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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Activation of Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 via Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Revealed a Isochaihulactone-Triggered Apoptotic Pathway in Human Lung Cancer A549 Cells

Graduate Institute of Biotechnology (Y.-L.C.) and Department of Applied Animal Science (C.-C.L.), National Ilan University, Ilan, Taiwan, Republic of China; Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, Republic of China (P.-C.L.); Institute of Medical Sciences, Buddhist Tzu-Chi University, Hualien, Taiwan, Republic of China (S.-P.C.); School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, Republic of China (N.-M.T.); Division of Thoracic Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China (Y.-L.C.); School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, Republic of China (W.-L.C.); Center for Neuropsychiatry (S.-Z.L.) and Department of Pathology (H.-J.H.), China Medical University and Hospital, Taichung, Taiwan, Republic of China; and Department of Pathology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, Republic of China (H.-J.H.)

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.

Address correspondence to: Dr. Horng-Jyh Harn, Neuro-Medical Scientific Center, Department of Pathology, Tzu-Chi General Hospital, 707, Section 3, Chung-Yang Rd., 970 Hualien, Taiwan, R.O.C. E-mail address: duke_harn{at}yahoo.com.tw