Abstract
The novel lignan isochaihulactone inhibits cell proliferation and is an effective inducer of apoptosis in a variety of carcinoma cell lines. To determine the mechanisms underlying these effects, we examined isochaihulactone-induced changes in gene expression using oligodeoxynucleotide-based microarray screening of a human lung carcinoma cell line, A549. Isochaihulactone-inducible genes included the early growth response gene-1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene (NAG-1). Isochaihulactone increased EGR-1 and then NAG-1 mRNA and protein expression. Pure isochaihulactone induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Isochaihulactone-induced increases in EGR-1 and NAG-1 expression were reduced by the mitogen-activated protein kinase kinase 1/2 inhibitor 2′-amino-3′-methoxyflavone (PD98059), and this effect was not blocked by the phosphatidylinositol 3-kinase/protein kinase B pathway inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). Inhibition of isochaihulactone-induced NAG-1 expression by EGR-1 small interfering RNA blocked isochaihulactone-induced apoptosis in A549 cells, suggesting that induction of EGR-1 expression decreases survival of A549 cells. RNA interference using double-stranded RNA specific for NAG-1 also inhibited isochaihulactone-induced apoptosis, and cells transfected to increased NAG-1 expression had a greater apoptotic response to isochaihulactone and reduced colony formation efficiency. In addition, treatment of nude mice with isochaihulactone increased the in vivo NAG-1 expression as examined by immunohistochemistry from tumor biopsy. Isochaihulactone treatment increased the luciferase activity of NAG-1 in A549 cells transfected with the NAG-1 promoter construct. This induction increased expression of NAG-1 that was p53-independent and Sp1-dependent. Our findings suggest that NAG-1 expression is up-regulated by isochaihulactone through an ERK-dependent pathway involving the activation of EGR-1.
Footnotes
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This work was supported by Grants 96-2320-B-197-001-MY2 and 96-2320-B-303-001-MY3 from National Science Council of the Republic of China. Y.-L.C. and P.-C.L. contributed equally as first authors, and S.-Z.L. and H.-J.H. share equal corresponding authorship.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.126193.
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ABBREVIATIONS: EGR-1, early growth response gene-1; TGF-β, transforming growth factor-β; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3-kinase; PPARγ, peroxisome proliferator-activated receptor-γ; ERK, extracellular signal-regulated kinase; NSAID, nonsteroidal anti-inflammatory drug; NAG-1, nonsteroidal anti-inflammatory drug-activated gene; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide; PKC, protein kinase C; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; PD98059, 2-amino-3-methoxyflavone; JNK, c-Jun NH2-terminal kinase; SP600125, anthra[1,9-cd] pyrazol-6 (2H)-one; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; AKT, protein kinase B; PTGF, placental transforming growth factor; GSK, glycogen synthase kinase; RT, reverse transcription; PCR, polymerase chain reaction; si, small interfering; PBS, phosphate-buffered saline; NRG-1, neuregulin-1; FGF-1, fibroblast growth factor-1; MEK, mitogen-activated protein kinase kinase; 5F203, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole.
- Received May 29, 2007.
- Accepted August 21, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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