Inhibitory effects of 9-(4-Thio-β-D-ribo-pentofuranosyl)guanine on tumor growth and angiogenesis

Article abstract of DOI:10.1248/bpb.27.520

Background: To find a nucleoside with anti-angiogenic activity, we tried to screen an active compound from our nucleoside library. Materials and Methods: The compound inhibiting the growth of human umbilical vein endothelial cell (HUVEC) induced by the conditioned medium of lung carcinoma cell line PC-9 was screened. The antitumor activity of the compound was evaluated against murine sarcoma S-180 implanted onto chick embryo chorioallantoic membrane (CAM). Results: 9-(4-Thio-β-D-ribo-pentofuranosyl)guanine (4′-thioguanosine) was found to be a potent inhibitor of the growth of HUVEC. The growth of S-180 implanted onto CAM was also inhibited by 4′-thioguanosine whereas the in vitro growth of S-180 was not inhibited. The administration of 4′-thioguanosine in mice caused unexpected side effect which suggested neurotoxicity. Conclusions: Antitumor effect of 4′-thioguanosine on S-180 was suggested to be due to inhibition of tumor angiogenesis. Because of toxicity of 4′-thioguanosine in mice, further development of the derivatives which have lower toxicity is required.

Full text of DOI:10.1248/bpb.27.520

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20
Biol. Pharm. Bull. 27(4) 520—523 (2004)
Vol. 27, No. 4
Inhibitory Effects of 9-(4-Thio-b-D-ribo-pentofuranosyl)guanine on Tumor
Growth and Angiogenesis
,
a
a
a
b
Shinji MIURA,* Kohei YAMADA, Fumitaka KANO, and Yuichi YOSHIMURA
a
b
Biochemicals Division, Yamasa Corporation; 2–10–1 Araoi-cho, Choshi, Chiba 288–0056, Japan: and School of
Pharmaceutical Sciences, Showa University; 1–5–8 Hatanodai, Shinagawa-ku, Tokyo 142–8555, Japan.
Received October 28, 2003; accepted December 22, 2003
Background: To find a nucleoside with anti-angiogenic activity, we tried to screen an active compound from
our nucleoside library. Materials and Methods: The compound inhibiting the growth of human umbilical vein
endothelial cell (HUVEC) induced by the conditioned medium of lung carcinoma cell line PC-9 was screened.
The antitumor activity of the compound was evaluated against murine sarcoma S-180 implanted onto chick em-
bryo chorioallantoic membrane (CAM). Results: 9-(4-Thio-b-D-ribo-pentofuranosyl)guanine (4؅-thioguanosine)
was found to be a potent inhibitor of the growth of HUVEC. The growth of S-180 implanted onto CAM was also
inhibited by 4؅-thioguanosine whereas the in vitro growth of S-180 was not inhibited. The administration of 4؅-
thioguanosine in mice caused unexpected side effect which suggested neurotoxicity. Conclusions: Antitumor ef-
fect of 4؅-thioguanosine on S-180 was suggested to be due to inhibition of tumor angiogenesis. Because of toxicity
of 4؅-thioguanosine in mice, further development of the derivatives which have lower toxicity is required.
Key words 4Ј-thioguanosine; angiogenesis; human umbilical vein endothelial cell (HUVEC); chorioallantoic membrane
(
CAM) assay; S-180
Malignant tumors induce angiogenesis to be supplied with furanose (1) was treated with silylated 6-chloropurine or 2-
nutrition and oxygen for the growth of themselves both in amino-6-chloropurine in the presence of trimethylsilyl triflate
