Effects of phosphorylation of immunomodulatory agent FTY720 (fingolimod) on antiproliferative activity against breast and colon cancer cells

Article abstract of DOI:10.1248/bpb.31.1177

FTY720 (fingolimod), a novel immunosuppressant, was found to become biologically activated by phosphorylation into FTY720-1-phosphate (FTY720-P), which is a high-affinity agonist for sphingosine-1-phosphate (sphingosine-1-P)-receptors. FTY720 has also been reported to have a strong antitumor activity. The association between the phosphorylation of FTY720 and the growth inhibition of FTY720 against cancer cells are still not completely understood. In this study, we investigated the effects of FTY720, sphingosine, and their related compounds on the proliferation of human breast cancer cell lines (MCF-7, MDA-MB-231 and Sk-Br-3) and human colon cancer cell lines (HCT-116 and SW620). Non-phosphorylated FTY720, sphingosine and an FTY720 derivative, ISP-I-55, showed significant growth inhibition against these cells, with IC ₅₀ values of 5-20 μM at 48 h post-drug treatment. We confirmed that FTY720 induces the activation of a major mitogen-activated protein kinase, JNK, without the activation of p38 and down-regulation of phospho-ERK in MCF-7 breast cancer cells. In contrast, the phosphorylated derivatives, FTY720-P and sphingosine-1-P, as well as a phosphinane FTY720 derivative, cFTY720-P, did not inhibit the growth of the cells in the concentration range of 5-50 μM, whereas FTY720-P and sphingosine-1-P slightly induced the growth of MCF-7 cells. Combining FTY720 with dimethylsphingosine, a sphingosine kinase inhibitor, augmented the inhibitory effect of FTY720. These results indicate that the antiproliferative activity of FTY720 does not result from its phosphorylation, either endogenous or exogenous.

Full text of DOI:10.1248/bpb.31.1177

June 2008
Biol. Pharm. Bull. 31(6) 1177—1181 (2008)
1177
Effects of Phosphorylation of Immunomodulatory Agent FTY720
(Fingolimod) on Antiproliferative Activity against Breast and Colon
Cancer Cells
a
Yasuo NAGAOKA, ,a Kota OTSUKI,a Tetsuro FUJITA,b and Shinichi UESATO
*
a Faculty of Chemistry, Materials and Bioengineering, Kansai University; Osaka 564–8680, Japan: and b Research
Institute for Production Development; Sakyo-ku, Kyoto 606–0805, Japan.
Received January 30, 2008; accepted March 5, 2008; published online March 7, 2008
FTY720 (fingolimod), a novel immunosuppressant, was found to become biologically activated by phospho-
rylation into FTY720-1-phosphate (FTY720-P), which is a high-affinity agonist for sphingosine-1-phosphate
(sphingosine-1-P)-receptors. FTY720 has also been reported to have a strong antitumor activity. The association
between the phosphorylation of FTY720 and the growth inhibition of FTY720 against cancer cells are still not
completely understood. In this study, we investigated the effects of FTY720, sphingosine, and their related com-
pounds on the proliferation of human breast cancer cell lines (MCF-7, MDA-MB-231 and Sk-Br-3) and human
colon cancer cell lines (HCT-116 and SW620). Non-phosphorylated FTY720, sphingosine and an FTY720 deriva-
tive, ISP-I-55, showed significant growth inhibition against these cells, with IC50 values of 5—20 mM at 48 h post-
drug treatment. We confirmed that FTY720 induces the activation of a major mitogen-activated protein kinase,
JNK, without the activation of p38 and down-regulation of phospho-ERK in MCF-7 breast cancer cells. In con-
trast, the phosphorylated derivatives, FTY720-P and sphingosine-1-P, as well as a phosphinane FTY720 deriva-
tive, cFTY720-P, did not inhibit the growth of the cells in the concentration range of 5—50 mM, whereas FTY720-
P and sphingosine-1-P slightly induced the growth of MCF-7 cells. Combining FTY720 with dimethylsphingo-
sine, a sphingosine kinase inhibitor, augmented the inhibitory effect of FTY720. These results indicate that the
antiproliferative activity of FTY720 does not result from its phosphorylation, either endogenous or exogenous.
