title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.
abstract: Polyphyllin I (PPI), a bioactive phytochemical extracted from the Rhizoma of Paris polyphylla, has been reported to exhibit anti-cancer activity. However, the detailed mechanism underlying this remains to be elucidated.
  
  
  
  Cell viability and cell cycle distribution were measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. The expression of enhancer of zeste homolog 2 (EZH2) mRNA was measured by quantitative real time PCR (qRT-PCR). Western blot analysis was performed to examine the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p65, DNA methyltransferase 1 (DNMT1) and EZH2. Exogenous expression of p65, DNMT1, and EZH2 were carried out by transient transfection assays. Promoter activity of EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. A xenografted tumor model in nude mice and bioluminescent imaging system were used to further test the effect of PPI in vivo.
  
  
  
  We showed that PPI significantly inhibited growth and induced cell cycle arrest of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Mechanistically, we found that PPI increased the phosphorylation of SAPK/JNK, reduced protein expression of p65 and DNMT1. The inhibitor of SAPK/JNK (SP600125) blocked the PPI-inhibited p65 and DNMT1 protein expression. Interestingly, exogenously expressed p65 overcame PPI-inhibited protein expression of DNMT1. Moreover, PPI reduced EZH2 protein, mRNA, and promoter activity; overexpression of EZH2 resisted the PPI-inhibited cell growth, and intriguingly, negative feedback regulation of SAPK/JNK signaling. Finally, exogenous expression of DNMT1 antagonized the PPI-suppressed EZH2 protein expression. Consistent with this, PPI inhibited tumor growth, protein expression levels of p65, DNMT1 and EZH2, and increased phosphorylation of SAPK/JNK in vivo.
  
  
  
  Our results show that PPI inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of p65 and DNMT1 protein levels, subsequently; this results in the reduction of EZH2 gene expression. The interactions among p65, DNMT1 and EZH2, and feedback regulation of SAPK/JNK by EZH2 converge on the overall responses of PPI. This study reveals a novel mechanism for regulating EZH2 gene in response to PPI and suggests a new strategy for NSCLC associated therapy.
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies of TCM, Guangdong Provincial Hospital of Chinese Medicine, No. 111, Dade Road, Guangzhou, Guangdong Province, 510120, China.|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, China.
authors.forename: Longmei
authors.initials: L
authors.lastname: Li
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies of TCM, Guangdong Provincial Hospital of Chinese Medicine, No. 111, Dade Road, Guangzhou, Guangdong Province, 510120, China.
authors.forename: JingJing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies of TCM, Guangdong Provincial Hospital of Chinese Medicine, No. 111, Dade Road, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies of TCM, Guangdong Provincial Hospital of Chinese Medicine, No. 111, Dade Road, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
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, China. wwanyin@126.com.
authors.forename: WanYin
authors.initials: W
authors.lastname: Wu
authors.affiliation: Laboratory of Tumor Molecular Biology and Targeted Therapies of TCM, Guangdong Provincial Hospital of Chinese Medicine, No. 111, Dade Road, Guangzhou, Guangdong Province, 510120, China. hann2012@outlook.com.|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, China. hann2012@outlook.com.
authors.forename: Swei Sunny
authors.identifier: 0000-0003-2030-5285
authors.initials: SS
authors.lastname: Hann
mesh_terms: D000072283:A549 Cells; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000076123:DNA (Cytosine-5-)-Methyltransferase 1 / Q000378:metabolism*; D004144:Diosgenin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D015536:Down-Regulation; D000071221:Enhancer of Zeste Homolog 2 Protein / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D010766:Phosphorylation; D051996:Transcription Factor RelA / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D051996:Transcription Factor RelA; C556217:polyphyllin I; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; C000619380:DNMT1 protein, human; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D048670:MAP Kinase Kinase 4; D004144:Diosgenin
keywords: DNMT1; EZH2; NF-kB/p65; NSCLC; PPI; SAPK/JNK