1
)
primary and metastatic stages. A drug inhibiting tumor an- (TMSOTf) in 1,2-dichloroethane at 100 °C to afford 4Ј-
giogenesis will be useful for cancer chemotherapy because it thionucleosides (2, 3) in ca. 50% yields as anomeric mix-
is expected to have lower toxicity than the other drugs which tures (b/aϭ 6.5, 3.1, respectively). 2-Amino-6-chloropurine
kill cancer cells directly. Many angiogenesis inhibitors such nucleoside (a,b-4) was obtained by the deprotection of 2 in
2
)
3)
4)
as fumagillin derivative TNP-470, SU-5416, angiostatin
79% yield. The a-isomer (a-4) could be removed by recrys-
5
)
and endostatin have been reported to exhibit antitumor or tallization from MeOH to give a pure b-4. 2,6-Diamino-
antimetastatic activities. Nucleoside compounds, however, purine nucleoside (a,b-5) was also synthesized from 2 by
have not been reported yet to have anti-angiogenic activity treatment with aqueous ammonia–MeOH at 80 °C in a sealed
6
)
except for 6-methylmercaptopurine riboside (6-MMPR).
tube. The a,b-anomers could be separated by reverse phase
Thus, we screened a compound which selectively suppresses ODS-column chromatography to afford b-5 in 46% yield. 4Ј-
the growth of vascular endothelial cells from our nucleoside Thioadenosine (6) was synthesized from a,b-3 by the similar
compound library. As a result, we identified 9-(4-thio-b-D- method to the preparation of diaminopurine derivative (5).
ribo-pentofuranosyl)guanine (4Ј-thioguanosine, 7) as a po- 4Ј-Thioguanosine (7) was synthesized from a,b-4 by the se-
tent inhibitor. We also describe the in vitro inhibitory effects lective enzymatic hydrolysis of the 6-chloro group of b-4
of derivatives of 4Ј-thioguanosine and the evaluation of in
vivo activity of 4Ј-thioguanosine.
MATERIALS AND METHODS
Cell Lines, Chemicals and Animals Human umbilical
vein endothelial cell (HUVEC) and EBM medium were pur-
chased from Clonetics (Walkersville, MD, U.S.A.). Human
lung carcinoma PC-9 was originally established by Professor
Y. Hayata (Tokyo Medical College, Tokyo, Japan). Human
basic fibroblast growth factor (bFGF) and vascular endothe-
lium growth factor (VEGF) were purchased from Progen
Biotechnik GmbH (Heidelberg, Deutschland) and PeproTech
EC Ltd. (London, U.K.), respectively. Fumagillin and 3-(3,4-
dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
MTT) were purchased from Sigma-Aldrich (St. Louis, MO,
U.S.A.). Severe combined immunodeficient (SCID) mice
were purchased from CLEA Japan, Inc. (Tokyo, Japan).
Syntheses of the Derivatives 4Ј-Thionucleosides used
Conditions: (a) Silylated purine base, TMSOTf, 1,2-dichloroethane, 100 °C, 2 (53%,
b/aϭ6.5), 3 (45%, b/aϭ3.0). (b) NH OH, MeOH, a,b-4 (79%). (c) NH OH, MeOH,
4
4
in this study were synthesized by the methods which reported 80 °C in a sealed tube, b-5 (46%). (d) NH , EtOH, 1,4-dioxane, 80 °C in a sealed tube,
3
7
)
8)
b-6 (44%). (e) Adenosine deaminase, Tris–HCl buffer (pH 7.0), b-7 (70%).
the syntheses of 4Ј-thioadenosine and 4Ј-thioguanosine
with our modification. Briefly, 1,2,3,5-tetraacetyl-4-thioribo-
Chart 1
∗
To whom correspondence should be addressed. e-mail: miura@yamasa.com
© 2004 Pharmaceutical Society of Japan

