Anti-angiogenesis and anti-tumor activity of recombinant anginex☆

https://doi.org/10.1016/j.bbrc.2006.08.154Get rights and content

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.

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Materials and methods

Expression and isolation of recombinant anginex. Expression and isolation of recombinant anginex was performed as described previously [6]. In short, 1L BMGY was inoculated with a 50 ml overnight culture of P. pastoris strain GS115 (Invitrogen) transformed with pPICZα-A-anginex and cultured for 24 h at 30 °C with shaking. These cells were harvested and resuspended in 2 L BMMY in order to induce expression. After 72 h, supernatant was collected and concentrated using Centricon-plus 80 biomax5

Results and discussion

Tumor growth is strongly dependent on the formation of new blood vessels, i.e., angiogenesis [10]. The discovery of angiogenesis inhibitors has confirmed the feasibility of angiogenesis inhibition for cancer treatment as postulated by Dr. Folkman in the early 1970s [11]. Unfortunately, the development of several promising angiogenesis inhibitors has been hampered by difficulties in production and activity [1]. Some of these limitations might be solved using a gene therapy approach whereby the

Acknowledgments

This study was supported by a generous research grant from the Technology Foundation STW, applied science division of NOW, and the technology program of the Ministry of Economic Affairs (MPG 5465 to A.W.G.).

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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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