ERK signaling pathway is involved in p15INK4b/p16INK4a expression and HepG2 growth inhibition triggered by TPA and Saikosaponin a.
The signal pathway mediating induction of p15(INK4b) and p16(INK4a) during HepG2 growth inhibition triggered by the phorbol ester tumor promoter TPA (12-O-tetradecanoylphorbol 13-acetate) and the Chinese herb Saikosaponin a was investigated. Western blot of three activated forms of mitogen-activated protein kinase (MAPK) (p-ERK, p-JNK and p-p38) demonstrated that phosphorylation of ERK is dramatically induced (11.6-fold ) by TPA during 15 min to 1 h and significantly induced (2.5-fold) by Saikosaponin alpha at 30 min, whereas phosphorylation of JNK was induced only by TPA during 30 min to 1 h. Phosphorylation of p38 was not induced by either drug. During this period, phosphorylation of one of the downstream transcriptional factors of MAPK cascade, ATF2, was 3.2- and 2.0-fold induced by TPA and Saikosaponin a, respectively, whereas that of another transcriptional factor, c-jun, was induced by TPA only. On the other hand, expressions of proto-oncogene c-jun, junB and c-fos were induced by TPA and Saikosaponin a during 30 min to 6 h of treatment. Pretreatment of 20 microg/ml PD98059, an inhibitor of MEK which is the upstream kinase of ERK, prevents the TPA- and Saikosaponin a-triggered HepG2 growth inhibition by 50 and 30%, respectively, accompanied by a 50 - 85% decrease of the p15(INK4b)/p16(INK4a) RNAs and proteins induced by both drugs. Inductions of c-fos RNA by both drugs and c-jun phosphorylation by TPA were also significantly reduced by PD98059 pretreatment. In addition, AP-1 DNA-binding assay using nonisotopic capillary electrophoresis and laser-induced fluorescence (CE/LIF) demonstrated that the AP-1-related DNA-binding activity was significantly induced by TPA and Saikosaponin a, which can be reduced by PD98059 pretreatment. These results suggested that activation of ERK together with its downstream transcriptional machinery mediated p15(INK4b) and p16(INK4a) expression that led to HepG2 growth inhibition.
Wen-Sheng
Wu
W
Department of Medical Technology, TZU CHI University, Hualien, Taiwan. wuws@mail.tcu.edu.tw
D051698:Activating Transcription Factor 2; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis* / Q000235:genetics; D003864:Depression, Chemical; D019075:Electrophoresis, Capillary; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016762:Genes, fos / Q000187:drug effects; D016758:Genes, jun / Q000187:drug effects; D019942:Genes, p16 / Q000187:drug effects*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D008113:Liver Neoplasms / Q000473:pathology; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics; D012503:Saponins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D048051:p38 Mitogen-Activated Protein Kinases
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D005419:Flavonoids; C000715470:MAS1 protein, human; D009363:Neoplasm Proteins; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012503:Saponins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D009828:Oleanolic Acid; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; C025759:saikosaponin D