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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-κB-dependent 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.
Figures and Tables
Figure 1
MG132 inhibits TNF-α induced NF-κB dependent transcriptional activation in both A549 and NCI-H1299 cells. A549 and NCI-H1299 cells which stably expressed an IgGκ-NF-κB luciferase reporter gene construct were pretreated with MG132 (3 uM) for 1 h followed by the addition of TNF-α (10 ng/ml) for 6 h. The cells were then harvested for analysis of luciferase activity. Values are mean of three experiments±SD.
Figure 2
Dose-dependent TRAIL-induced cytotoxicity in A549 and NCI-H1299 lung cancer cells. Cells were incubated with TRAIL for 24 h followed by analysis of cell viability by an MTT assay. Values are mean of three experiments±SD.
Figure 3
TRAIL induces NF-κB activation in A549 cells. (A) Luciferase assay: Stable A549 IgGκ-NF-κB luciferase cells were treated with TRAIL (100 ng/ml) for different time points. Cells were harvested for luciferase assay. The fold luciferase activation was calculated relative to a normalized value of one given to control (untreated) cells. Data represent the mean luciferase value from triplicates in one experiment, which was used to calculate the mean+SD. from two experiments. (B) Western blot for IκBα degradation: A549 cells were treated with TRAIL (100 ng/ml) for various times and cellular proteins was harvested followed by loading of equal amounts of protein on an SDS-PAGE gel which was probed with an anti-IκBα antibody after transfer to nitrocellulose. (C) EMSA with supershift. A549 cells were treated with TRAIL (100 ng/ml) for various times and harvested for nuclear extraction. Electromobility shift assay was done with a radiolabeled IgGκ-NF-κB probe. Equal amounts (7.5 ug) of nuclear protein was loaded in each lane. In 5th lane, an 100x excess of unlabeled oligonucleotide was added 5 min before the addition of radiolabeled probe. In 6th lane, a rabbit polyclonal antibody p65 was added to the nuclear extract 10 min before the addition of radiolabeled probe.
Figure 4
(A) MG132 sensitizes A549 and NCI-H1299 cells to TRAIL-induced cytotoxicity. MG132 (3 uM) was pretreated 1 h before the addition of TRAIL (100 ng/ml) and after 24 h incubation cell viability test by an MTT assay was done. Values are mean of three experiments±SD. (B) Annexin V assay shows the sensitization effect of MG132 to TRAIL-induced apoptosis in A549 cells. Treatment schedule was the same with Figure 3A. and cells were harvested and followed by the analysis of apoptosis by FACS scan. (a: control, b: MG132 3µM, c: TRAIL 100 ng/ml, d: MG132 1 hr pre-treatment+TRAIL).
Figure 5
MG132 inhibits TRAIL-induced NF-κB transcriptional activity in A549 and NCI-H1299 cells. A549 and NCI-H1299 cells which stably expressed an IgGκ-NF-κB luciferase reporter gene construct were pretreated with MG132 (3 uM) for 1 h followed by the addition of TRAIL (100 ng/ml) for 24 h. The cells were then harvested for analysis of luciferase activity. Values are mean of three experiments±SD.
Figure 6
MG132 inhibits TRAIL-induced DNA binding of NF-κB in A549 cells and supershift assay shows NF-κB complex containing p65. A549 cells were treated with TRAIL (100 ng/ml) for various times and harvested for nuclear extraction. Electromobility shift assay was done with a radiolabeled IgGκ-NF-κB probe. Equal amounts (7.5 ug) of nuclear protein was loaded in each lane. In 4th lane, an 100x excess of unlabeled oligonucleotide was added 5 min before the addition of radiolabeled probe. In 3rd and 7th lanes, a rabbit polyclonal antibody p65 was added to the nuclear extract 10 min before the addition of radiolabeled probe (lane 1: control, lane 2: TRAIL 100 ng/ml for 2 hr, lane 3: TRAIL+p65 Ab, lane 4: TRAIL+x100 (cold competition), lane 5: MG132 3µM, lane 6: MG132+TRAIL, lane 7: MG132+TRAIL+p65 Ab).
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