Key words FTY720; fingolimod; antiproliferation; breast cancer; colon cancer; MCF-7
A group of our co-workers (Fujita et al.) discovered a po- MATERIALS AND METHODS
tent immunosuppressant, ISP-I1) (identical with the antifun-
gal antibiotics myriocin2,3) and thermozymocidin4,5)), in the
General Procedure IR spectra were recorded on Shi-
entomopathogenic fungus Isaria sinclairii, and were led to madzu FTIR-8400 infrared spectrophotometer. FAB-MS
FTY720 (fingolimod), which is in the late stages of a clinical spectra were measured on a JEOL JMS-HX 100 instrument.
trial for multiple sclerosis treatment.6) FTY720 inhibits the Electrospray ionization mass spectra (ESI-MS) were meas-
sphingosine-1-phosphate (sphingosine-1-P) receptor–de- ured on an API-3000 mass spectrometer (Applied Biosys-
pendent egress of lymphocytes from lymph nodes into effer- tems) fitted with a TurboionsprayTM interface. 1H-NMR spec-
ent lymphatics and blood. In this process, FTY720 was found tra were recorded on JEOL EX-400 (400 MHz) instruments
to be monophosphorylated by sphingosine kinase type 2 into using tetramethylsilane as an internal standard. Analytical
FTY720-1-phosphate (FTY720-P), which down-modulates TLC and preparative TLC were performed using Silica gel
the sphingosine-1-P receptor on lymphocytes.7—9)
60 F254 (Merck, 0.25 and 0.5 mm, respectively) glass plates.
FTY720 has also been reported to have selective cytotoxi- Column chromatography was performed using Silica Gel 60
city against human cancer cell lines including multiple (70—230 mesh ASTM). All extracted solvents were dried
myeloma,10) prostate cancer,11) bladder cancer,12) hepatoma,13) over Na2SO4, followed by evaporation in vacuo.
breast cancer14) and leukemia cells.15) The cells incubated
Materials and Chemicals Sphingosine and DMS were
with FTY720 demonstrated features characteristic of apopto- purchased from Wako Pure Chemical Industries, Ltd. (Osaka,
sis. However, the association between the phosphorylation of Japan) and sphingosine-1-P was obtained from the Kanto
FTY720 and its cytotoxicity against cancer cells is not clear. Chemical Co., Inc. (Tokyo, Japan). ISP-I-55 was prepared
Thus, we tested the effect of the phosphorylation of FTY720 previously by one of our co-workers (T. Fujita).17) FTY720
and its related compounds (Fig. 1) on the proliferation of
human breast cancer cell lines (MCF-7, MDA-MB-231 and
Sk-Br-3) and human colon cancer cell lines (HCT-116 and
SW620). We also examined the influence of endogenous
sphingosine kinases on the cytotoxicity of FTY720 using
N,N-dimetylsphingosine (DMS),16) an inhibitor of sphingo-
sine kinase. Furthermore, we examined the influences of
FTY720 and sphingosine on the activation of stress activated
mitogen-activated protein kinases (MAPKs), c-Jun NH2-ter-
minal kinase (JNK) and p38, as well as a survival MAPK,
extracellular signal-regulated kinase (ERK), in MCF-7 cells.
Fig. 1. FTY720 (Fingolimod) and Related Compounds Used in This Study
∗ To whom correspondence should be addressed. e-mail: ynagaoka@ipcku.kansai-u.ac.jp
© 2008 Pharmaceutical Society of Japan

1178
Vol. 31, No. 6
m/z: 592.3210 (Calcd for C35H47NO5P: 592.3192).
FTY720-P20) A solution of 2 (16.5 mg, 0.028 mmol) in
methanol/acetic acidꢁ1/1 (4.0 ml) was stirred over 10% pal-
ladium-on-charcoal (16 mg) under H2 atmosphere for 7 h at
rt. After removal of the catalyst by filtration, the filtrate
was concentrated. The residue was dissolved in conc
HCl/ethanolꢁ1/10 (0.18 ml) and the solution was stirred at
50 °C for 6 h. 0.47 ml of water was added and stirred at room
temperature (rt) for over night to form a precipitate of
HCl·FTY-720-P. The precipitate was collected by filtration,
washed with EtOAc. The compound (8.3 mg, 76%) was used
1
for the biological tests without further purification. H-NMR
(CD3OD) d: 0.871 (3H, t, Jꢁ3.20 Hz), 1.22—1.29 (10H, m),
1.58 (2H, s), 1.92—1.98 (2H, m), 2.54—2.65 (2H, m),
3.47—3.74 (2H, m), 4.08—4.30 (2H, m), 7.00—7.18 (4H,
m). ESI-MS m/z: 388.5 (MꢂHꢂ).
