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Znt. J. Cancer: 61,268-271 (1995)
0 1995 Wiley-Liss, Inc.
Publicationof the InternationalUnion Against Cancer
Publicationde I Union InternationaleContre le Cancer
COMPARISON OF THE INHIBITORY EFFECT OF THE ANGIOGENESIS
INHIBITOR, TNP-470, AND MITOMYCIN C ON THE GROWTH
AND LIVER METASTASIS OF HUMAN COLON CANCER
Hiroyuki KONNO',Tatsuo TANAKA,Iwao MATSUDA,Toshikazu KANAI,Yuji MAKUO,Nobuhiko NISHINO,
Satoshi NAKAMUKA
and Shozo BABA
Second Department of Surgery, Hamarnatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431-31,Japan.
Angiogenesis inhibitors have attracted considerable interest.
The anti-tumor and anti-metastatic effects of TNP-470, an
angiogenesis inhibitor, and mitomycin C (MMC), a representative anti-neoplastic agent, were investigated using a xenotransplanted human colon cancer, TK-4. Suturing of small pieces of
TK-4 tumors to the cecal wall in nude mice (orthotopic
transplantation) induced liver metastasis. Mice were randomly
divided into 3 groups; a control group given saline solution, a
group receiving TNP-470 and a group receiving MMC. TNP-470
was given S.C. on alternate days for 5 weeks from day 10 after
cecal transplantation and MMC was administered intraperitoneally (i.p.) once a week from day 10 after cecal transplantation.
MMC significantly inhibited cecal tumor growth. In the control
group, liver metastases developed in 9 out of 10 mice, including
3 with more than 20 metastatic foci. Liver metastasis also
developed in 8 out of 10 mice receiving MMC, 2 of which had
many metastases. In contrast, liver metastasisdeveloped in only
2 out of 8 mice in the TNP-470 group and neither of these
animals had numerous metastases.
o 1995 Wiley-Liss,Inc.
Angiogenesis is essential for the growth of solid tumors
(Folkman, 1972, 1985), especially in the early phase. This
suggests that the process of tumor metastasis requires angiogenesis at the metastatic site and that inhibition of angiogenesis
may become an effective treatment for metastasis of solid
tumor.
An arginine-rich basic protein, protamine, was previously
reported to suppress tumor growth (Taylor and Folkman,
1982), but no clinical trials were performed because of its
severe adverse effects. A synthetic analogue of fumagillin,
TNP-470, has also been shown to have an anti-angiogenic
effect in vitro and in vivo (Ingber et a/., 1990; Kusaka et a/.,
1991). Furthermore, it was reported that TNP-470 could have
an inhibitory effect on the growth and metastasis of rodent
tumors (Yanase et al., 1993; Yamaoka et al., 1993). Antineoplastic agents are used clinically to prevent the liver
metastasis of colon cancer, for instance, by intraportal administration (Tsujitani et al., 1991), but the therapeutic effect has
been uncertain. Comparison of the therapeutic effects of a
representative anti-neoplastic drug, mitomycin C (MMC), and
an anti-angiogenic agent, TNP-470, for the prevention of liver
metastasis should be performed prior to the clinical application of TNP-470.
We established a liver metastatic model of human colon
cancer based on that of Fu et al. (1991), who reported that
orthotopic transplantation of intact human tumor tissue into
the corresponding organ of nude mice achieved a high metastatic rate without the disruption of tumor integrity. We have
already established several colon cancers in this way and TK-4
is the most highly metastatic of them. In the present study,
TK-4 tumors are used to compare the inhibitory effect on liver
metastasis of TNP-470 with that of MMC, a representative
anti-neoplastic agent for colon cancer.
MATERIAL AND METHODS
Preparation of TNP-470
The angiogenesis inhibitor TNP-470 was kindly provided by
Takeda (Osaka, Japan). TNP-470 was suspended in a vehicle
of 1 % ethanol and 5% gum arabic in saline.
MMC was purchased from Kyowa Hakko (Tokyo, Japan)
and dissolved in saline.
Animals
Male BALBic nuinu mice, obtained at 4 weeks of age from
Clea (Tokyo), were used for this study at 5 weeks of age.
