Fluorinated pyrimidines are agents that have been widely used to
treat a variety of cancers. Their continually expanding role in
cancer chemotherapy is related to the introduction of new treatment
regimens and combinations with other agents, and to the development
of oral formulations suitable for prolonged administration. UFT, an
anticancer agent composed of tegafur and uracil in a molar ratio of
1:4, is a second-generation, fluorinated pyrimidine-based drug that
is widely used in Japan and is now undergoing intensive clinical
evaluation in the United States. Oral administration of UFT
produces fluorouracil (5-FU) plasma levels similar to those observed
in cancer patients after continuous intravenous infusion.
Early clinical reports indicate that UFT inhibits the growth not only
of primary tumor but also metastatic sites.[2-4] Several studies[5,6]
have demonstrated that UFT prevents or inhibits metastasis in
tumor-bearing animals and is closely associated with increased
survival. However, the mechanism of inhibition remains unknown. Our
recent studies aimed to clarify the antimetastatic effect of UFT and
elucidate a possible mechanism involved in this phenomenon.
In the process of cancer metastasis, one of the crucial steps is
angiogenesis, the process of delivery of essential energetic factors
and oxygen to a newly growing tumor mass.[7-9] The inhibition of the
formation of microvessels entering or developing in a tumor affects
the expansion of micrometastases and may lead to disappearance of the
tumor and, consequently, increased survival. Tumor-celldriven
angiogenesis is controlled by vascular endothelial growth factor
(VEGF), fibroblast growth factor-2, interleukin-8, and several other
growth factors produced by tumor mass.[10-12] It seems reasonable,
therefore, to consider their role in the antimetastatic and
antiangiogenic activity of a drug, specifically UFT.
In this study, we attempted to (1) demonstrate an inhibitory effect
of UFT on spontaneously forming metastasis and its role in increased
survival, (2) find a correlation between antitumor (antimetastatic)
effect and inhibition of an expanding tumor microvasculature, and (3)
identify growth factors affected by UFT and related compounds in angiogenesis.
Spontaneous Metastasis to the Lung in Mice
The murine renal cancer cell line (RENCA) was kindly provided by Dr.
I. J. Fidler, University of Texas M. D. Anderson Cancer Center,
Houston, Texas. On day 0, RENCA cells 2 ´ 104
were implanted into the left kidney of BALB/cA mice. Ten days later,
the primary tumor growing in the kidney was surgically removed. The
anticancer effect was evaluated based on prolonged survival of
tumor-bearing animals, which were dying of intensive metastasis to
the lungs. The survival effect of drug-treated animals was evaluated
as the ratio of the mean survival time of treated animals to that of
control animals. The density of microvessels in the lung metastatic
foci was determined by immunostaining with an antiCD-31
Dorsal Air Sac Assay in Mice
This experiment was performed exactly as described previously.
Briefly, the Millipore chambers containing either tumor cells or
recombinant human VEGF (rhVEGF) were implanted into the preformed air
sac in the dorsum of a male BALB/cA mouse.
Five days after implantation, the chambers were removed from
subcutaneous fascia and the angiogenic response was assessed with
dissecting microscope photographs to determine the number of newly
formed blood vessels. The extent of angiogenesis was scored as an
index of 0, 1, 2, 3, 4, and 5 for the numbers of newly formed blood
vessels showing zigzagging characteristics in the area attached to
the chamber of 0, 1, 2, 3, 4, and 5 or more.
Life-Prolonging Effects of UFT in Lung Metastasis Model
This study examined the life-prolonging effects of several anticancer
drugs, including UFT, 5-FU, and TNP-470, in the lung spontaneous
metastasis model of murine RENCA cells. In this metastasis model,
there was a 100% incidence of spontaneous metastasis to the lung.
The average survival time for the control animals was 41.3 ±
2.9days (mean ± SEM). The UFT-treated (20 mg/kg/day) animal
group showed a significant life-prolonging effect as manifested by a
corresponding treated to control response ratio (T/C) value of 160.8% (P
< .05). The group of animals treated with 5-FU (13 mg/kg/day) also
experienced the life-prolonging effect, although 5-FU was less potent
(T/C = 125.7%, P < .01). Conversely, TNP-470, a known
antiangiogenic agent, showed only a marginal effect on the survival
of treated animals (Figure 1).
In a separate experiment, the antimetastatic effects of test
compounds were evaluated 34 days after implantation of cancer cells
by determining the total weight of cancer-invaded lungs. The average
weight of normal lungs of the age-matched animals was 0.13 g, while
cancer-invaded lungs weighed 0.45 g, indicating the presence of
intensive metastases. The weights of invaded lungs were significantly
reduced with UFT, TNP-470, and, to lesser extent, 5-FU, as
demonstrated by corresponding TC values of 44.5%, 51.3%, and 64.9%,
respectively (data not shown).
