jlc Journal of Lung Cancer J Lung Cancer 1598-7809 Korean Association for the Study of Lung Cancer 10.6058/jlc.2006.5.2.102 jlc-5-102 Original Article Down-regulation of IL-1 ガ-induced COX-2 Expression in A549 Lung Cancer Cells at Transcriptional Level by Lepto- mycin Β Involves Inhibition of the I jcB- /NF- jcB Pathway but Independent of CRM1 ParkChang-KwonM.D. 1 KimJae-BumM.D. 1 KeumDong-YunM.D. 1 JangByeong-ChurlPh.D. 2 Dartment of Thoracic and Cardiovascular Suraerv. Chronic Disease Research (CDR) Center & Institute for Medical Science, School of Medicine, Keimyung University, Daegu, Korea Address for correspondence Bveong-Churl Jang, PhD. Chronic Disease Research (CDR) Center and Institute for Medical Science, School of Medicine, Keimvunq University, 194, Donqsan-dong, Jung-gu, Daegu 700-712, Korea Tel: 82-53-250-7032 Fax; 82-53-255-1398 E-mail; jangbc12@kmu.aakr 122006 18122006 5 2 102 110 04122006 12122006 Copyright © 2006 Korean Association for the Study of Lung Cancer 2006 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Purpose

Overexpression of COX-2t an enzyme responsible fro the synthesis of prostaglandins, is well linked to human chronic lung diseases. The mechanism by which COX-2 expression is increased or enhanced in cancer cells remains largely unknown. Any compound which can reduce COX-2 expression may be considered as an anti-cancer agent.

 Materials and Methods:

Leptomycin Β (LMB) is a metabolite of Streptomyces and a specific inhibitor of CRM1 nuclear export receptor A549 is a human lung cancer cell line. To evaluate the effect of LMB on COX-2 expression induced by IL-1 β, a pro-inflammatory cytokine, in A549 cells, Western blot and RT-PCR assays were applied to measure COX-2 protein and mRNA expressions in response to IL-1 β, respectively. Luciferase experiments were done to measure promoter activity of COX-2, NF- a B or AP-1. CRM1 siRNA trasfection experiment was performed to knock-down endogenous CRM! Biochemical protein fractionation method was also carried out to see intracellular localization of proteins.

 Results:

LMB at 9 nM strongly suppressed IL-1 β-induced expression of COX-2 protein that was attributable to decreased COX-2 transcript and promoter activity, but not mRNA stability. Distinctly, knock-down of CRM1 had no effect on COX-2 expression by IL-1 β. Moreover, LMB did not affect IL-1 -induced phosphorylation of ERK-1/2, JNK- 1/2, and ρ3δ MAPK or AP-1 promoter activity. In contrast, LMB blocked IL-1 β- mediated cytosolic Ia:B-« degradation, p65 NF-a-B nuclear translocation, and NF-a:B promoter activity.

 Conclusion:

LMB potently down-regulates IL-1 β- induced COX-2 at transcriptional level in A549 cells, in part, through modulation of the I a’B- a/NF-A-B pathway but independent of CRM1ôMAPKs and AP-1 (J Lung Cancer 2006;5(2):102-110)