Human colon cancer
The human colon cancer strain, TK-4, was established in our
department from a metastatic liver lesion of a SO-year-old
Japanese male with sigmoid colon cancer, and was maintained
by passage in nude mice (BALBlc nuinu males) for about 3
years. The tumor was a well-differentiated adenocarcinoma
that maintained its original features after inoculation.
Experimenta 1 protocol
Small pieces of TK-4 tumor tissue (5 mm in diameter) were
resected from S.C. lesions in the exponential growth phase.
These tumor pieces were sutured to the wall of the cecum in
nude mice with 6-0 Dexon (Davis-Geck, Manati, PR) after
removal of the serosa. Mice were divided into 3 groups: control
group (n = lo), TNP-470 group (n = 8) and MMC group
(n = 10). Animals in the TNP-470 group received 0.2 ml of
TNP-470 solution (30 mgikg) S.C. and those in the control
group received saline s.c., in each case on alternate days from
day 10 after tumor transplantation. Animals in the MMC
group received 0.2 ml of MMC (2 mgikg) i.p. once a week from
day 10 after transplantation. Treatment was continued until
animals were killed at 6 weeks after tumor transplantation.
Liver metastases were evaluated macroscopically and confirmed histologically. The transplanted tumors were also removed and weighed.
Statistical analysis
Student's t-test was used for comparison of the actual tumor
weight and body weight. The x2 test was used to compare the
number of mice with liver metastasis in each group.
RESULTS
Primary tumor growth
Figure 1 shows the actual tumor weight at killing. The actual
tumor weights were 499 f 247 mg in the control group, 478 5
'To whom correspondence and reprint requests should be sent. Fax:
053 435 2273.
Received: November 14, 1994 and in revised form December 20,
1994.
TNP-470 INHIBITS THE LIVER METASTASIS
269
FIGURE1 -Actual tumor weight at killing. *Significant difference from control or TNP-470 group ( p < 0.01).
185 mg in the TNP-470 group and 276 ? 80 mg in the MMC
group. TNP-470 had no effect on the primary tumors transplanted in the cecal wall, but MMC significantly inhibited
primary tumor growth. The histological appearance of the
primary tumors in the 3 groups is demonstrated in Figure 2. No
histological evidence of a therapeutic effect of MMC or
TNP-470, such as tumor-cell necrosis, was observed.
Liver metastasis
Table I demonstrates the effect of TNP-470 and MMC on
liver metastasis. Administration of TNP-470 significantly inhibited liver metastasis. In contrast, MMC showed little antimetastatic effect, although the number of metastatic foci was
lower in the MMC group than in the control group (Table 11).
All the metastatic lesions were confirmed histologically. Figure
3 (a,b,c) shows the histological appearance of liver metastasis.
Many metastaseswere observed in some mice of the control or
MMC groups, but not in the TNP-470 group.
Body weight
The body weights at the time of killing were 20.37 & 2.02 in
the control group, 20.12 ? 1.24 in the MMC group and 19.41 5
1.21 in the TNP-470 group. Mice treated with TNP-470
showed some weight loss, but this was not statistically significant.
DISCUSSION
The present oirthotopic transplantation model employing
nude mice has proved useful in clarifying the mechanism of
metastasis and in (developinganti-metastatic therapy. Metastasis occurs when human cancer strains of many kinds are
transplanted orthotopically (Furukawa et al., 1993 a,b) and we
have already established 6 human colon cancers in this
manner. Of these, TK-4 has the highest metastatic potential,
and genetic analysis has demonstrated that it possesses mutants of@, K-ras (data not shown).
The present study showed that TNP-470 had no effect on the
primary tumor growth of TK-4, although a significant antimetastatic effect was demonstrated. TNP-470 has also shown
an anti-metastatic effect on other types of colon cancer
transplanted in the cecum, despite having no inhibitory effect
on the primary tumors (Tanaka et al., in press). On the other
hand, TNP-470 slriows an inhibitory effect on S.C. transplanted
primary tumors in which the doubling time was about half of
FIGURE
2 - Histological appearance of the TK-4 primary tumors
~ 2 0 0. (a) Control group; (b) MMC group; (c) TNP-470 group.
dell-Jifferentiated adenocarcinoma is demonstrated in a, b and c.
