The Proteasome Inhibitor MG132 Sensitizes Lung Cancer Cells to TRAIL-induced Apoptosis by Inhibiting NF-kappaB Activation.
10.4046/trd.2008.65.6.476
- Author:
Pil Won SEO
1
;
Kye Young LEE
Author Information
1. Department of Thoracic Surgery, Dankook University College of Medicine, Cheonan, Korea.
- Publication Type:Original Article
- Keywords:
TRAIL;
NF-kappaB;
Lung cancer;
Proteasome inhibitor;
Apoptosis
- MeSH:
Annexin A5;
Apoptosis;
Blotting, Western;
Cell Death;
Cell Line;
Cell Survival;
DNA;
Genes, Reporter;
Homicide;
Humans;
I-kappa B Proteins;
Leupeptins;
Luciferases;
Lung;
Lung Neoplasms;
NF-kappa B;
Phenotype;
Proteasome Endopeptidase Complex;
Proteasome Inhibitors;
Transcriptional Activation
- From:Tuberculosis and Respiratory Diseases
2008;65(6):476-486
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
BACKGROUND: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates NF-kappaB in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate NF-kappaB in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of NF-kappaB activation using proteasome inhibitor MG132 which blocks I kappa B alpha degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. METHODS: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study NF-kappaB-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgGkappa-NF-kappaB luciferase construct. To investigate DNA binding of NF-kappaB activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of I kappa B alpha degradation. RESULTS: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 (3microM) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced NF-kappaB transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of NF-kappaB activated by TRAIL and supershift with p65 antibody. I kappa B alpha degradation was proven by western blot. MG132 completely blocked both TRAIL-induced NF-kappaB dependent luciferase activity and DNA binding of NF-kappaB. CONCLUSION: This results suggest that inhibition of NF-kappaB can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.
