title: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells.
abstract: BACKGROUND
  Ursolic acid (UA), a natural pentacyclic triterpenoid, exerts anti-tumor effects in various cancer types including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying this remain largely unknown.
  
  
  METHODS
  Cell viability and cell cycle were examined by MTT and Flow cytometry assays. Western blot analysis was performed to measure the phosphorylation and protein expression of p38 MAPK, insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and forkhead box O3A (FOXO3a). Quantitative real-time PCR (qRT-PCR) was used to examine the mRNA levels of IGFBP1 gene. Small interfering RNAs (siRNAs) method was used to knockdown IGFBP1 gene. Exogenous expressions of IGFBP1 and FOXO3a were carried out by transient transfection assays. IGFBP1 promoter activity was measured by Secrete-Pair™ Dual Luminescence Assay Kit . In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings in vitro.
  
  
  RESULTS
  We showed that UA stimulated phosphorylation of p38 MAPK. In addition, UA increased the protein, mRNA levels, and promoter activity of IGFBP1, which was abrogated by the specific inhibitor of p38 MAPK (SB203580). Intriguingly, we showed that UA increased the expression of FOXO3a and that overexpressed FOXO3a enhanced phosphorylation of p38 MAPK, all of which were not observed in cells silencing of endogenous IGFBP1 gene. Moreover, exogenous expressed IGFBP1 strengthened UA-induced phosphorylation of p38 MAPK and FOXO3a protein expression, and more importantly, restored the effect of UA-inhibited growth in cells silencing of endogenous IGFBP1 gene. Consistent with these, UA suppressed tumor growth and increased phosphorylation of p38 MAPK, protein expressions of IGFBP1 and FOXO3a in vivo.
  
  
  CONCLUSIONS
  Collectively, our results show that UA inhibits growth of HCC cells through p38 MAPK-mediated induction of IGFBP1 and FOXO3a expression. The interactions between IGFBP1 and FOXO3a, and feedback regulatory loop of p38 MAPK by IGFBP1 and FOXO3a resulting in reciprocal pathways, contribute to the overall effects of UA. This in vitro and in vivo study corroborates a potential novel mechanism by which UA controls HCC growth and implies that the rational targeting IGFBP1 and FOXO3a can be potential for the therapeutic strategy against HCC.
authors.affiliation: Laboratory of Tumor Biology and Target Therapy, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Li Jun
authors.initials: LJ
authors.lastname: Yang
authors.affiliation: Laboratory of Tumor Biology and Target Therapy, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Laboratory of Tumor Biology and Target Therapy, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Laboratory of Tumor Biology and Target Therapy, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Laboratory of Tumor Biology and Target Therapy, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.affiliation: Department of Radiation Therapy, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China.
authors.forename: Zhenhui
authors.initials: Z
authors.lastname: Dai
authors.affiliation: Laboratory of Tumor Biology and Target Therapy, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. hann2012@outlook.com.|No. 55, Neihuan West Road, Higher Education Mega Center, Panyu District, Guangzhou, Guangdong Province, 510006, P. R. China. hann2012@outlook.com.
authors.forename: Swei Sunny
authors.initials: SS
authors.lastname: Hann
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000008:administration & dosage* / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000071316:Forkhead Box Protein O3 / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D015398:Signal Transduction / Q000187:drug effects; D014315:Triterpenes / Q000008:administration & dosage* / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays; D000097245:Ursolic Acid
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D000071316:Forkhead Box Protein O3; D018970:Insulin-Like Growth Factor Binding Protein 1; D014315:Triterpenes; D000097245:Ursolic Acid; C499517:FOXO3 protein, human; C032231:IGFBP1 protein, human
keywords: FOXO3a; HCC; IGFBP1; UA; p38 MAPK