NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drugReportar como inadecuado




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Journal of Translational Medicine

, 10:252

Combination strategies

Abstract

BackgroundMost 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.

MethodsAKT 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.

ResultsTMZ 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.

ConclusionNF-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.

KeywordsNuclear factor κB AKT Temozolomide NEMO binding domain peptide Cell proliferation Cell senescence AbbreviationsATMAtaxia-Telangiectasia Mutated

BGO-benzylguanine

Chk1Checkpoint Kinase 1

CTRLControl

DAPI4’-6-Diamidino-2-phenylindol

ECLEnhanced chemiluminescence

ELISAEnzyme-linked immunosorbent assay

FCSFetal calf serum

IC50Concentration producing 50% inhibition

IκBsInhibitors of κB

IKKIκB kinase

IL-8Interleukin 8

mAbMonoclonal antibody

MAPKMitogen activated protein kinase

MCP-1Monocyte chemoattractant protein-1

MGMTO-methylguanine-DNA methyltransferase

MMRMismatch repair

MTT3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide

NBDNEMO-binding domain

NEMONF-κB essential modulator

NF-κBNuclear factor κB

O-MeGO-methylguanine

PBSPhosphate buffered saline

PI3KPhosphatidylinositol-3-kinase

SA-β-GalSenescence associated β-galactosidase

SAHFSenescence-associated heterochromatin foci

SDSSodium dodecyl sulfate

SEMStandard error of the mean

siRNASmall interfering RNA

TMZTemozolomide

TNFTumor necrosis factor.

Electronic supplementary materialThe online version of this article doi:10.1186-1479-5876-10-252 contains supplementary material, which is available to authorized users.

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Autor: Simona Caporali - Lauretta Levati - Grazia Graziani - Alessia Muzi - Maria Grazia Atzori - Enzo Bonmassar - Giuseppe Palmi

Fuente: https://link.springer.com/







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