April 2004
521
9
)
with adenosine deaminase.
were administered i.v. twice per day for 4 consecutive days
The structures of compounds 4—7 were confirmed by de- from the day when the tumor had reached a diameter of 5—
tailed NMR and FAB mass spectroscopy and elemental 9 mm, and the body weight and the tumor volume of the
analysis.
Evaluation of the Inhibitory Effects on the Growth of activity of the drugs.
Vascular Endothelial Cell and Cancer Cell Line Panel in
mice were measured for evaluation of toxicity and antitumor
6
Vitro Human lung carcinoma PC-9 cells (2.3ϫ10 cells/75- RESULTS
2
cm culture flask) were seeded in RPMI1640 medium con-
taining 10% fetal bovine serum (FBS) and incubated at 37 °C
Inhibitory Effects of 4؅-Thioguanosine and Its Deriva-
for 24 h, then medium were replaced with fresh one and incu- tives on the Growth of Vascular Endothelial Cells To
bated for an additional 24 h. The conditioned medium was find a selective inhibitor which affects tumor-induced angio-
harvested and used to induce the growth of HUVEC. genesis, we established the screening system in which the
HUVEC were seeded in a 96-well plates at a density of growth of HUVEC was induced by the addition of the condi-
1
000 cells/well in 50 ml of EBM medium containing 1 mg/ml tioned medium of lung carcinoma cell line PC-9. Screening
of hydrocortisone, 50 mg/ml of gentamicin and 50 mg/ml of of our chemical library which mainly consists of nucleoside
amphotericin B. Then, 50 ml of conditioned medium of PC-9 compounds resulted in an identification of 4Ј-thioguanosine
was added to the each well. For the determination of the (7) as a potent inhibitory compound. As shown in Fig. 1A, it
“
basal” growth, the medium incubated without PC-9 was inhibited the HUVEC growth induced by PC-9 conditioned
added instead of PC-9 conditioned medium. The plate was medium (“PC-9” column in Fig. 1A) in a dose-dependent
incubated for 72 h and cell viability was determined by the manner. Fumagillin, a fungal metabolite which was reported
1
0)
2,13)
MTT assay. In the experiment in which bFGF or VEGF to have anti-angiogenic activity in vitro and in vivo,
was
was used to induce the growth of HUVEC, they were seeded found to be less active at concentrations of 0.01—1 mg/ml in
as described above with 50 ml of the medium incubated with- comparison with 4Ј-thioguanosine (Fig. 1A). We then also
out PC-9. Each growth factor was added at a final concentra-
tion of 10 ng/ml. The inhibitory effects of the compound on
3
2
9 cell lines were determined by sulforhodamine B assay at
-d drug exposure. The experimental details and the origin of
1
1)
the cell line were described in the reference.
Evaluation in the Chick Embryo Chorioallantoic Mem-
brane (CAM) Assay The CAM assay was performed by
6
)
the method reported previously with some modifications.
Briefly, murine sarcoma S-180 cells were implanted s.c. to
ICR mice. After 2 weeks, the solid tumor was resected, cut
into pieces of 1—2 mm and implanted onto CAM of 7-d old
chick embryos (Day 0). The 10 ml of drug solution in phos-
phate-buffered saline were pippeted directly onto the tumor
on Days 1, 3, 5 and 7. The tumors were resected from CAM
on Day 9 and their weights were measured.
Evaluation of Inhibitory Effects on S-180 Tumor
Growth in Vitro in the Collagen Gel Matrix Assay The
collagen gel matrix assay was performed according to the
1
2)
previous method with some modifications. Briefly, murine
sarcoma S-180 cells were implanted s.c. to ICR mouse. After
2
2
weeks, the solid tumor was resected, cut into pieces of 1—
mm and placed onto sterilized collagen gel matrix (Koken
Co. Ltd., Tokyo, Japan) which were cut into fragments (10ϫ
1
1
0ϫ5 mm) and immersed in RPMI1640 medium containing
0% FBS in 24-well plates. After incubating them at 37 °C
for 3 d in 5% CO incubator, the medium in the well and col-
2
lagen gel were removed by aspiration, and 300 ml of drug-
containing fresh medium was added to each well. After addi-
tional incubation for 3 d, 30 ml of MTT solution (5 mg/ml)
and 50 ml of 1% collagenase solution in phosphate-buffered
saline were added to each well and incubated at 37 °C for 3 h.
The formed formazan was solubilized by the addition of
4
00 ml of 0.02 N HCl/50% N,N-dimethylformamide/20%
SDS, and the viability of the tumor cells were determined by Fig. 1. The Inhibitory Effects of 4Ј-Thioguanosine (7) and Fumagillin on
measuring the optical density at 570 nm.
the Growth of HUVEC Induced by PC-9 Conditioned Medium (A), bFGF
(
B) and VEGF (C)
Evaluation of Antitumor Effects in Vivo in SCID Mice
7
The growth of HUVEC was determined by the MTT assay and shown as the optical
density (OD) at 570 nm. Each value represents the average of triplicate determinations
Cultured human lung carcinoma PC-9 cells (2.8ϫ10 cells)
were transplanted s.c. into 6-week-old SCID mice. Drugs and each bar indicates the standard deviation.

