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The Proteasome Inhibitor MG132 Sensitizes Lung Cancer Cells to TRAIL-induced Apoptosis by Inhibiting NF-κB Activation
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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-κB in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate NF-κB in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of NF-κB activation using proteasome inhibitor MG132 which blocks IκBα 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-κBdependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgGκ-NF-κB luciferase construct. To investigate DNA binding of NF-κB 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 IκBα 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 (3μM) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced NF-κB transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of NF-κB activated by TRAIL and supershift with p65 antibody. IκBα degradation was proven by western blot. MG132 completely blocked both TRAIL-induced NF-κB dependent luciferase activity and DNA binding of NF-κB. Conclusion: This results suggest that inhibition of NF-κB can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer
- keywords
- TRAIL, NF-κB, Lung cancer, Proteasome inhibitor, Apoptosis
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