No histological findings of a therapeutic effect of MMC were
observed. Scale bar = 10 ym.
that of orthotopically transplanted tumors (data not shown).
These results suggest that this anti-angiogenic agent may show
its anti-proliferative effect on rapidly growing tumors. This is
consistent with the hypothesis that rapidly proliferating tumors
are more angiogenesis-dependent (Kim et aL, 1993). It has
been reported that the cytostatic action of TNP-470 is more
important than its cytotoxic action in vivo, because TNP-470
exerts its anti-proliferative effect on endothelial cells through
its cytostatic action (Yamaoka et al., 1993). This suggests that
TNP-470 can exert its effect on any tumors that require
270
KONNO ETAL.
TABLE I - INHIBITORY EFFECT OF TNP-470AND MMC ON LIVER
METASTASIS
Group
Number of mice with liver metastasis
Control
MMC
TNP-470
81 10
9/10
2/8*
"Significantly different from control ( p < 0.01) and from MMC
group(p < 0.05).
rABLE 11 - hlJMRER OF MFTASTA7 IC L I V t R FO('I
< foci < 5
Group
0
Control
MMC
TNP-470
1
2
3
6
6
1
0
6 < foci < 20
2
0
1
I
20<
3
2
0
neovascularization, even if they are resistant to TNP-470 in
vitro. TNP-470 causes an increase in the proportion of endothelial cells in the Go/GIphase and a decrease in the proportion
of cells in the G*/M and S phases, suggesting that this agent
induces Go/G1 arrest (Kusaka et QL, 1994). It has been also
reported that the labeling index of endothelial cells is not
decreased by treatment with chemotherapeutic agents, but is
significantly decreased by TNP-470 (Yamamoto et al., 1994).
It is well known that MMC shows a strong cytotoxic effect on
, arrest.
tumor cells both in vitro and in vivo by inducing G_
MMC was selected as the chemotherapeutic agent for the
present study because it is frequently used to treat liver
metastasis of colon cancer and because Iigo et al. (1992)
reported that it was more effective against liver metastasis
induced by the intrasplenic injection of colon 26 cells than
against S.C. tumors. In the present study, only a minimal
anti-metastatic effect of MMC was demonstrated. In contrast,
MMC significantly inhibited primary tumor growth to about
half of that observed in the TNP-470 group. The orthotopic
transplantation model used in the present study may reflect
clinical liver metastasis more precisely than the model of Iigo ef
al., and our findings suggest that the anti-metastatic effect of
MMC is limited, although its anti-proliferative effect on liver
metastasis may be considerable. As anti-metastatic therapy,
anti-angiogenic agents are more likely than chemotherapeutic
agents to be useful, because neovascularization is essential for
tumor micro-metastases to become established and grow. The
present study suggests that liver metastasis was prevented by
TNP-470 because the growth of TK-4 tumor cells arriving in
the liver from primary tumors was inhibited by its antiangiogenic action.
The combination of TNP-470 with a chemotherapeutic
agent which is strongly cytotoxic may be useful in treating solid
tumors, because the anti-neoplastic actions of the 2 agents are
clearly different. The efficacy of such combination therapy is
currently under investigation.
Before clinical use, the toxicity of an anti-angiogenic agent
also needs to be evaluated. In general, anti-angiogenic agents
are less toxic than chemotherapeutic agents. The major adverse effect of TNP-470 appears t o be weight loss. In the
present study, 30 mg/kg of TNP-470 induced weight loss in the
mice with cecal tumors, but the decrease was not significant.
appearance of the liver metastases
FIGURE
( ~ 2 0 0 ) fa)
. Control group; (b) MMC group; (c) TNP-470 group.
Massive metastasis is observed in the control and MMC groups,
.
but not in the TNP-470 group.
In conclusion, the anti-angiogenesis agent TNP-470 appears
to be potentially useful for preventing liver metastasis of
colorectal cancer.
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