RelA核转位。AKT 1沉默抑制TMZ诱导的IκB-α降解和NF-κB2/p52生成。TMZ抑制了NF-κB转录活性的上调，抑制了RelA和NF-κB2/p52的核转位。在HCT 116/3-6和M10细胞中RelA沉默增加TMZ诱导的生长抑制。在M10细胞中，NBD肽降低了基础NF-κB活性，消除了TMZ诱导的NF-κB活性上调，并增加了对TMZ的敏感性。在HCT 116/3-6细胞中，NBD肽和TMZ联合处理产生了相加的生长抑制作用。NF-κB以MMR和AKT依赖性方式响应TMZ而被激活，并赋予针对药物诱导的细胞生长抑制的保护作用。我们的研究结果表明，TMZ与NF-κB抑制剂联合应用可获得临床获益。
Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ. AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA. TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects. NF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors.
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