Tuberc Respir Dis > Volume 65(6); 2008 > Article
Tuberculosis and Respiratory Diseases 2008;65(6):476-486.
DOI: https://doi.org/10.4046/trd.2008.65.6.476    Published online December 1, 2008.
The Proteasome Inhibitor MG132 Sensitizes Lung Cancer Cells to TRAIL-induced Apoptosis by Inhibiting NF-kappaB Activation.
Pil Won Seo, Kye Young Lee
1Department of Thoracic Surgery, Dankook University College of Medicine, Cheonan, Korea.
2Department of Internal Medicine, Konkuk University School of Medicne, Seoul, Korea. kyleemd@kuh.ac.kr
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.
Key Words: TRAIL, NF-kappaB, Lung cancer, Proteasome inhibitor, Apoptosis
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