Benzyl 2-(2-Chlorophenoxy)-5-[2-(4-octylphenyl)ethyl]-
2-oxido-1,3,2-dioxaphosphinan-5-ylcarbamate (4) To a
solution of 320) (200 mg, 0.45 mmol) in dioxane (2.72 ml)
was added 2-chlorophenylphosphordichloridate (0.117 ml,
0.68 mmol) and pyridine (0.110 ml, 1.36 mmol). Being stirred
at rt for 22 h, the solution was concentrated and dissolved in
CHCl3 (15 ml). The solution was washed with water and
dried. Concentration and subsequent separation with prepara-
Fig. 2. Synthetic Scheme for FTY720-P (A) and cFTY720-P (B)
Conditions: (a) triethyl orthoacetate, DIEA; (b) (BnO)2PN(iso-Pro)2, 1H-tetrazole
and then I2; (c) H2, 10% Pd/C; (d) conc HCl; (e) Z-Cl; (f) 2-chrolophenylphospho-
rodichroridate; (g) 2-nitrobenzoaldxime, 1,1,3,3-tetramethylguanidine; (h) H2, 10%
Pd/C.
tive SiO2 TLC (CHCl3/methanolꢁ9/1) gave 4 (171.6 mg,
1
was synthesized according to a method previously de- 62%) as a colorless oil. H-NMR (CDCl3) d: 0.875 (3H, t,
tailed.18,19) FTY720-P20) and cFTY720-P21) were synthesized Jꢁ6.5 Hz), 1.14—1.41 (10H, m), 1.57 (2H, t, Jꢁ7.3 Hz),
starting from FTY720, as shown in Fig. 2. The spectroscopic 2.00 (2H, t, Jꢁ8.1 Hz), 2.22 (2H, t, Jꢁ8.1 Hz), 2.54 (4H, t,
data were identical to that reported previously. Precise syn- Jꢁ7.8 Hz), 4.34 (2H, dd, Jꢁ11.1, 17.3 Hz), 4.42 (2H, d,
thetic methods for the preparation of FTY720-P, cFTY720-P Jꢁ11.3 Hz), 4.59 (2H, dd, Jꢁ11.3, 24.0 Hz), 4.79 (2H, dd,
and the intermediates are described hereafter.
Jꢁ8.9, 11.1 Hz), 5.04 (2H, s), 5.06 (1H, s), 5.10 (2H, s),
{2-Methyl-4-[2-(4-octylphenyl)ethyl]-4,5-dihydro-1,3- 6.94—7.49 (13H, m). 13C-NMR (CDCl3) d: 14.2, 22.7, 28.7,
oxazol-4-yl}methanol (1)20) A solution of FTY720 (1.30 g, 29.3, 29.5, 31.6, 31.8, 31.9, 32.7, 35.5, 53.0 and 53.1 (con-
4.2 mmol), N,Nꢀ-diisopropylethylamine (1.09 ml, 6.3 mmol) former), 53.6 and 53.7 (conformer), 66.9 and 67.0 (con-
and triethyl orthoacetate (0.93 ml, 5.1 mmol) in DMF former), 72.4 (JC–Pꢁ6.7 Hz) and 73.8 (JC–Pꢁ7.8 Hz, con-
(10 ml) was stirred at 120 °C for 2 h. The solution was former), 120.9, 121.2, 126.0, 127.9, 128.0, 128.1, 128.2,
extracted with EtOAc, washed with sat. NaCl and dried. 128.4, 128.5, 130.5, 135.7, 135.9, 137.2, 137.3, 140.9, 146.0
Concentration and subsequent SiO2 column chromatography and 146.1 (conformer), 145.7 and 145.8 (conformer), 154.4,
(chloroform/methanolꢁ9/1) gave 1 (1.20 g, 86%) as a color- 154.7. IR (KBr) cmꢃ1: 1236, 1309, 1497, 1724, 2854, 2926,
1
less oil. H-NMR (CDCl3) d: 7.08 (4 H, s), 4.22 (1H, d, 3292, 3568, 3587. FAB-MS m/z: 614 (MꢂHꢂ), 277, 255,
Jꢁ8.4 Hz), 4.06 (1H, d, Jꢁ8.4 Hz), 3.68 (1H, d, Jꢁ11.0 Hz), 209, 185, 143, 117, 91. HR-FAB-MS m/z: 614.2431 (Calcd
3.44 (1H, dd, Jꢁ11.0, 7.8 Hz), 2.61—2.49 (4H, m), 2.10— for C33H42ClNO6P: 614.2438).
