Anat Cell Biol.  2017 Dec;50(4):293-300. 10.5115/acb.2017.50.4.293.

Celecoxib induces cell death on non-small cell lung cancer cells through endoplasmic reticulum stress

• Kim B
• Kim J
• Kim YS

• KMID: 2399898
• DOI: http://doi.org/10.5115/acb.2017.50.4.293

Abstract

Cyclooxygenase-2 (COX-2) is an enzyme induced by various proinflammatory and mitogenic stimuli. Celecoxib is a selective inhibitor of COX-2 that have been shown to affect cell growth and apoptosis. Lung cancer cells expressing COX-2 is able to be a target of celecoxib, this study focuses on investigating that celecoxib induces apoptosis via endoplasmic reticulum (ER) stress on lung cancer cells. We investigated whether celecoxib induced apoptosis on non-small cell lung cancer cell line, A549 and H460. The 50 µM of celecoxib increased apoptotic cells and 100 µM of celecoxib significantly induced apoptosis. To check involvement of caspase cascade, pretreatment of z-VAD-fmk blocked celecoxib-induced apoptosis. However, caspase-3, -8, and -9 were not activated, but cleavage of non-classical caspase-4 was detected using western blot. As checking ER stress associated molecules, celecoxib did not increase expressions of growth arrest and DNA damage inducible protein 34, activating transcription factor 4, and spliced X-box binding protiens-1, but increase of both glucose-regulated protein 78 (GRP78) and C/EBP homologous transcription factor were detected. Salubrinal, inhibitor of eIF2 and siRNA for IRE1 did not alter celecoxib-induced apoptosis. Instead, celecoxib-induced apoptosis might be deeply associated with ER stress depending on GRP78 because siRNA for GRP78 enhanced apoptosis. Taken together, celecoxib triggered ER stress on lung cancer cells and celecoxib-induced apoptosis might be involved in both non-classical caspase-4 and GRP78.

Keyword

Celecoxib; Lung neoplasms; ER stress; Apoptosis

MeSH Terms

Activating Transcription Factor 4
Apoptosis
Blotting, Western
Carcinoma, Non-Small-Cell Lung*
Caspase 3
Celecoxib*
Cell Death*
Cell Line
Cyclooxygenase 2
DNA Damage
Endoplasmic Reticulum Stress*
Endoplasmic Reticulum*
Eukaryotic Initiation Factor-2
Lung Neoplasms
RNA, Small Interfering
Transcription Factors
Activating Transcription Factor 4
Caspase 3
Celecoxib
Cyclooxygenase 2
Eukaryotic Initiation Factor-2
RNA, Small Interfering
Transcription Factors

Figure

• 

  Fig. 1 Viability of celecoxib-treated A549 (A) and H460 (B). A549 and H460 were treated with celecoxib (0, 25, 50, 100, and 200 µM) for 16 hours. Cell viability was measured by WST-1 assay as described in materials and methods.

• 

  Fig. 2 Flow cytometry analysis for celecoxib-induced apoptosis on A549 and H460. A549 and H460 were treated with 50 and 100 µM of celecoxib for 16 hours. Then, cells were harvested and analyzed using flow cytometry after staining with fluorescein isothiocyanate (FITC)-labeled annexin V. Indicated percentile numbers mean cell proportion in gated region. A representative example of five independent experiments is shown.

• 

  Fig. 3 Effect of z-VAD-fmk on celecoxib-induced apoptosis. Z-VAD-fmk (Merck, Darmstadt, Germany) was treated on A549 for 1 hour before celecoxib treatment. A549 was washed with phosphate buffered saline, and then treated with 100 µM of celecoxib for 16 hours. Cells were harvested and analyzed using flow cytometry after staining with fluorescein isothiocyanate (FITC)-labeled annexin V. Indicated percentile numbers mean cell proportion in gated region. A representative example of three independent experiments is shown.

• 

  Fig. 4 Western blot for caspase-3, 4, 8, and 9 in celecoxib-treated A549. A549 was treated with celecoxib (0, 25, 50, 75, and 100 µM) for 16 hours. After washing with phosphate buffered saline, cell lysates were obtained using lysis buffer. Western blot for caspase-3, -4, -8, and -9 were performed using antibodies against each caspase, and visualized as described in Materials and Methods. A representative example of six independent experiments is shown. NC, negative control.

