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.
- DOI - The Journal of pharmacology and experimental therapeutics (DOI)
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JournalThe Journal of pharmacology and experimental therapeutics
J Pharmacol Exp Ther (0022-3565)
The Journal of pharmacology and experimental therapeutics
Graduate Institute of Biotechnology, National Ilan University, Ilan, Taiwan, Republic of China.
J Pharmacol Exp Ther. 2007 Nov;323(2):746-56
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