Regulation of tumour-angiogenesis by protease inhibitors and receptor antagonists.
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Date
2012
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Abstract
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.
Description
Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.
Keywords
Neovascularization., Blood-vessels--Growth., Protease inhibitors., Carcinogenesis., Theses--Therapeutics and medicines management.