Combination therapy targeting integrins reduces glioblastoma tumor growth through antiangiogenic and direct antitumor activity and leads to activation of the pro-proliferative prolactin pathway

Leticia Oliveira-Ferrer,Jasmin Wellbrock,Jessica Hauschild,Marianne Klokow,Gunter Schuch,Carsten Bokemeyer,Eva Maria Murga Penas,Walter Fiedler,Udo Bartsch

doi:10.1186/1476-4598-12-144

Abstract

BackgroundTumors may develop resistance to specific angiogenic inhibitors via activation of alternative pathways. Therefore, multiple angiogenic pathways should be targeted to achieve significant angiogenic blockade. In this study we investigated the effects of a combined application of the angiogenic inhibitors endostatin and tumstatin in a model of human glioblastoma multiforme.ResultsInhibitors released by stably transfected porcine aortic endothelial cells (PAE) showed anti-angiogenic activity in proliferation and wound-healing assays with endothelial cells (EC). Interestingly, combination of endostatin and tumstatin (ES + Tum) also reduced proliferation of glioma cells and additionally induced morphological changes and apoptosis in vitro. Microencapsulated PAE-cells producing these inhibitors were applied for local therapy in a subcutaneous glioblastoma model. When endostatin or tumstatin were applied separately, in vivo tumor growth was inhibited by 58% and 50%, respectively. Combined application of ES + Tum, in comparison, resulted in a significantly more pronounced inhibition of tumor growth (83%). cDNA microarrays of tumors treated with ES + Tum revealed an up-regulation of prolactin receptor (PRLR). ES + Tum-induced up-regulation of PRLR in glioma cells was also found in in vitro. Moreover, exogenous PRLR overexpression in vitro led to up-regulation of its ligand prolactin and increased proliferation suggesting a functional autocrine growth loop in these cells.ConclusionOur data indicate that integrin-targeting factors endostatin and tumstatin act additively by inhibiting glioblastoma growth via reduction of vessel density but also directly by affecting proliferation and viability of tumor cells. Treatment with the ES + Tum-combination activates the PRLR pro-proliferative pathway in glioblastoma. Future work will show whether the prolactin signaling pathway represents an additional target to improve therapeutic strategies in this entity.

Highlights

Tumors may develop resistance to specific angiogenic inhibitors via activation of alternative pathways
We have recently shown that cilengitide inhibits integrin-dependent signaling and induces apoptosis in endothelial and in glioma cells thereby explaining the profound activity of integrin inhibitors in this disease [17]
After cell encapsulation [22,23], cells in the alginate microbeads were cultured for several weeks, and the conditioned medium (CM) analysed by Western blot after different culture periods to confirm continuous release of angiogenic inhibitors (Figure 1B)

Summary

Introduction

Tumors may develop resistance to specific angiogenic inhibitors via activation of alternative pathways. Human glioblastoma multiforme (GBM) is the most common and malignant type of brain tumors. We have recently shown that cilengitide inhibits integrin-dependent signaling and induces apoptosis in endothelial and in glioma cells thereby explaining the profound activity of integrin inhibitors in this disease [17]. These data suggest that anti-angiogenic molecules directed towards integrins may have a multi-targeting effect on both endothelial and glioma cells

Methods

Results

Discussion

Conclusion