Recent Advances in Angiogenesis and ... - Bentham Science

Role of Thymidine Recent Advances in Angiogenesis and Antiangiogenesis, 2009 69
cells transfected with three enzymatically inactive TP
mutants, K115E, L148R and R202S did not possess
angiogenic activity in contrast to wild-type
transfectants [13].
Also the role of TP substrate (thymidine) and
enzymatic products were extensively studied. TP, its
catalytic product 2dR-1P, and the subsequent
metabolite 2dR promoted endothelial tubulogenesis
in vitro, and the regeneration of a wounded
monolayer of ECs without exerting any mitogenic
effect. In vivo, 2dR promoted blood vessel formation
in an avascular sponge and in the CAM assay
[12,16].
TP, 2dR-1P and 2dR were also found to induce
chemotaxis of human umbilical vein endothelial cells
(HUVEC). Migration induced by 2dR-1P was
blocked by an alkaline phosphatase inhibitor,
suggesting that the chemotactic activity of 2dR-1P
first requires its conversion to 2dR. In a co-culture
assay, U937 and THP1 human monocyte cells, which
constitutively express high levels of TP, and TPoverexpressing
carcinoma cells strongly induced
HUVEC migration. Cell-stimulated HUVEC
migration was inhibited by the TP inhibitor 5-chloro-
6(1-imidazolylmethyl)uracil but not by a neutralizing
antibody to TP, although the latter completely
blocked migration induced by purified TP [23].
Together, these experimental data indicate that the
angiogenesis-stimulating properties of TP (i) require
its enzymatic activity and (ii) are mediated by the
degradation product 2dR. So, although a receptor for
TP has never been identified and TP is mainly
retained intracellularly, the protein can elicit its
angiogenic activity via the intracellular metabolism
of thymidine and subsequent extracellular release of
2dR, which forms a chemotactic gradient.
Interestingly, several of the biological effects induced
by 2dR were found to be inhibited by its stereoisomer
2-deoxy-L-ribose [24-26].
Only recently, molecular mediators of TP and
signaling pathways associated with TP-induced
angiogenesis have been identified. Both TP and 2dR
were found to stimulate the formation of focal
adhesions and the phosphorylation of focal adhesion
kinase (FAK) at tyrosine 397 in HUVEC and this
effect was blocked by antibodies to either integrin
51 or v3. TP and 2dR also increased the cell
surface expression of integrin 51 and the
association of FAK and vinculin with 51 [27].
A complementary DNA microarray was used to
identify genes that are induced during in vitro
migration of TP-overexpressing DLD-1 colon cancer
cells. Rho-associated coiled-coil domain kinase
(ROCK1) was found to be significantly
overexpressed in TP transfectants compared with
mock-transfected cells. TP transfectants also showed
higher cell motility than control cells. Also addition of
recombinant TP increased cell migration of wild-type
DLD-1 cells, and motility was blocked by a
neutralizing antibody to TP and by Y-27632, a specific
inhibitor of ROCK1. Moreover, actin fiber
polymerization, which is a marker of activation of
ROCK1, was higher in TP transfectants than in control
cells [28].
Addition of TP to AGS and MKN-45 gastric carcinoma
cell lines resulted in increased cancer cell invasion
through matrigel, which was accompanied by actin
filament remodeling and increased activation of the
phosphatidylinosital-3-kinase (PI3K) pathway. In
addition, TP-overexpressing MKN-45 cells showed
increased activity of mammalian target of rapamycin
(mTOR) and p70 ribosomal S6 kinase (p70 S6K )
compared with control cells, suggesting the
involvement of the PI3K/mTOR/ p70 S6K pathway in
TP-induced migration and invasion of gastric
carcinoma cells [19].
Transfection of RT112 human bladder carcinoma cells
with TP resulted in the secretion of vascular endothelial
growth factor (VEGF), interleukin (IL)-8, and matrix
metalloproteinase (MMP)-1 [29]. Secretion of these
angiogenic factors was only evident after
supplementing the culture medium with thymidine,
which recapitulates the tumor microenvironment in
which thymidine concentrations are raised due to the
hydrolysis of DNA from necrotic cells in hypoxic areas.
Thymidine was shown to increase cellular levels of
heme-oxygenase (HO-1), a marker for oxidative stress,
in RT112/TP cells. This effect was abrogated by
thymine, which may reverse thymidine catabolism by
capturing 2dR-1P. These data suggest that 2dR-1P is
responsible for the TP-induced oxidative stress,
possibly by generating oxygen radical species during
the early stages of protein glycation [29]. A timedependent
increase in HO-1 expression was also
observed during monolayer regeneration of RT112/TP
cells [16].
Increased mRNA expression and activity of MMP-9
were observed in KB/TP cell cultures and tumors
compared with their mock-transfected counterparts and
this was suggested to reflect the increased invasive
potential of KB/TP cells/tumors [26]. KK47 bladder
cancer cells that overexpress TP had higher levels of
MMP-7 and MMP-9 than control cells, whereas
overexpression of TP in prostate cancer cells resulted in
increased expression of MMP-1 and MMP-7.
Moreover, in bladder cancers from 72 randomly
selected patients, the expression level of TP was found
to correlate with that of urokinase-type plasminogen
activator (uPA), MMP-1, MMP-9, plasminogen
activator inhibtor (PAI)-1 and VEGF. Taken together,
these data indicate that the TP-induced production and