 Leptomycin B COX-2 IL-1 β CRM1 UB-i?/p65 NF-a’B A549 cells REFERENCES Smith WL: DeWitt DL Garavito R Cyclooxygenases: structural, cellular, and molecular biology, Annu Rev Biochem 2000 69 145182 Mamett LJ DuBois RN COX-2: a target for colon cancer prevention Annu Rev Pharmacol Toxicol 2002 42 5580 Hla T Bishop-Bailey D: Liu CH Schaefers HJ: Trifan OC Cyclooxygenase-1 and -2 isoenzymes, Int J Biochem Cell Biol 1999 31 551557 Hawk ET Viner JL Dannenberg A DuBois RN COX-2 in cancer-a player that's defining the rules J Natl Cancer Inst 2002 94 545546 Herschman HR Reddy ST Xie W Function and regulation of prostaglandin synthase-2 Adv Exp Med Biol 1997 407 6166 Newton R: Kuitert LM: Bergmann M Adcock IM: Barnes PJ Evidence for involvement of NF-kappaB in the transcriptional control of COX: 2 gene expression by IL-lbeta Biochem Biophys Res Commun 1997 237 2832 Inoue H Yokoyama C Ham S Tone Y Tanabe T Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element J Biol Chem 1995 270 2496524971 Ristimaki A Garfinke】S: Wessendorf J Maciag T Hla T Induction of cyclooxygenase-2 by interleukin-1 alpha. Evidence for post-transcriptional regulation J Biol Chem 1994 269 1176911775 Srivastava SK Tetsuka T Daphna-Iken D: Morrison AR IL-1 beta stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region Am J Physiol 1994 267 504508 Jang BC Munoz-Najar U Paik JH Claffey K Yoshida M Hla T Leptomycin B, an inhibitor of the nuclear export receptor CRM1: inhibits COX-2 expression J Biol Chem 2003 278 27732776 Chen W: Tang Q Gonzales MS Bowden GT Role of p38 MAP kinases and ERK in mediating ultraviolet-B induced cyclooxygenase-2 gene expression in human keratinocytes Oncogene 2001 20 39213926 Hunot S: Vila M: Teismann P JNK-mediated induction of cyclooxygenase-2 is required for neurodegeneration in a mouse model of Parkinson's disease, Proc Natl Acad Sci USA 2004 101 665670 Hamamoto T: Gunji S Tsuji H: Beppu T Leptomycins A and B, new antifungal antibiotics. I. Taxonomy of the producing strain and their fermentation, purification and characterization J Antibiot (Tokyo) 1983 36 3945 Yoshida M Nishikawa M Nishi K: Abe K: Horinouchi S Beppu T Effects of leptomycin B on the cell cycle of fibro-Wasts and fission yeast cells Exp Cell Res 1990 187 150156 Jang BC Paik JH Jeong HY Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells Biochem Pharmacol 2004 68 263274 Lecane PS Kiviharju TM: Sellers RG Peehl DM Leptomycin B stabilizes and activates p53 in primary prostatic epithelial cells and induces apoptosis in the LNCaP cell line Prostate 2003 54 258267 Komiyama K: Okada K Tomisaka S: Umezawa I: Hamamoto T: Beppu T Antitumor activity of leptomycin B J Antibiot (Tokyo) 1985 38 427429 Nishi K Yoshida M Fujiwara D Nishikawa M Horinouchi S: Beppu T Leptomycin B targets a regulatory cascade of crml, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression J Biol Chem 1994 269 63206324 Fomerod M: Ohno M: Yoshida M Mailaj IW CRM1 is an export receptor for leucine-rich nuclear export signals Cell 1997 90 10511060 Brennan CM Gallouzi IE Steitz JA Protein ligands to HuR modulate its interaction with target mRNAs in vivo J Cell Biol 2000 151 114 Giannini A Mazor M: Orme M: Vivanco M Waxman J: Kypta R Nuclear export of alpha-catenin: overlap between nuclear export signal sequences and the beta-catenin binding site Exp Cell Res 2004 295 150160 Huang TT: Kudo N Yoshida M: Miyamoto S A nuclear export signal in the N-terminal regulatory domain of Ikappa-Balpha controls cytoplasmic localization of inactive NF-kappa B/1 kappa Bal pha complexes∗ Proc Natl Acad Sci 2000 97 10141 이9. Jang BC Sung SH Park JG Leptomycin B, a metabolite of Streptomyces, inhibits the expression of inducible nitric oxide synthase in BV2 microglial cells Int J Oncol 2006 29 15091515 Simeonidis S: Stauber D Chen G Hendrickson WA: Thanos D Mechanisms by which IkappaB proteins control NF-kappaB activity Proc Natl Acad Sci 1999 96 4954 Liu W Reinmuth N Stoeltzing O Cyclooxygenase-2 is up-regulated by interleukin-1 beta in human colorectal cancer cells via multiple signaling pathways Cancer Res 2003 63 36323636 Sawano H Haneda M: Sugimoto T Inoki K Koya D Kikkawa R 15-Deoxy-Deltal2∗ 14-prostaglandin J2 inhibits IL-1 beta-induced cyclooxygenase-2 expression in mesangial cells Kidney Int 2002 61 19571967 Erkmann JA Kutay U Nuclear export of mRNA: from the site of transcription to the cytoplasm Exp Cell Res 2004 296 1220 Rodriguez MS Thompson J: Hay RT Dargemont C Nuclear retention of IkappaBalpha protects it from signal-induced degradation and inhibits nuclear factor kappaB transcriptional activation. J Biol Chem 1999 274 91089015 Han Y Weinman S: Boldogh I: Walker RK: Brasier AR Tumor necrosis factor-alpha-inducible IkappaBalpha proteolysis mediated by cytosolic m-calpain. A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappab activation J Biol Chem 1999 274 787794 Figures and Tables

Effect of ᄂMB on Iᄂ-1 㽐-induced COX-2 protein and mRNA and promoter activity in A549 lung cancer cells, (Α, Β) A549 celts were pre-treated with the indicated concentrations of I一MB for 1 h. Cells were then exposed to IL-1 β for 4 h. Total cell lysates and RNA were prepared, and analyzed for COX-2 immunoblot (A) and RT-PCR (B), respectively. Actin or GAPDH was used to evaluate the relative expression of COX-2 protein or mRNA, (C) A549 cells were transfected with COX-2 promoter/luciferase DNA along with control pRL-TK DNA for 24 h and then exposed to IL-1 for 4 h in absence or presence of LMB, Cell lysates were prepared and used for reporter gene activity. Data are mean 士 S.E. of three independent experiments.

Effect of LMB on IL-1 -induced proteolysis of U-Β-α and NF-/cB nuclear localization and its promoter activity in A549 cells. (A) A549 cells were pre-treated with LMB for 1 h. Cells were then exposed to L-0 for 05 h in absence or presence of LMB. Total cell lysates were prepared, and used to measure total protein of ϊκΒ-α, p65 NF-λ’ΒôHuRôor actin by immunoblot with respective antibody. NS indicates nonspecific protein band. (B) Cytosolic and η䴸clear proteins were prepared and used to measure the level of cytosolic κΒ-α and nuclear p65 NF-^B by immunoblot with respective antibody. (C) A549 cells were transfected with NF-a:B promoter/luciferase DNA along with control pRL-TK DNA for 24 h and then exposed to IL- 1 for 4 h in absence or presence of LMB. Cell lysates were prepared and used for reporter gene activity. Data are mean 土 S.E of three independent experiments. (D) The same as in (C) except HuR immunoblot.

Effect of LMB on IL-1 㻐-mediated activation of MAPKs and AP-1 promoter activity in A549 cells, (A) A549 cells were pre-treated with LMB for 1 h. Cells were then exposed to IL-1 β for 0.5 h in absence or presence of LMB, Total cell tysates were prepared, and used to measure the extent of phosphorylation of ERK-1/2, JNK-1/2, or p38 MAPK. Total protein level of each protein was confirmed with striping and reprobing the membrane by immunoblot 䴸sing respective antibody, (Β) A549 cells were transfected with AP-1 promoter/luciferase DNA along with control pRL-TK DNA for 24 h and then exposed to IL-1 㻐 for 4 h in absence or presence of LMB. Cell lysates were prepared and used for reporter gene activityôData are mean 士 SôE. of three independent experiments.

No dependence of CRM1 and COX-2 mRNA stability in ᄂMB inhibition of IL-1 -induced COX-2 expression in A549 cells. (A) A549 cells were transfected with 80 nM of control or CRM1 siRNA. Cells were then exposed to IL-1 β in absence or presence of LMB for 4 h. Cell lysates were prepared and used to measure the expression level ᄋf CRM 1ôCOX-2, or actin by immunoblot with respective antibody. (Β) A549 cells were primarily treated without (lane 1) or with IL-1 㽐 (lanes 2〜 10) for 4 h to highly induce COX-2 mRNA. Cells were then exposed to Iᄂ-1/3 in the presence of actinomycin D (Act D)’ a transcription inhibitor, (lanes 3 〜 10) in absence (lane 3-6) or presence (lanes 7-10) of LMB for the indicated times. At each time, total RNA was prepared, and 䴸sed for COX-2 or GAPDH RT- PCR to measure the amount of respective RNA remained in the cells.