title: High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells.
abstract: BACKGROUND
  Pseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated.
  
  
  METHODS
  Cell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated.
  
  
  RESULTS
  The strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth.
  
  
  CONCLUSIONS
  PFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs.
authors: Department of Medical Pharmacy, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan. sato-y@yasuda-u.ac.jp.
authors: Yuichiro
authors: Y
authors: Sato
authors: Department of Life Sciences, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan.
authors: Takanori
authors: T
authors: Kubo
authors: Department of Medical Pharmacy, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan.
authors: Kinjiro
authors: K
authors: Morimoto
authors: Department of Life Sciences, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan.|Present address; Division of Translational Research, National Cancer Center Research Institute, 6-5-1 Kashiwanoha, Kashiwa, Chiba, Japan.
authors: Kazuyoshi
authors: K
authors: Yanagihara
authors: Department of Life Sciences, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan.
authors: Toshio
authors: T
authors: Seyama
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D016023:Integrins / Q000096:biosynthesis* / Q000378:metabolism; D037601:Mannose-Binding Lectin / Q000008:administration & dosage* / Q000737:chemistry; D051379:Mice; D011551:Pseudomonas fluorescens / Q000737:chemistry; D011799:Quinazolines / Q000008:administration & dosage; D034741:RNA, Small Interfering; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: D016023:Integrins; D037601:Mannose-Binding Lectin; D011799:Quinazolines; D034741:RNA, Small Interfering; C512478:EGFR protein, human; D066246:ErbB Receptors; D000077156:Gefitinib