• 

  Fig. 5 Expressions of endoplasmic reticulum stress markers in A549 and H460 after celecoxib treatment. Celecoxib 75 µM was treated on A549 and H460 cells for 16 hours and RNA and protein were isolated respectively. (A) Glucose-regulated protein 78 (GRP78), spliced X-box binding protiens-1 (sXBP-1), growth arrest and DNA damage inducible protein 34 (GADD34), activating transcription factor 4 (ATF4), and C/EBP homologous transcription factor (CHOP) mRNA were measured by reverse transcription polymerase chain reaction. A representative example of three independent experiments is shown. (B) GRP78 and CHOP protein were measured using western blot. A representative example of four independent experiments is shown.

• 

  Fig. 6 Analysis for celecoxib-induced apoptosis after transfection of siRNA for glucose-regulated protein 78 (GRP78) or IRE1 in A549. Two hundred pmol of siRNA for GRP78 or inositol-requiring enzyme 1 (IRE1) was transfected in A549 cells and stabilized overnight. And then celecoxib 75 µM was treated for 16 hours. Cells were harvested and analyzed using flow cytometry after staining with fluorescein isothiocyanate (FITC)-labeled annexin V. Indicated percentile numbers mean cell proportion in gated region. A representative example of four independent experiments is shown.

• 

  Fig. 7 Analysis for celecoxib-induced apoptosis after treatment of salubrinal in A549. The 10 µM of salubrinal, eIF2 inhibitor was preincubated with A549 for 1 hour and then then celecoxib 75 µM was treated for 16 hours. Cells were harvested and analyzed using flow cytometry after staining with fluorescein isothiocyanate (FITC)-labeled annexin V. Indicated percentile numbers mean cell proportion in gated region. A representative example of three independent experiments is shown.

Reference

1. Hirsch FR, Lippman SM. Advances in the biology of lung cancer chemoprevention. J Clin Oncol. 2005; 23:3186–3197.

2. Keith RL. Lung cancer chemoprevention. Proc Am Thorac Soc. 2012; 9:52–56.

3. Dela Cruz CS, Tanoue LT, Matthay RA. Lung cancer: epidemiology, etiology, and prevention. Clin Chest Med. 2011; 32:605–644.

4. Sandler AB, Dubinett SM. COX-2 inhibition and lung cancer. Semin Oncol. 2004; 31:2 Suppl 7. 45–52.

5. Salgia R, Skarin AT. Molecular abnormalities in lung cancer. J Clin Oncol. 1998; 16:1207–1217.

6. Collins LG, Haines C, Perkel R, Enck RE. Lung cancer: diagnosis and management. Am Fam Physician. 2007; 75:56–63.

7. Dy GK, Adjei AA. Novel targets for lung cancer therapy: part I. J Clin Oncol. 2002; 20:2881–2894.

8. Gasparini G, Longo R, Sarmiento R, Morabito A. Inhibitors of cyclo-oxygenase 2: a new class of anticancer agents? Lancet Oncol. 2003; 4:605–615.

9. Breyer MD, Harris RC. Cyclooxygenase 2 and the kidney. Curr Opin Nephrol Hypertens. 2001; 10:89–98.

10. Frungieri MB, Calandra RS, Mayerhofer A, Matzkin ME. Cyclooxygenase and prostaglandins in somatic cell populations of the testis. Reproduction. 2015; 149:R169–R180.

11. Vosooghi M, Amini M. The discovery and development of cyclooxygenase-2 inhibitors as potential anticancer therapies. Expert Opin Drug Discov. 2014; 9:255–267.

12. Ghosh N, Chaki R, Mandal V, Mandal SC. COX-2 as a target for cancer chemotherapy. Pharmacol Rep. 2010; 62:233–244.

13. Rizzo MT. Cyclooxygenase-2 in oncogenesis. Clin Chim Acta. 2011; 412:671–687.

14. Grösch S, Maier TJ, Schiffmann S, Geisslinger G. Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst. 2006; 98:736–747.

15. Kismet K, Akay MT, Abbasoglu O, Ercan A. Celecoxib: a potent cyclooxygenase-2 inhibitor in cancer prevention. Cancer Detect Prev. 2004; 28:127–142.

16. Jendrossek V. Targeting apoptosis pathways by celecoxib in cancer. Cancer Lett. 2013; 332:313–324.

17. Ko SH, Choi GJ, Lee JH, Han YA, Lim SJ, Kim SH. Differential effects of selective cyclooxygenase-2 inhibitors in inhibiting proliferation and induction of apoptosis in oral squamous cell carcinoma. Oncol Rep. 2008; 19:425–433.

