Journal List > Tuberc Respir Dis > v.65(6) > 1001314

Tuberc Respir Dis. 2008 Dec;65(6):476-486. Korean.
Published online December 31, 2008.  https://doi.org/10.4046/trd.2008.65.6.476
Copyright © 2008 The Korean Academy of Tuberculosis and Respiratory Diseases
The Proteasome Inhibitor MG132 Sensitizes Lung Cancer Cells to TRAIL-induced Apoptosis by Inhibiting NF-κB Activation
Pil Won Seo, M.D.,1 and Kye Young Lee, M.D.2
1Department of Thoracic Surgery, Dankook University College of Medicine, Cheonan, Korea.
2Department of Internal Medicine, Konkuk University School of Medicne, Seoul, Korea.

Address for correspondence: Kye Young Lee, M.D., Ph.D. Department of Internal Medicine, Konkuk University School of Medicine, 4-12, Hwayang-dong, Gwangjin-gu, Seoul 143-729, Korea. Phone: 82-2-2030-7521, Fax: 82-2-2030-7748, Email: kyleemd@kuh.ac.kr
Received November 18, 2008; Accepted December 16, 2008.

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.

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

Figures


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.
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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.
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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.
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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).
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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.
Click for larger image


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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