title: Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells.
abstract: BACKGROUND
  Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown.
  
  
  METHODS
  Cell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)-methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays.
  
  
  RESULTS
  We showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth.
  
  
  CONCLUSIONS
  Our results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells.
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, 510120, Guangdong Province, P. R. China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, 510120, Guangdong Province, P. R. China.
authors.forename: Shunyu
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, 510120, Guangdong Province, P. R. China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, 510120, Guangdong Province, P. R. China.
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, 510120, Guangdong Province, P. R. China.
authors.forename: YuQin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, 510120, Guangdong Province, P. R. China.
authors.forename: LiJun
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Medical Oncology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, P. R. China.
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Medical Oncology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, P. R. China.
authors.forename: Liuning
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Medical Oncology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, P. R. China.
authors.forename: WanYin
authors.initials: W
authors.lastname: Wu
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, 510120, Guangdong Province, P. R. China. swhan2010@live.com.|, No. 55, Neihuan West Road, Higher Education Mega Center, Panyu District, Guangzhou, Guangdong Province, 510006, P. R. China. swhan2010@live.com.
authors.forename: Swei Sunny
authors.initials: SS
authors.lastname: Hann
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy*; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004354:Drug Screening Assays, Antitumor; D000071221:Enhancer of Zeste Homolog 2 Protein; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D014315:Triterpenes / Q000494:pharmacology*; D000097245:Ursolic Acid
chemical_list: D000970:Antineoplastic Agents; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000071221:Enhancer of Zeste Homolog 2 Protein; D020928:Mitogen-Activated Protein Kinases; D063151:Polycomb Repressive Complex 2; D014315:Triterpenes; D000097245:Ursolic Acid; C000619380:DNMT1 protein, human; C498409:EZH2 protein, human