Laboratory of Molecular Oncology, Istituto Dermopatico dell'Immacolata-IRCCS, Rome, Italy.
J Transl Med. 2012 Dec 21;10:252. doi: 10.1186/1479-5876-10-252.
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.
大多数 DNA 损伤化学治疗剂激活转录因子核因子 κB（NF-κB）。然而，NF-κB 的激活既可以保护药物，也可以促进药物的生长抑制作用。我们之前表明，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 AKT，AKT 是 NF-κB 的正调节剂。在这里，我们研究了 TMZ 是否激活 NF-κB，以及 AKT 是否参与这一分子事件。我们还评估了抑制 NF-κB 对肿瘤细胞对 TMZ 反应的功能后果。
在 MMR 缺陷（HCT116、293TLα-）和/或 MMR 功能正常（HCT116/3-6、293TLα+、M10）细胞中研究 TMZ 处理后 AKT 磷酸化、NF-κB 转录活性、IκB-α 降解、NF-κB2/p52 生成以及 RelA 和 NF-κB2/p52 核易位。在 HCT116/3-6 和 M10 细胞中转染 AKT1 靶向 siRNA 或使用表达野生型或激酶失活突变体 AKT1 的同基因 MMR 功能正常细胞系 pUSE2 和 KD12，研究 AKT 在 TMZ 诱导的 NF-κB 激活中的作用。在 HCT116/3-6 和 M10 细胞中使用 NF-κB 抑制剂 NEMO 结合结构域（NBD）肽或抗 RelA siRNA 研究抑制 NF-κB 对 TMZ 敏感性的影响。
TMZ 增强了 HCT116/3-6 和 M10 但不是 HCT116 细胞的 NF-κB 转录活性、激活 AKT、诱导 IκB-α 降解和 RelA 核易位。在 M10 细胞中，TMZ 促进了 NF-κB2/p52 的生成和核易位，并增强了 IL-8 和 MCP-1 的分泌。TMZ 还诱导了 293TLα+但不是 293TLα-细胞中的 RelA 核易位。AKT1 沉默抑制了 TMZ 诱导的 IκB-α 降解和 NF-κB2/p52 的生成。在 KD12 细胞中，NF-κB 转录活性和 RelA 和 NF-κB2/p52 的核易位对 TMZ 的反应受到损害。在 HCT116/3-6 和 M10 细胞中，RelA 沉默增加了 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 HCT116/3-6 细胞中，NBD 肽与 TMZ 的联合治疗产生了相加的生长抑制作用。
NF-κB 以 MMR 和 AKT 依赖性方式对 TMZ 作出反应并赋予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，通过联合 TMZ 和 NF-κB 抑制剂可能会获得临床益处。
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