5
22
Vol. 27, No. 4
examined whether 4Ј-thioguanosine inhibits the HUVEC Table 1. The Inhibitory Effects of 4Ј-Thionucleosides on the Growth of
HUVEC Induced by PC-9 Conditioned Medium
growth induced by bFGF or VEGF. As shown in Figs. 1B
and C, bFGF- or VEGF-induced HUVEC growth was not in-
hibited by 4Ј-thioguanosine. Interestingly, fumagillin po-
tently inhibited HUVEC growth in contrast to the results of
the experiment using PC-9 conditioned medium.
To examine the selectivity of the 4Ј-thioguanosine effect
on HUVEC, we investigated the inhibitory effects on the
growth of 39 cell lines originating from breast, ovary, lung,
kidney, colon, stomach, brain, prostate cancer and
melanoma. 4Ј-Thioguanosine was almost inactive against the
a)
Compound
IC50 (mg/ml)
b-4
0.027
0.017
7.1
0.021
2.8
b-5
6
7
Fumagillin
a) Inhibitions of HUVEC growth (%) were calculated as the following: inhibition
%)ϭ100Ϫ(TϪB)/(CϪB)ϫ100, where C was the mean optical density of the control
PC-9 conditioned medium) group, T is that of the drug-treated group and B is that of
(
(
cancer cell line panel; its IC50 value was Ͼ50 mM in all cell the basal group. The IC50 were determined from the dose–inhibition curves.
lines tested.
Then we examined the inhibitory effects of the derivatives
of 4Ј-thioguanosine on the HUVEC growth induced by PC-9
conditioned medium. As shown in Table 1, 2-amino-6-
chloro-9-(4-thio-b-D-ribo-pentofuranosyl)purine (b-4) and
2
,6-diamino-9-(4-thio-b-D-ribo-pentofuranosyl)purine (b-5)
were equally inhibitory as 7 against the HUVEC growth.
Since b-4 and b-5 are predicted to be converted to 7 by
adenosine deaminase of the cell, the active metabolite was
suggested to be 4Ј-thioguanosine. On the other hand,
HUVEC growth induced by PC-9 conditioned medium was
not potently inhibited by 4Ј-thioadenosine (b-6); its IC was
5
0
similar to that of fumagillin. Pyrimidine 4Ј-thionucleosides
such as 4Ј-thiocytidine were inactive (data not shown). The
Fig. 2. The Inhibitory Effects of 4Ј-Thioguanosine (7) and Fumagillin on
the Growth of Murine S-180 Tumor Implanted on Chick Embryo CAM
4
Ј-oxy congeners of the active 4Ј-thionucleosides and 2Ј-
Each value represents the average of 7—9 CAM determinations and each bar indi-
cates the standard deviation. ∗ : pϽ0.05 versus control (Dunnett’s multiple compar-
isons).
deoxy- or 2Ј-ara-thionucleosides did not show potent activity
against HUVEC growth without inhibiting cancer cell
growth (data not shown).
In Vivo Effects of 4؅-Thioguanosine on the Tumor
Growth To evaluate the in vivo anti-angiogenic effect of
4
Ј-thioguanosine, we preformed the CAM assay. First, the
methylcellulose disc containing 4Ј-thioguanosine was im-
planted onto chick embryo CAM and the inhibitory effect on
the physiological angiogenesis was determined according to
1
4)
the method previously reported, but no effects were ob-
served (data not shown). In the next experiment, therefore,
we implanted the murine S-180 tumor onto CAM which was
grown in mice by subcutaneous implantation and the in-
hibitory effect of 4Ј-thioguanosine on the growth of tumor
was then examined, because 6-MMPR was reported to inhibit
the tumor growth by anti-angiogenic action in similar experi-
Fig. 3. The Effects of 4Ј-Thioguanosine (7) and Fumagillin on the Growth
of Murine S-180 Tumor in in Vitro Collagen Gel Matrix Assay
6
)
mental system. The tumor growth was inhibited by the ad-
The viability of the tumor cells was determined by the MTT assay and the optical
density (OD) at 570 nm was corrected by the weight of tested tumor fragments. Each
value represents the average of triplicate determinations and each bar indicates the stan-
dard deviation.
ministrations of 0.001—0.01 mg/d of 4Ј-thioguanosine (7) at
1
, 3, 5 and 7 d after implantation (Fig. 2). Fumagillin also in-
hibited the tumor growth in same doses as 4Ј-thioguanosine
0.001—0.01 mg/d, Fig. 2).
To determine whether the above inhibitory effect of 4Ј- (SCID) mice and evaluated the 4Ј-thioguanosine effect on the
(
thioguanosine was due to the inhibition of the angiogenesis tumor growth by i.v. administration. 4Ј-Thioguanosine exhib-
or the direct inhibition of tumor cell growth, the in vitro cyto- ited unexpected toxicity; the mice received 4Ј-thioguanosine
toxicity of the drugs against S-180 cells was determined by at doses of 20 and 50 mg/kg/d i.v. for 4 consecutive days re-
1
2)
the collagen gel matrix method because S-180 cells were duced their body weight and 1 of 4 (20 mg/kg/d) and 2 of 4
unable to grow in culture in single cell suspensions. As (50 mg/kg/d) mice died after suffering from paralysis in their
shown in Fig. 3, 4Ј-thioguanosine (7) and fumagillin did not legs, whereas the inhibition of tumor growth by 4Ј-
inhibit the growth of S-180 even at a concentration of thioguanosine was not recognized at these doses (data not
1
mg/ml, which is same as that of the drug solution adminis- shown).
tered to the “0.01 mg/d/CAM” group shown in Fig. 2, by con-
tinuous exposure for 3 d.
DISCUSSION
For further evaluation, we implanted PC-9 lung carcinoma
cells subcutaneously to severe combined immunodeficient
To find a specific inhibitor which suppresses the angiogen-

April 2004
523
esis induced by tumors, we established an assay system in Agents supported by Grant-in-Aid for Scientific Research on
which the compound inhibiting HUVEC growth induced by Priority Area “Cancer” from the Ministry of Education, Cul-
the conditioned medium of PC-9 could be screened. Though ture, Sports, Science and Technology.
the identified active compound 4Ј-thioguanosine inhibited the
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