title: Andrographolide decreased VEGFD expression in hepatoma cancer cells by inducing ubiquitin/proteasome-mediated cFos protein degradation.
abstract: BACKGROUND
  Andrographolide (Andro) is reported to inhibit hepatoma tumor growth in our previous studies. This study aims to further search the critical signals involved in such Andro-induced inhibition.
  
  
  METHODS
  The anti-tumor effect of Andro was evaluated in vivo. Cancer PathwayFinder RT(2) Profiler™ PCR array was used to find the altered genes. Real-time PCR was used to detect the mRNA expression. Protein expression was detected by Western-blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. Activator protein-1 (AP-1) transcriptional activity was detected by luciferase reporter assay.
  
  
  RESULTS
  Andro (10mg/kg) inhibited hepatoma tumor growth in vivo. The expression of four genes decreased in Andro-treated tumor tissues. Among which, vascular endothelial growth factor (VEGFD) was the highest decreased gene. The decreased VEGFD expression was further confirmed by real-time PCR and immunohistochemical staining assay. Andro decreased VEGFD mRNA and protein expression in hepatoma Hep3B and HepG2 cells. Andro also decreased VEGFD amount in Hep3B cell supernatant. Andro decreased cFos protein expression and its translocation into nucleus, and also reduced AP-1 luciferase activity. Further results showed that Andro induced polyubiquitination of cFos. Proteasome inhibitor MG132 reversed the decreased expression of cFos protein, and the decreased mRNA and protein expression of VEGFD. SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), reversed the decreased expression of cFos and VEGFD induced by Andro.
  
  
  CONCLUSIONS
  Andro decreased VEGFD expression in hepatoma cancer cells via inducing c-fos protein degradation, which will contribute to its anti-cancer activity, and JNK plays some roles in regulating this process.
authors.affiliation: Shanghai Key Laboratory of Complex Prescription and The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address: lichenyue1307@126.com.
authors.forename: Lili
authors.initials: L
authors.lastname: Ji
authors.affiliation: Shanghai Key Laboratory of Complex Prescription and The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Shanghai Key Laboratory of Complex Prescription and The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Shi
authors.affiliation: Shanghai Key Laboratory of Complex Prescription and The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors.forename: Yinjie
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Shanghai Key Laboratory of Complex Prescription and The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Lu
authors.affiliation: Shanghai Key Laboratory of Complex Prescription and The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address: ztwang@shutcm.edu.cn.
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004224:Diterpenes / Q000494:pharmacology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*; D042643:Vascular Endothelial Growth Factor D / Q000235:genetics*
chemical_list: D004224:Diterpenes; D016760:Proto-Oncogene Proteins c-fos; D014452:Ubiquitins; D042643:Vascular Endothelial Growth Factor D; C030419:andrographolide; D048031:JNK Mitogen-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex
keywords: Andrographolide; Hepatoma cancer; Protein degradation; VEGFD; cFos