2.04 (1H, m), 2.02 (3H, s), 1.88 (1H, ddd, Jꢁ13.6, 11.4,
Benzyl 2-Hydroxy-5-[2-(4-octylphenyl)ethyl]-2-oxido-
5.8 Hz), 1.75 (1H, ddd, Jꢁ13.6, 11.0, 5.8 Hz), 1.58 (2H, 1,3,2-dioxaphosphinan-5-ylcarbamate (5) A solution of
quint, Jꢁ7.5 Hz), 1.34—1.23 (10H, m), 0.87 (3H, t, Jꢁ 2-nitrobenzaldoxime (39.7 mg, 0.24 mmol), 1,1,3,3-tetrameth-
6.8 Hz). ESI-MS m/z: 332 (MꢂHꢂ).
ylguanidine (59.7 ml, 0.48 mmol) in dioxane (0.5 ml) was
Dibenzyl {2-Methyl-4-[2-(4-octylphenyl)ethyl]-4,5-dihy- added to a solution of 4 (69.6 mg, 0.12 mmol) in dioxane
dro-1,3-oxazol-4-yl}methyl Phosphate (2) To a solution (0.6 ml) and stirred for 44 h at rt. After removal of the solvent
of 1 (57.7 mg, 0.174 mmol) in CH2Cl2 (1.1 ml) was added by evaporation, the residue was dissolved in methanol and
0.45 M 1H-tetrazole in CH3CN (0.77 ml) and N,Nꢀ-diiso- treated with SEPHADEX IR-120 ion exchange resins. Con-
propyldibenzylphospholamidite (74 ml, 0.23 mmol). After centration and subsequent separation with preparative SiO2
being stirred for 1 h the reaction mixture was added to TLC (CHCl3/methanolꢁ7/3) gave 5 (43 mg, 75%) as a color-
a 10% solution of I2 in THF/pyridine/H2Oꢁ66/1/33. 5% less oil. 1H-NMR (CD3OD) d: 0.89 (3H, t, Jꢁ6.8 Hz),
Na2SO3 aq. was added and extract with CHCl3, wash with 1.19—1.40 (10H, m), 1.57 (2H, t, Jꢁ6.8 Hz), 1.93 (2H, t,
brine and dried. Concentration and subsequent separation Jꢁ6.8 Hz), 2.53 (4H, t, Jꢁ6.8 Hz), 4.03—4.41 (4H, m), 5.07
with preparative SiO2 TLC (EtOAc/methanolꢁ9/1) gave 2 (2H, s), 6.91—7.07 (4H, m), 7.22—7.43 (5H, m). 13C-NMR
(44.7 mg, 44%) as a colorless oil. 1H-NMR (CDCl3) d: 0.856 (CD3OD) d: 14.5, 23.7, 29.9, 30.3, 30.4, 30.6, 32.8, 33.0,
(3H, t, Jꢁ7.19 Hz), 1.25—1.50 (10H, m), 1.862 (2H, d, 33.6, 36.5, 55.0, 67.0, 72.0, 128.7, 128.8, 129.1, 129.2,
Jꢁ9.59 Hz), 1.91—1.99 (2H, m), 2.02 (3H, s), 2.45—2.58 129.3, 140.1, 141.3, 157.1. IR (KBr) cmꢃ1: 122.8, 1506.3,
(4H, m), 3.65—4.25 (2H, m), 4.77—4.86 (2H, m), 6.86— 1712.2, 2358.8, 2854.5, 2927.7, 3018.4, 3647.1, 3674.1.
7.23 (14H, m). ESI-MS m/z: 592.8 (MꢂHꢂ), HR-FAB-MS FAB-MS m/z: 504 (MꢂHꢂ), 440, 414, 392, 370, 255, 239,

June 2008
1179
Table 1. Inhibitory Effects of FTY720, Sphingosine and ISP-I-55 on Can-
cer Cell Proliferation, Determined by WST-1 Assay
223, 207, 155, 115, 91. HR-FAB-MS m/z: 504.2516 (Calcd
for C27H39NO6P: 504.2515).
