Regulation of tumour-angiogenesis by protease inhibitors and receptor antagonists.
Introduction Angiogenesis, the growth of new blood vessels from the pre-existing vasculature, is a pre-requisite for tumour growth and metastasis. Tumour-angiogenesis is regulated by various pro- and anti-angiogenic factors released by both endothelial and tumour cells, as well as by the micro-environment. Numerous studies have implicated various systems in the acquisition of the angiogenic phenotype. The present study sought to investigate the role of the kallikrein-kinin system (KKS) in tumour-angiogenesis. The kallikreins consist of two serine proteases, plasma and tissue kallikrein (TK), involved in the release of kinin peptides by enzymatic cleavage of kininogens. Stimulation of the cognate bradykinin receptors (BKR), B1R and B2R, mediates the mitogenic and vasoactive properties of kinins. In addition, TK activates matrix metallo-proteinases (MMPs) involved in extracellular matrix (ECM) degradation. The expression profiles of TK and kinins have been found to be dys-regulated in numerous human cancers, and several studies have demonstrated the involvement of the KKS in growth and metastasis of prostate tumours. Further, previous in vitro models in our laboratory have established an association between the KKS and prostate tumour-angiogenesis. In those studies it was postulated that the up-regulated TK (produced by endothelial and tumour cells) stimulated endothelial cell proliferation. Thus, the aim of the present study was to define the effects of the KKS and seek a direct correlation with angiogenesis using in vitro models with tumour conditioned medium (CM), kinin receptor agonists and antagonists. Methods Ethical approval for this project was granted by the Biomedical Research Ethics Committee, University of KwaZulu-Natal (reference number BE152/08). Micro-vascular endothelial cells represent a suitable in vitro angiogenic model and dermal micro-vascular endothelial cells (dMVECs) were obtained commercially for this purpose. The tumour model used in this study was an immortalised prostate cancer (DU145) cell line. The CM model involves the treatment of one cell line with the metabolites of another. In the angiogenic model, dMVECs were exposed to increasing concentrations of DU145 CM. Stimulation was further augmented with BKR agonists. Specific BKR antagonists were used to test the specificity of stimulation. In addition, vascular endothelial growth factor (VEGF) was tested as a positive proliferation control. The potential of these agents to induce proliferation and migration was determined using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and a modified Boyden chamber assay, respectively. Previous studies investigating the pro-angiogenic effects of CM differed, in many respects, in terms of their models and methodologies. In an attempt to fully explore the pro-mitogenic effects of CM on endothelial cells, various modifications, as well as alternate endothelial and tumour cell types, were employed in the present study. The mitogenic and migratory effect of BKR agonists and antagonists on DU145 cells was also assessed. Further, the tumour model was expanded to investigate the autocrine potential of the KKS, by investigating the effect of DU145 CM on DU145 migration. Results In the angiogenic model, although the addition of DU145 CM elicited a statistically significant increase in micro-vascular endothelial cell proliferation, this increase was very small (<10%) and not dose-dependent. Pre-incubation of dMVECs with a B1R or B2R antagonist did not influence this small effect of CM on proliferation. In addition, neither B1R nor B2R agonists, at any concentration, produced any significant proliferative effect on endothelial cells. In contrast to these findings VEGF, a well-known mitogen, was able to stimulate proliferation of dMVECs. Migration assays revealed that DU145 CM failed to stimulate endothelial cell motility. Further, neither BKR agonist displayed any chemo-attractant potential in those assays. The most important finding was in the tumour model, where stimulation with a B1R agonist significantly enhanced proliferation and especially migration of DU145 cells. In addition, pre-treatment with a B1R antagonist abolished both these effects. B2R agonists could not produce the same positive effect as the B1R agonist on growth and migration of prostate tumour cells. DU145 CM did not prove to be a migratory stimulus for DU145 cells at any concentration. Discussion Previous studies in our laboratory have shown prostate-tumour CM to promote proliferation of endothelial cells and have postulated that TK up-regulation may be the reason for this. However, the present study could not reproduce this effect of CM. Further, BKR antagonists had no notable or consistent effect on the minimal promotion of proliferation that had been produced by DU145 CM. In addition, selective BKR agonists failed to induce proliferation or migration of endothelial cells, key events in the angiogenic cascade. Although in contrast to some studies, the present study was unable to implicate the KKS in angiogenesis, tumour neo-vascularisation is a consequence of several angiogenic factors functioning together as opposed to a single, isolated factor. For example, we were able to demonstrate a positive mitogenic effect of VEGF on endothelial cells and it may be this as well as other factors in the CM that are responsible for the small proliferation we observed. Up-regulation of kallikreins and kinins in tumours may enhance fundamental events in tumourigenesis in an autocrine manner, and bradykinin (BK) has previously been shown to promote tumour growth in mouse models. Our study supported the involvement of the KKS in tumourigenesis. Although CM from DU145 cells did not self-stimulate the migration of these cells, a B1R agonist enhanced both proliferation and migration, an effect that was also abrogated by the relevant antagonist, indicating a role for kinins. In contrast to the findings of another study, stimulation of the B2R failed to significantly promote tumour growth or motility. However, this is not an unexpected finding because it is thought that the ubiquitous B2R mediates physiological effects in the prostate while the inducible B1R plays a role in prostate cancer pathology. In summary, this study lends support to the ongoing exploration of BKR antagonists as possible candidates in the development of alternate approaches to cancer therapy. This may be particularly beneficial to hormone-independent tumours, such as those of the prostate, for which there exists few effective treatment options.