Место издания:Blackwell Publishing Inc United Kingdom
Первая страница:501
Последняя страница:501
Аннотация:Nowadays the important role of cell adhesion molecules in invasion
of metastatic tumor in surrounding tissues as well as in the
growth of tumor blood vessels is well known. In particular the
increased invasiveness and metastasizing that is typical for vertically
growing melanoma is known to be mediated by specific integrins.
So, integrin avb3 which is overexpressed in melanoma
cells with high metastatic potential as well as in endothelial cells
of tumor blood vessels and exhibits low expression level in normal
melanocytes used to be an attractive target for melanoma
diagnostics and therapy. Integrin avb3 is known to recognize the
RGD peptide sequence which has been found in a wide variety
of its natural ligands.
A number of expression vectors analogous to constructs
described in [1, 2] has been constructed. These vectors provide
the production of the streptavidin fused with RGD-bearing peptides
(SR10, SR13, SR15) in E.coli. These proteins were demonstrated
to bind selectively to murine (B16F10) and human
(MeWo) melanoma cells as well as to endothelial cells (HUVEC).
In order to estimate kinetics of binding of each protein with
receptors on the MeWo and HUVEC cell surface we’ve analyzed
fluorescence intensity of cell population after incubation with
FITC-labeled proteins SR10, SR13, SR15. We noted a characteristic
feature – sharp decline in fluorescence intensity after 45 minutes
of incubation. Probably this effect is due to the
internalization of integrin receptors bound with SR10, SR13 or
SR15. Internalization was confirmed by confocal fluorescent
imaging of fixed MeWo and HUVEC cells. Luminous spots were
detected on the membrane and in the compartment which is typical
for endosome location. Moreover, it was noted that increase
in incubation time leads to increase in fluorescence intensity both
on the cell surface and in cytoplasm. Internalization observed
might be caused by activation of the integrin receptors after
binding with ligand. It’s known that activated receptors undergo
endocytosis with the following recycling [3].
An influence of different concentrations of SR proteins on cell
viability was estimated by XTT-test. It was demonstrated that
incubation of HUVEC in the presence of 1.5 lM SR15 leads to
two-fold decrease in cell proliferation rate. The maximum
decrease in MeWo proliferation rate (up to 18%) was achieved
by exposition of cells to SR13 in 0.075 lM concentration.