title: Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.
abstract: OBJECTIVE
  The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4).
  
  
  METHODS
  hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay.
  
  
  RESULTS
  Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity.
  
  
  CONCLUSIONS
  Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.
authors.affiliation: R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine.
authors.forename: O L
authors.initials: OL
authors.lastname: Gerashchenko
authors.forename: E V
authors.initials: EV
authors.lastname: Zhuravel
authors.forename: O V
authors.initials: OV
authors.lastname: Skachkova
authors.forename: N N
authors.initials: NN
authors.lastname: Khranovska
authors.forename: V V
authors.initials: VV
authors.lastname: Filonenko
authors.forename: P V
authors.initials: PV
authors.lastname: Pogrebnoy
authors.forename: M A
authors.initials: MA
authors.lastname: Soldatkina
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014410:Tumor Stem Cell Assay; D023083:beta-Defensins / Q000235:genetics* / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C106641:DEFB4A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins