title: A recombinant human RNASET2 glycoprotein with antitumorigenic and antiangiogenic characteristics: expression, purification, and characterization.
abstract: BACKGROUND
  Human RNASET2 is a T2-RNase glycoprotein encoded by the RNASET2 gene, which is located on chromosome 6 (6q27). Deletion in 6q27 is associated with several human malignancies.
  
  
  METHODS
  A synthetic RNASET2 gene that was optimized for expression in the yeast Pichia pastoris was designed according to the cDNA sequence and was cloned under the control of the methanol-induced promoter fused to the alpha-mating secretion peptide. The recombinant protein was purified from the culture supernatant of transformed P. pastoris through an affinity Sepharose-concanavalin A column. Actin-binding activity was examined by membrane blotting using monoclonal mouse antiactin immunoglobulin M and by cross-linking in solution to G-actin using 1-[3-(dimethylamino)propyl]-3-ethyl-carboimide methiodide. The antiangiogenic activity of RNASET2 (from 0.5 microM to 10 microM) was assessed by a human umbilical vein endothelial (HUVE) cell assay in the presence of 1 microg/mL angiogenin, basic fibroblast growth factor (bFGF), or recombinant human vascular endothelial growth factor (VEGF). Cell colony formation was examined in human colon HT29 cancer cells to assess the antitumorigenic activity of RNASET2 or the enzymatic-inactivated RNASET2 (EI-RNASET2) (1 microM each). In an athymic mouse xenograft model, LS174T human cancer cells were injected subcutaneously. When tumors were palpable, the mice were treated for 3 weeks with RNASET2 (1 mg/kg), paclitaxel (10 mg/kg or 15 mg/kg), or a combination of the 2 drugs.
  
  
  RESULTS
  The recombinant RNASET2 was identified as a 27-kilodalton glycoprotein that possessed the ability to bind actin in vitro. RNASET2 significantly inhibited clonogenicity in HT29 cells. EI-RNASET2 produced a similar effect, suggesting that its antitumorigenic activity is unrelated to its RNase activity. In HUVE cells, RNASET2 inhibited angiogenin-, bFGF-, and VEGF-induced tube formation in a dose-dependent manner. In athymic mice, RNASET2 inhibited the development of an LS174T-derived xenograft by 40%. A synergistic effect was obtained with combined RNASET2 and paclitaxel treatments.
  
  
  CONCLUSIONS
  The current results suggested that RNASET2 represents a new class of antitumorigenic and antiangiogenic drugs, and the findings of this study emphasize the advantage of using agents like RNASET2 in combined therapy.
authors.affiliation: Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Jerusalem, Rehovot, Israel.
authors.forename: Patricia
authors.initials: P
authors.lastname: Smirnoff
authors.forename: Levava
authors.initials: L
authors.lastname: Roiz
authors.forename: Boaz
authors.initials: B
authors.lastname: Angelkovitch
authors.forename: Betty
authors.initials: B
authors.lastname: Schwartz
authors.forename: Oded
authors.initials: O
authors.lastname: Shoseyov
mesh_terms: D020533:Angiogenesis Inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001681:Biological Assay; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000187:drug effects; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D017239:Paclitaxel / Q000494:pharmacology; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012260:Ribonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D006023:Glycoproteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D012260:Ribonucleases; C425232:RNASET2 protein, human; D017239:Paclitaxel