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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.
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Fig. 1.
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.
Fig. 2.
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.
Fig. 3.
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.
Fig. 4.
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.
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