title: Apicularen A induces cell death through Fas ligand up-regulation and microtubule disruption by tubulin down-regulation in HM7 human colon cancer cells.
abstract: OBJECTIVE
  Apicularen A has been shown to cause growth inhibition and apoptosis in several cancer cell lines. However, the mechanisms of apicularen A-induced cell death and in vivo effects remain unclear. In this study, we investigated the molecular mechanisms of apicularen A-induced cell death in HM7 human colon cancer cells in vitro and anticancer activity in vivo.
  
  
  METHODS
  We tested cytotoxicity with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis with DNA fragmentation assay, mitochondrial membrane potential, and cell cycle with fluorescence-activated cell sorting. Caspase activation was done by fluorometry. Alterations of microtubule structure, tubulin protein, and mRNA level were assessed by immunofluorescence, Western blot, and reverse transcription-PCR. In vivo studies were assessed using nude mice tumor cell growth in xenograft model and liver colonization assay.
  
  
  RESULTS
  Apicularen A treatment of HM7 cells inhibited cell growth and this inhibition was partially rescued by z-VAD-fmk. Apicularen A caused accumulation of sub-G(1)-G(0), DNA fragmentation, Fas ligand induction, and activation of caspase-8 and caspase-3, but mitochondrial membrane potential was not changed. Furthermore, beta-tubulin protein and mRNA were decreased by apicularen A, but in vitro polymerization of tubulin was not affected. Concurrently, apicularen A-treated cell showed disruption of microtubule architecture. In in vivo studies, apicularen A reduced tumor volume by approximately 72% at the end of a 15-day treatment. Moreover, apicularen A reduced liver colonization as much as 95.6% (50 microg/kg/d).
  
  
  CONCLUSIONS
  Apicularen A induces cell death of HM7 cells through up-regulating Fas ligand and disruption of microtubule architecture with down-regulation of tubulin level. These findings indicate that apicularen A is a promising new microtubule-targeting compound.
authors.affiliation: Department of Biochemistry, College of Medicine, Chungnam National University, Daejeon, Republic of Korea.
authors.forename: Jong-Seok
authors.initials: JS
authors.lastname: Kim
authors.forename: Young-Chul
authors.initials: YC
authors.lastname: Lee
authors.forename: Ho-Tak
authors.initials: HT
authors.lastname: Nam
authors.forename: Ge
authors.initials: G
authors.lastname: Li
authors.forename: Eun-Jin
authors.initials: EJ
authors.lastname: Yun
authors.forename: Kyoung-Sub
authors.initials: KS
authors.lastname: Song
authors.forename: Kang-Sik
authors.initials: KS
authors.lastname: Seo
authors.forename: Ji-Hoon
authors.initials: JH
authors.lastname: Park
authors.forename: Jong-Woong
authors.initials: JW
authors.lastname: Ahn
authors.forename: Okpyo
authors.initials: O
authors.lastname: Zee
authors.forename: Jong-Il
authors.initials: JI
authors.lastname: Park
authors.forename: Wan-Hee
authors.initials: WH
authors.lastname: Yoon
authors.forename: Kyu
authors.initials: K
authors.lastname: Lim
authors.forename: Byung-Doo
authors.initials: BD
authors.lastname: Hwang
mesh_terms: D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D053938:DNA Fragmentation; D004789:Enzyme Activation; D053222:Fas Ligand Protein / Q000378:metabolism*; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008099:Liver / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008819:Mice, Nude; D008870:Microtubules / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D053222:Fas Ligand Protein; D014404:Tubulin; C117813:apicularen A; D053148:Caspase 3; D053181:Caspase 8