title: Restoration of klotho expression induces apoptosis and autophagy in hepatocellular carcinoma cells.
abstract: OBJECTIVE
  Klotho has been identified as a tumor suppressor in several human malignancies including hepatocellular carcinoma (HCC). However, the signaling pathways involved in the tumor suppressive role of klotho in HCC have not been reported. Here, we investigated the role of klotho in HCC cell proliferation, apoptosis, autophagy, and invasion, as well as its associated signal transduction pathways.
  
  
  METHODS
  Restoration of klotho gene expression was established by delivering a klotho gene expression vector into the human HCC cell lines HepG2 and MHCC-97-H. Cell viability was measured using a cell counting (CCK-8) assay and apoptosis was analyzed through flow cytometry. Autophagy was measured via LC3-I and LC3-II protein expression levels and tumor cell invasion was assessed using a Matrigel invasion chamber assay. Expression and phosphorylation of several apoptosis and survival related proteins were assessed using Western blot assays.
  
  
  RESULTS
  Exogenous klotho gene expression significantly inhibited HCC cell proliferation, induced HCC cell apoptosis, increased LC3-I and LC3-II protein expression in HCC cells, and decreased migration of HCC cells in a Matrigel invasion chamber assay. Exogenous klotho gene expression also down-regulated the phosphorylation levels of the IGF-1 receptor, and the downstream Akt, ERK, and p70S6K proteins. Both apoptosis and autophagy inhibitors decreased klotho-induced apoptosis and autophagy.
  
  
  CONCLUSIONS
  Klotho is a tumor suppressor that, through the regulation of IGF-1R phosphorylation and subsequent activation of downstream Akt-p70S6K and ERK signaling, regulates HCC tumor cell proliferation, apoptosis, autophagy and invasion.
authors.affiliation: Departemt of Geriatric Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, China.
authors.forename: Guoshun
authors.initials: G
authors.lastname: Shu
authors.forename: Biao
authors.initials: B
authors.lastname: Xie
authors.forename: Feng
authors.initials: F
authors.lastname: Ren
authors.forename: Dong-cai
authors.initials: DC
authors.lastname: Liu
authors.forename: Jiapeng
authors.initials: J
authors.lastname: Zhou
authors.forename: Qinglong
authors.initials: Q
authors.lastname: Li
authors.forename: Jinhui
authors.initials: J
authors.lastname: Chen
authors.forename: Lianwen
authors.initials: L
authors.lastname: Yuan
authors.forename: Jianping
authors.initials: J
authors.lastname: Zhou
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001343:Autophagy / Q000187:drug effects / Q000235:genetics*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D000090265:Klotho Proteins; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014162:Transfection
chemical_list: D000590:Amino Acid Chloromethyl Ketones; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C025946:3-methyladenine; D017526:Receptor, IGF Type 1; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D048049:Extracellular Signal-Regulated MAP Kinases; D005966:Glucuronidase; D000090265:Klotho Proteins; D000225:Adenine