title: Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation.
abstract: BACKGROUND
  Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism.
  
  
  METHODS
  HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface beta1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined.
  
  
  RESULTS
  A low concentration of arecoline (<or= 100 microg/ml) caused cytoskeletal changes in HA22T/VGH cells, but not hepatocytes, and this was accompanied by decreased beta1-integrin expression and followed by apoptosis, indicating that HA22T/VGH cells undergo anoikis after arecoline treatment. IL-6 expression and phosphorylation of STAT3, which provides protection against anoikis, were inhibited and levels of downstream signaling proteins, including Bcl-XL and Bcl-2, were decreased, while Bax expression, mitochondrial cytochrome c release, and caspase-3 activity were increased. In addition, phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and of its upstream regulator, SHP2, was inhibited by arecoline treatment, while Rho/Rock activation was increased. Addition of the RhoA inhibitor attenuated the effects of arecoline.
  
  
  CONCLUSIONS
  This study demonstrated that arecoline induces anoikis of HA22T/VGH cells involving inhibition of STAT3 and increased RhoA/Rock activation and that the STAT3 and RhoA/Rock signaling pathways are connected.
authors.affiliation: Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Hsiao-Ling
authors.initials: HL
authors.lastname: Cheng
authors.forename: Shu-Jem
authors.initials: SJ
authors.lastname: Su
authors.forename: Li-Wen
authors.initials: LW
authors.lastname: Huang
authors.forename: Bau-Shan
authors.initials: BS
authors.lastname: Hsieh
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Hu
authors.forename: Thu-Ching
authors.initials: TC
authors.lastname: Hung
authors.forename: Kee-Lung
authors.initials: KL
authors.lastname: Chang
mesh_terms: D000818:Animals; D023102:Anoikis / Q000187:drug effects*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001115:Arecoline / Q000494:pharmacology*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D002469:Cell Separation; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D020287:In Situ Nick-End Labeling; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000187:drug effects / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D050796:STAT3 Transcription Factor; D001115:Arecoline; D020742:rhoA GTP-Binding Protein