title: Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells.
abstract: BACKGROUND
  Toll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB).
  
  
  METHODS
  TLR3 expression in human NB samples was examined by immunohistochemical analysis. Quantitative RT-PCR and western blot was used to determine TLR3 expression in three human NB cell lines. The effect of TLR3 agonist, polyinosinic-polycytidylic acid (poly(I:C)), on the growth of human NB cells was evaluated by WST-1 cell proliferation assay, flow cytometry analysis, and immunoblot analysis. Blockade of TLR3 signaling was achieved using TLR3 neutralizing antibody, small interference RNA, and 2-aminopurine (2-AP), an inhibitor of protein kinase R (PKR), an interferon-induced, double-stranded RNA-activated protein kinase.
  
  
  RESULTS
  In immunohistochemical studies, TLR3 mainly expressed in the cytoplasm of ganglion cells and in some neuroblastic cells, but not in the stromal cells in human NB tissues. Among three human NB cell lines analyzed, TLR3 was significantly up-regulated in SK-N-AS cells at mRNA and protein level compared with other two low TLR3- expressing NB cells. Treatment with poly(I:C) elicited significant growth inhibition and apoptosis only in high TLR3-expressing SK-N-AS cells, but not in low TLR3-expressing SK-N-FI and SK-N-DZ cells. Moreover, poly(I:C) treatment significantly stimulated the activities of PKR, interferon regulatory factor 3 (IRF-3) and caspase-3 in SK-N-AS cells. Application of TLR3 neutralizing antibody or small interference RNA (siRNA) reduced the poly(I:C)-induced inhibition of cell proliferation and apoptosis in SK-N-AS cells. On the contrary, ectopic TLR3 expression enhanced the sensitivity of low TLR3-expressing NB cells to poly(I:C). Finally, application of 2-AP attenuated the poly(I:C)-induced IRF-3 and caspase-3 activation in SK-N-AS cells.
  
  
  CONCLUSIONS
  The present study demonstrates that TLR3 is expressed in a subset of NB cells. Besides, TLR3/PKR/IRF-3/capase-3 pathway is implicated in the selective cytotoxicity of TLR3 agonist towards high TLR3-expressing NB cells.
authors.affiliation: Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
authors.forename: Jiin-Haur
authors.initials: JH
authors.lastname: Chuang
authors.forename: Hui-Ching
authors.initials: HC
authors.lastname: Chuang
authors.forename: Chao-Cheng
authors.initials: CC
authors.lastname: Huang
authors.forename: Chia-Ling
authors.initials: CL
authors.lastname: Wu
authors.forename: Yung-Ying
authors.initials: YY
authors.lastname: Du
authors.forename: Mei-Lang
authors.initials: ML
authors.lastname: Kung
authors.forename: Chih-Hao
authors.initials: CH
authors.lastname: Chen
authors.forename: San-Cher
authors.initials: SC
authors.lastname: Chen
authors.forename: Ming-Hong
authors.initials: MH
authors.lastname: Tai
mesh_terms: D015075:2-Aminopurine / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D009447:Neuroblastoma / Q000378:metabolism*; D011070:Poly I-C / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D051196:Toll-Like Receptor 3 / Q000819:agonists / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D015075:2-Aminopurine; D011070:Poly I-C