Death receptor regulation and celecoxib-induced apoptosis in human lung cancer cells.
BACKGROUND
Celecoxib, a cyclooxygenase 2 inhibitor, has chemopreventive and therapeutic activities toward lung cancer and other epithelial malignancies. Celecoxib can induce apoptosis in various cancer cell lines through a mechanism that is independent of its cyclooxygenase 2 inhibitory activity but is otherwise largely uncharacterized. We investigated the mechanism of celecoxib-induced apoptosis further.


METHODS
All experiments were conducted in human non-small-cell lung carcinoma (NSCLC) cell lines; results in celecoxib-treated and untreated cells were compared. Cell survival was assessed with a sulforhodamine B assay. Apoptosis was assessed by DNA fragmentation with an enzyme-linked immunosorbent assay, by terminal deoxynucleotidyltransferase-mediated dUTP nick-end-labeling (TUNEL) assay, and by western blot analysis of caspase activation. Death receptor gene and protein expression was detected by northern and western blot analysis, respectively. Gene silencing was achieved with small interfering RNA (siRNA) technology.


RESULTS
Celecoxib treatment decreased cell survival, activated caspase cascades, and increased DNA fragmentation, all of which were abrogated when caspase 8 expression was silenced with caspase 8 siRNA. Celecoxib treatment induced the expression of death receptors, particularly that of DR5. Overexpression of a dominant negative Fas-associated death domain mutant, but not of BCL2, reduced the level of celecoxib-induced apoptosis, and silencing of DR5 expression by DR5 siRNA suppressed celecoxib-induced caspase 8 activation and apoptosis. Combination treatment with celecoxib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced additional apoptosis. For example, survival of A549 cells was decreased with 50 muM celecoxib alone by 38.7% (95% confidence interval [CI] = 35.2% to 42.2%), with TRAIL alone by 29.3% (95% CI = 25.1% to 33.6%), but with their combination by 77.5% (95% CI = 74.5% to 79.5%), a greater than additive effect.


CONCLUSIONS
Celecoxib appears to induce apoptosis in human NSCLC through the extrinsic death receptor pathway.
Liu
Xiangguo
X
Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA.
Yue
Ping
P
Zhou
Zhongmei
Z
Khuri
Fadlo R
FR
Sun
Shi-Yong
SY
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D015152:Blotting, Northern; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D000068579:Celecoxib; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D004789:Enzyme Activation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020287:In Situ Nick-End Labeling; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins; D011451:Prostaglandin-Endoperoxide Synthases; D011720:Pyrazoles; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology*; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000378:metabolism; D015854:Up-Regulation
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011720:Pyrazoles; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D013449:Sulfonamides; D053221:TNF-Related Apoptosis-Inducing Ligand; C505639:TNFRSF10B protein, human; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D000068579:Celecoxib