title: Repression of phosphoinositide-dependent protein kinase 1 expression by ciglitazone via Egr-1 represents a new approach for inhibition of lung cancer cell growth.
abstract: BACKGROUND
  Peroxisome proliferator-activated receptors gamma (PPARγ) ligands have been shown to inhibit the growth of non-small cell lung cancer (NSCLC) cells. However, the mechanisms underlying this effect remain incompletely elucidated.
  
  
  METHODS
  Cell proliferation and apoptosis were measured by cell viability, MTT and caspase3/7 activity assays. Phosphorylation/protein expression and gene silence/overexpression of AMPKα, phosphoinositide-dependent protein kinase 1 (PDK1), Egr-1 and PPARγ were performed by Western blot and siRNA/transfection assays. Dual-Luciferase Reporter Kit was used to measure the PPAR response elements (PPRE) reporter and PDK1 promoter activities, and ChIP assay was used to detect the Egr-1 protein binding to the DNA site in the PDK1 gene promoter.
  
  
  RESULTS
  We found that ciglitazone, one synthetic PPARγ ligand, inhibited growth and induced apoptosis of NSCLC cells through decreased expression of PDK1, which was not blocked by GW9662 (a specific PPARγ antagonist). Overexpression of PDK1 overcame the effect of ciglitazone on cell growth and caspase 3/7 activity. Ciglitazone increased the phosphorylation of AMPKα and c-Jun N-terminal kinase (JNK), and the inhibitor of AMPK (compound C), but not JNK (SP600125), reversed the effect of ciglitazone on PDK1 protein expression. Ciglitazone reduced PDK1 gene promoter activity, which was not observed in cells exposed to compound C, but not silenced of PPARγ siRNA. Combination of ciglitazone and metformin further reduced PDK1 expression and promoter activity. Furthermore, we showed that ciglitazone induced the protein expression of Egr-1, which was not observed in cells silencing of AMPKα. Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth. On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity. ChIP assays demonstrated that ciglitazone induced Egr-1 protein bind to the specific DNA site in the PDK1 gene promoter.
  
  
  CONCLUSIONS
  Collectively, our results demonstrate that ciglitazone inhibits PDK1 expression through AMPKα-mediated induction of Egr-1 and Egr-1 binding to the specific DNA site in the PDK1 gene promoter, which is independent of PPARγ. Activation of AMPKα by metformin enhances the effect of ciglitazone. In turn, this leads to inhibition of NSCLC cell proliferation.
authors.affiliation: University of Guangzhou Traditional Chinese Medicine, Guangdong Academy of Traditional Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, Guangdong Province, China 510120. swhan2010@live.com.
authors.forename: Swei Sunny
authors.initials: SS
authors.lastname: Hann
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.forename: Shunyu
authors.initials: S
authors.lastname: Zhao
authors.forename: Jianping
authors.initials: J
authors.lastname: Chen
authors.forename: ZhiYu
authors.initials: Z
authors.lastname: Wang
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases / Q000096:biosynthesis* / Q000235:genetics; D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D051766:Early Growth Response Protein 1 / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008687:Metformin / Q000008:administration & dosage; D047495:PPAR gamma / Q000235:genetics; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D045162:Thiazolidinediones / Q000008:administration & dosage
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D047495:PPAR gamma; D045162:Thiazolidinediones; D008687:Metformin; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D055372:AMP-Activated Protein Kinases; C039671:ciglitazone