doi:10.1016/j.bbrc.2006.08.154
Copyright © 2006 Elsevier Inc. All rights reserved.
Anti-angiogenesis and anti-tumor activity of recombinant anginex
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Ricardo J.M.G.E. Brandwijka, Ruud P.M. Dingsb, Edith van der Lindena, Kevin H. Mayob, Victor L.J.L. Thijssena and Arjan W. Griffioena, 
, 
aAngiogenesis Laboratory, Research Institute Growth and Development (GROW), Department of Pathology, Maastricht University and University Hospital, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
bDepartment of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA
Received 22 August 2006.
Available online 5 September 2006.
Abstract
Anginex, a synthetic 33-mer angiostatic peptide, specifically inhibits vascular endothelial cell proliferation and migration along with induction of apoptosis in endothelial cells. Here we report on the in vivo characterization of recombinant anginex and use of the artificial anginex gene for gene therapy approaches. Tumor growth of human MA148 ovarian carcinoma in athymic mice was inhibited by 80% when treated with recombinant anginex. Histological analysis of the tumors showed an approximate 2.5-fold reduction of microvessel density, suggesting that angiogenesis inhibition is the cause of the anti-tumor effect. Furthermore, there was a significant correlation between the gene expression patterns of 16 angiogenesis-related factors after treatment with both recombinant and synthetic anginex. To validate the applicability of the anginex gene for gene therapy, stable transfectants of murine B16F10 melanoma cells expressing recombinant anginex were made. Supernatants of these cells inhibited endothelial cell proliferation in vitro. Furthermore, after subcutaneous injection of these cells in C57BL/6 mice, an extensive delay in tumor growth was observed. These data show that the artificial anginex gene can be used to produce a recombinant protein with similar activity as its synthetic counterpart and that the gene can be applied in gene therapy approaches for cancer treatment.
Keywords: Recombinant anginex; Gene therapy; Tumor inhibition; Angiogenesis; Endothelial cells
Fig. 1. Anti-tumor activity of recombinant anginex. (A) Tumor growth curves of the xenograft MA148 ovarian carcinoma mouse model. Treatment was started in palpable tumors by IP injection every three days (10 mg anginex/kg and equivalent dose for recombinant anginex). *p < 0.01 treatment vs. control. (B) Immunohistochemical stainings of endothelial cells in the different tumors. (C) Quantification of microvessel density in the different treatment groups. *p < 0.03 treatment vs. control.
Fig. 2. Effect of treatment on angiogenesis expression profile. Scatter plots of response in gene expression after treatment with recombinant or synthetic anginex. A total of 16 angiogenesis factors was included in the analysis. By using species-specific primers a distinction could be made between the response in the tumor compartment (A, human) and the response in the vascular compartment (B, mouse) of the xenograft tumor tissues.
Fig. 3. Anti-tumor activity of the artificial recombinant anginex gene. (A) Agarose gel electrophoresis showing PCR genotyping of B16F10 cells stably transfected with pcDNA3.1 and pcDNA3.1-anginex. bp = basepairs (B) proliferation assay on HUVEC with culture supernatants of transfected cell lines. Proliferation was determined using the tritium thymidine incorporation assay. *p < 0.01 treatment vs. control. (C) Tumor growth curves of B16F10 melanoma cells stably transfected with the anginex gene and B16F10 treated with synthetic anginex or saline. Dotted line represents mice that had no visible tumor at day 16 and therefore were not sacrificed until day 40. (D) Expression level of recombinant anginex in excised tumors as determined by real-time RT-PCR with anginex specific primers.
Abbreviations: CAM, chorioallantoic membrane; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; PF4, platelet factor 4; r-anginex, recombinant anginex; IL8, interleukin 8.
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