Requirement for ERK activation in acetone extract identified from Bupleurum scorzonerifolium induced A549 tumor cell apoptosis and keratin 8 phosphorylation.
We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (AE-BS) 60 microg/ml has anti-proliferation activity and apoptosis effects to A549 human lung cancer cells. They can also cause tumor cell arrest in G2/M phase. To better understand its target protein in A549 cell, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry were applied. The modification of keratin 8 was identified. By immunoblot, the expression of phosphorylated keratin 8 at Ser-73 was increased from 2.0 to 3.0-fold after AE-BS treatment 24 to 48 hr respectively as compared with untreated A549 control cells. Furthermore, the A549 cells were pretreated with 50 microM PD98059, a specific inhibitor of the upstream regulator of ERK1/2, or with the p38 kinase inhibitor 20 microM SB203580 or JNK inhibitor 20 microM SP600125 for 30 min, followed by 24 h of incubation with AE-BS, PD98059 can inhibit K8-Ser-73 hyperphosphorylation and prevented cell apoptosis which was induced by AE-BS significantly. By immunoblot, AE-BS also can induce ERK 1/2 phosphorylation. In conclusion, our data indicate that the AE-BS induced tumor apoptosis in A549 cells was related to ERK 1/2 activation. The molecular mechanism of hyperphosphorylation of K8 on Ser-73 was associated with ERK 1/2 activation rather than JNK and p38 kinase. The apoptosis induced by AE-BS may be related to K8 phosphorylation.
Chen
Yi-Lin
YL
Graduate Institution of Medical Science, Tzu-chi University, Hualian, Taiwan.
Lin
Shinn-Zong
SZ
Chang
Wen-Liang
WL
Cheng
Yeung-Leung
YL
Harn
Horng-Jyh
HJ
D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D029972:Bupleurum / Q000737:chemistry*; D002853:Chromatography, Liquid; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D053533:Keratin-8; D007633:Keratins / Q000378:metabolism*; D013058:Mass Spectrometry; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000873:Anthracenes; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; C426203:KRT8 protein, human; D053533:Keratin-8; D011725:Pyridines; C432165:pyrazolanthrone; D007633:Keratins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one