18. Wu T, Leng J, Han C, Demetris AJ. The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells. Mol Cancer Ther. 2004; 3:299–307.

19. Liggett JL, Zhang X, Eling TE, Baek SJ. Anti-tumor activity of non-steroidal anti-inflammatory drugs: cyclooxygenase-independent targets. Cancer Lett. 2014; 346:217–224.

20. Wang WA, Groenendyk J, Michalak M. Endoplasmic reticulum stress associated responses in cancer. Biochim Biophys Acta. 2014; 1843:2143–2149.

21. Chevet E, Hetz C, Samali A. Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis. Cancer Discov. 2015; 5:586–597.

22. Huang KH, Kuo KL, Chen SC, Weng TI, Chuang YT, Tsai YC, Pu YS, Chiang CK, Liu SH. Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells. PLoS One. 2012; 7:e33615.

23. Tsutsumi S, Namba T, Tanaka KI, Arai Y, Ishihara T, Aburaya M, Mima S, Hoshino T, Mizushima T. Celecoxib upregulates endoplasmic reticulum chaperones that inhibit celecoxib-induced apoptosis in human gastric cells. Oncogene. 2006; 25:1018–1029.

24. Sano R, Reed JC. ER stress-induced cell death mechanisms. Biochim Biophys Acta. 2013; 1833:3460–3470.

25. McGuckin MA, Eri RD, Das I, Lourie R, Florin TH. ER stress and the unfolded protein response in intestinal inflammation. Am J Physiol Gastrointest Liver Physiol. 2010; 298:G820–G832.

26. Tsutsumi S, Gotoh T, Tomisato W, Mima S, Hoshino T, Hwang HJ, Takenaka H, Tsuchiya T, Mori M, Mizushima T. Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis. Cell Death Differ. 2004; 11:1009–1016.

27. Zhou XM, Wong BC, Fan XM, Zhang HB, Lin MC, Kung HF, Fan DM, Lam SK. Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak. Carcinogenesis. 2001; 22:1393–1397.

28. Duncan K, Uwimpuhwe H, Czibere A, Sarkar D, Libermann TA, Fisher PB, Zerbini LF. NSAIDs induce apoptosis in nonproliferating ovarian cancer cells and inhibit tumor growth in vivo. IUBMB Life. 2012; 64:636–643.

29. Li M, Wu X, Xu XC. Induction of apoptosis in colon cancer cells by cyclooxygenase-2 inhibitor NS398 through a cytochrome c-dependent pathway. Clin Cancer Res. 2001; 7:1010–1016.

30. Ramer R, Walther U, Borchert P, Laufer S, Linnebacher M, Hinz B. Induction but not inhibition of COX-2 confers human lung cancer cell apoptosis by celecoxib. J Lipid Res. 2013; 54:3116–3129.

31. Gentz SH, Bertollo CM, Souza-Fagundes EM, da Silva AM. Implication of eIF2alpha kinase GCN2 in induction of apoptosis and endoplasmic reticulum stress-responsive genes by sodium salicylate. J Pharm Pharmacol. 2013; 65:430–440.

32. Chen ST, Thomas S, Gaffney KJ, Louie SG, Petasis NA, Schönthal AH. Cytotoxic effects of celecoxib on Raji lymphoma cells correlate with aggravated endoplasmic reticulum stress but not with inhibition of cyclooxygenase-2. Leuk Res. 2010; 34:250–253.

33. Mandegary A, Torshabi M, Seyedabadi M, Amirheidari B, Sharif E, Ghahremani MH. Indomethacin-enhanced anticancer effect of arsenic trioxide in A549 cell line: involvement of apoptosis and phospho-ERK and p38 MAPK pathways. Biomed Res Int. 2013; 2013:237543.

34. Liu X, Yue P, Zhou Z, Khuri FR, Sun SY. Death receptor regulation and celecoxib-induced apoptosis in human lung cancer cells. J Natl Cancer Inst. 2004; 96:1769–1780.

35. Hitomi J, Katayama T, Eguchi Y, Kudo T, Taniguchi M, Koyama Y, Manabe T, Yamagishi S, Bando Y, Imaizumi K, Tsujimoto Y, Tohyama M. Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J Cell Biol. 2004; 165:347–356.