Year: 2012
Type: dissertation
Abstract: Introduction \nAngiogenesis, the growth of new blood vessels from the pre-existing vasculature, is a \npre-requisite for tumour growth and metastasis. Tumour-angiogenesis is regulated by \nvarious pro- and anti-angiogenic factors released by both endothelial and tumour cells, as \nwell as by the micro-environment. Numerous studies have implicated various systems in the \nacquisition of the angiogenic phenotype. The present study sought to investigate the role of \nthe kallikrein-kinin system (KKS) in tumour-angiogenesis. \nThe kallikreins consist of two serine proteases, plasma and tissue kallikrein (TK), involved in \nthe release of kinin peptides by enzymatic cleavage of kininogens. Stimulation of the \ncognate bradykinin receptors (BKR), B1R and B2R, mediates the mitogenic and vasoactive \nproperties of kinins. In addition, TK activates matrix metallo-proteinases (MMPs) involved \nin extracellular matrix (ECM) degradation. \nThe expression profiles of TK and kinins have been found to be dys-regulated in numerous \nhuman cancers, and several studies have demonstrated the involvement of the KKS in growth \nand metastasis of prostate tumours. Further, previous in vitro models in our laboratory have \nestablished an association between the KKS and prostate tumour-angiogenesis. In those \nstudies it was postulated that the up-regulated TK (produced by endothelial and tumour cells) \nstimulated endothelial cell proliferation. Thus, the aim of the present study was to define the \neffects of the KKS and seek a direct correlation with angiogenesis using in vitro models with \ntumour conditioned medium (CM), kinin receptor agonists and antagonists. \nMethods \nEthical approval for this project was granted by the Biomedical Research Ethics Committee, \nUniversity of KwaZulu-Natal (reference number BE152/08). Micro-vascular endothelial \ncells represent a suitable in vitro angiogenic model and dermal micro-vascular endothelial \ncells (dMVECs) were obtained commercially for this purpose. The tumour model used in \nthis study was an immortalised prostate cancer (DU145) cell line. The CM model involves \nthe treatment of one cell line with the metabolites of another. In the angiogenic model, \ndMVECs were exposed to increasing concentrations of DU145 CM. Stimulation was further \naugmented with BKR agonists. Specific BKR antagonists were used to test the specificity of \nstimulation. In addition, vascular endothelial growth factor (VEGF) was tested as a positive \nproliferation control. The potential of these agents to induce proliferation and migration was \ndetermined using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) \nassay and a modified Boyden chamber assay, respectively. Previous studies investigating the \npro-angiogenic effects of CM differed, in many respects, in terms of their models and \nmethodologies. In an attempt to fully explore the pro-mitogenic effects of CM on endothelial \ncells, various modifications, as well as alternate endothelial and tumour cell types, were \nemployed in the present study. The mitogenic and migratory effect of BKR agonists and \nantagonists on DU145 cells was also assessed. Further, the tumour model was expanded to \ninvestigate the autocrine potential of the KKS, by investigating the effect of DU145 CM on \nDU145 migration. \nResults \nIn the angiogenic model, although the addition of DU145 CM elicited a statistically \nsignificant increase in micro-vascular endothelial cell proliferation, this increase was very \nsmall (<10%) and not dose-dependent. Pre-incubation of dMVECs with a B1R or B2R \nantagonist did not influence this small effect of CM on proliferation. In addition, neither \nB1R nor B2R agonists, at any concentration, produced any significant proliferative effect on \nendothelial cells. In contrast to these findings VEGF, a well-known mitogen, was able to \nstimulate proliferation of dMVECs. Migration assays revealed that DU145 CM failed to \nstimulate endothelial cell motility. Further, neither BKR agonist displayed any \nchemo-attractant potential in those assays. \nThe most important finding was in the tumour model, where stimulation with a B1R agonist \nsignificantly enhanced proliferation and especially migration of DU145 cells. In addition, \npre-treatment with a B1R antagonist abolished both these effects. B2R agonists could not \nproduce the same positive effect as the B1R agonist on growth and migration of prostate \ntumour cells. DU145 CM did not prove to be a migratory stimulus for DU145 cells at any \nconcentration. \nDiscussion \nPrevious studies in our laboratory have shown prostate-tumour CM to promote proliferation \nof 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 \nhad no notable or consistent effect on the minimal promotion of proliferation that had been \nproduced by DU145 CM. In addition, selective BKR agonists failed to induce proliferation \nor migration of endothelial cells, key events in the angiogenic cascade. Although in contrast \nto some studies, the present study was unable to implicate the KKS in angiogenesis, tumour \nneo-vascularisation is a consequence of several angiogenic factors functioning together as \nopposed to a single, isolated factor. For example, we were able to demonstrate a positive \nmitogenic effect of VEGF on endothelial cells and it may be this as well as other factors in \nthe CM that are responsible for the small proliferation we observed. \nUp-regulation of kallikreins and kinins in tumours may enhance fundamental events in \ntumourigenesis in an autocrine manner, and bradykinin (BK) has previously been shown to \npromote tumour growth in mouse models. Our study supported the involvement of the KKS \nin tumourigenesis. Although CM from DU145 cells did not self-stimulate the migration of \nthese cells, a B1R agonist enhanced both proliferation and migration, an effect that was also \nabrogated by the relevant antagonist, indicating a role for kinins. In contrast to the findings \nof another study, stimulation of the B2R failed to significantly promote tumour growth or \nmotility. However, this is not an unexpected finding because it is thought that the ubiquitous \nB2R mediates physiological effects in the prostate while the inducible B1R plays a role in \nprostate cancer pathology. \nIn summary, this study lends support to the ongoing exploration of BKR antagonists as \npossible candidates in the development of alternate approaches to cancer therapy. This may \nbe particularly beneficial to hormone-independent tumours, such as those of the prostate, for \nwhich there exists few effective treatment options.
Author: Naressa Naidu
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1574676112