The tumor mass reduction in the lungs of treated animals was
associated with a decrease in the number of blood vessels in
metastatic nodules, as determined by an immunochemical staining with
an antiCD-31 monoclonal antibody. The number of blood vessels
in the cancer nodules was reduced by 35.5%, 20.6%, and 61.5%
following treatment with UFT, 5-FU, and TNP-470, respectively.
Antiangiogenic Activity of UFT in DAS Assay
The decreased blood vessel density in lung metastatic foci of
drug-treated animals indicated a possible inhibition of cancer-induced
angiogenesis. Therefore, we examined whether UFT treatment directly
affected the angiogenesis, or was an indirect effect resulting from
tumor shrinkage. The evaluation of antiangiogenic activity was
performed in a dorsal air sac (DAS) assay, in which the process of
angiogenesis was induced by RENCA cells in suspension, embedded
between semi- permeable membranes. UFT showed a marked inhibition of
RENCA cell-induced angiogenesis, exceeding that observed after
treatment with 5-FU.
UFT is composed of uracil and tegafur; the latter is further
metabolized in vivo to 5-FU and g-hydroxybutyric
acid (GHB) (Figure 2).[14,15]
Therefore, we looked for an active chemical entity originating from
UFT that might be responsible for antiangiogenic activity. We
examined the effects of uracil and tegafur as well as the metabolites
of tegafur: 5-FU, GHB, and g-butyrolactone
(GBL) in a DAS assay.
The angiogenic response induced by RENCA cells was abrogated both by
5-FU and tegafur, while uracil remained completely inactive.
Surprisingly, GHB and GBL, which are biotransformation products of
tegafur, showed pronounced inhibition of angiogenesis (Figure
To examine whether the antiangiogenic effect of UFT is limited to a
particular cell line or is universal, further studies were performed
using human cancer cell lines such as gastric, lung, and colon
cancers. This experiment revealed a wide spectrum of antiangiogenic
activity with UFT: five of five tested cell lines, including RENCA
cells, responded to the antiangiogenic activity of UFT and 5-FU.
Effects on VEGF-Dependent Responses in HUVEC
Several cancer cell lines in vitro and in vivo reportedly produce
VEGF and fibroblast growth factor-2 (FGF-2), known
angiogenesis-promoting factors. In our experiments, we found VEGF to
be predominantly produced and secreted by cancer cells, while FGF-2
was present in minimal quantities. We suspected, therefore, that the
antiangiogenic activity of UFT might be related to the suppression of
VEGF-dependent response of vascular endothelial cells by 5-FU or GHB.
The experimental results on the proliferation of VEGF-stimulated
human umbilical vein endothelial cells (HUVECs) revealed
concentration-dependent inhibition of HUVEC cell proliferation by
5-FU, while no inhibition was observed in the case of GHB. On the
other hand, 5-FU and GHB abrogated chemotactic migration and tube
formation of HUVECs stimulated with VEGF (data not shown). These
results indicate that UFT suppresses angiogenesis, partly by an
antiproliferative effect of 5-FU, a cytotoxic metabolite, and
predominantly by the inhibition of the VEGF-dependent responses of
vascular endothelial cells mediated by GHB, a completely noncytotoxic
metabolite of UFT.
These effects, observed in vitro, were reproduced in the experiment
performed in vivo in a DAS assay, when the angiogenesis was induced
by rhVEGF (Table 1). The UFT
treatment resulted in 94.2% inhibition of VEGF-induced angiogenesis,
while the treatment with an equimolar dose of GHB was associated with
a complete lack of any angiogenic response. This effect was also seen
with 5-FU but was less pronounced.
This study attempted to confirm previously reported cases of the
antimetastatic activity of UFT and to demonstrate its implication to
the prolongation of the life span of tumor-bearing animals. A
significant increase in survival time demonstrates that UFT is a
potent drug in the model of murine cancer that spontaneously
metastasized to the lungs. Moreover, the metastasis-reducing
potential of UFT is accompanied by a decreased number of microvessels
in lung metastatic nodules. Also, the inhibition of cancer-induced
angiogenesis appears to be closely associated with UFT (shown by a
DAS assay allowing us to trace the formation of new blood vessels).
It is of great interest that the antiangiogenic effect of UFT is
related to tegafur, and especially to its metabolites 5-FU, GBL, and
GHB, the latter being noncytotoxic.
The angiogenesis inhibitory effect of UFT is not limited to murine
cancer. It was also observed in various human cancer cell lines. In
vitro experiments revealed the ability of UFT-derived compounds to
inhibit VEGF-mediated vascular endothelial cell proliferation and
their chemotactic migration and tube formation. However, inhibition
of cell proliferation was only observed with 5-FU, a cytotoxic
metabolite of UFT. These data indicate that UFT has an antiangiogenic
potential due to its ability to disrupt a VEGF-mediated process
controlling the proliferation of vascular endothelial cells.
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