5-Amino-5-[2-(4-octylphenyl)ethyl]-1,3,2-dioxaphosphi-
nan-2-ol 2-oxide (cFTY720-P)21) A solution of 5 (45.8 mg,
0.091 mmol) in ethanol (5 ml) was stirred under H2 atmo-
sphere on 10% palladium-on-charcoal (4.5 mg) for 21 h at
rt. After removal of the catalyst by filtration, the filtrate was
concentrated to afford cFTY720-P (28.9 mg, 86%) as a col-
IC50 (mM)
Cell line
FTY720
Sphingosine
ISP-I-55
MCF-7
MDA-MB-231
Sk-Br-3
HCT-116
SW620
6.8ꢄ0.6
6.7ꢄ0.5
5.8ꢄ1.3
5.7ꢄ1.2
6.7ꢄ0.6
12.6ꢄ1.4
14.7ꢄ0.8
18.2ꢄ0.7
17.0ꢄ0.9
6.8ꢄ1.2
8.5ꢄ1.5
12.3ꢄ0.7
17.1ꢄ0.7
16.4ꢄ1.4
6.6ꢄ1.2
1
orless oil. H-NMR (CD3OD) d: 0.798 (3H, t, Jꢁ5.95 Hz),
1.16—1.69 (26H, m), 3.72 (2H, dd, Jꢁ10.8, 18.1 Hz), 4.04
(2H, dd, Jꢁ6.2, 10.8 Hz). 13C-NMR (CD3OD) d: 14.5, 23.6,
23.8, 30.5, 30.6, 30.7, 30.8, 31.5, 33.1, 35.1, 40.1, 51.6, 75.4
(JC–Pꢁ5.6 Hz). IR (KBr) cmꢃ1: 1218, 1503.3, 2358.8,
2854.5, 2927.7, 3018.4, 3637.1, 3574.1. FAB-MS m/z: 370
(MꢂHꢂ), 286, 255, 203, 185, 155, 143, 115, 93, 91.
IC50 values, the concentrations of tested compounds causing 50% growth inhibition
in the WST-1 assay 48 h after treatment, were determined by linear interpolation
between the values immediately above and below the 50% inhibition. Values are
meansꢄS.D. from at least three independent experiments.
Cell Lines and Culture Conditions All of the cell lines Arlington Heights, IL, U.S.A.).
were purchased from the American Type Culture Collection
(ATCC, Manassas, Virginia). The human breast carcinoma RESULTS
cell lines MCF-7, MDA-MB-231 and Sk-Br-3 were main-
tained in an RPMI-1640 medium (Sigma-Aldrich Co., St.
Inhibitory Effect of FTY720, Sphingosine and ISP-I-55
Louis, MO, U.S.A.) with 10% fetal bovine serum (Gibco on Cancer Cell Proliferation A WST-1 assay was per-
BRL, Grand Island, NY, U.S.A.) and 50 U/ml ampicillin at formed to determine the IC50 values of FTY720 and sphingo-
37 °C in a humidified atmosphere containing 5% CO2. The sine against the proliferation of human breast cancer cell
human colon carcinoma cell lines HCT-116 and SW620 were lines, MCF-7, MDA-MB-231, and Sk-Br-3 as well as human
maintained in the same conditions except that McCoy’s 5A colorectal cancer cell lines, HCT-116 and SW620. The re-
medium (Invitrogen, Carlsbad, CA, U.S.A.) was exploited.
sults demonstrated that treatment of the compounds caused
In Vitro Proliferation Assay Cells were seeded onto 96- cell death in a dose-dependent manner. As shown in Table 1,
well plates at the densities of 3000 cells/well. After 24 h of FTY720 exhibited comparatively low IC50 values within the
pre-cultivation, appropriate concentrations of the samples concentration range of 5—7 mM for all of the cells tested in
were added and the cells were cultured for 2 d. The relative this study. Judging from the values, the effects of sphingo-
growth ratio was determined by WST-1 assay using a cell sine were two to three times lower than those of FTY720, ex-
counting kit (Dojindo Laboratories, Kumamoto, Japan) ac- cept for the comparative effects on SW620 cells. We also
cording to the manufacturer’s instructions. The IC50 value tested an analog of FTY720, ISP-I-55, which lacks an aro-
was determined from the dose response curve. At least three matic ring in the aliphatic chain region. ISP-I-55 induced
independent experiments were performed. The proliferation dose-dependent growth inhibition against breast cancer cell
assay that tested the effects of DMS was performed using lines and colon cancer cell lines with IC50 values comparable
identical methods. Compounds with or without DMS were to those of sphingosine.
added to the wells and the plates were incubated for 78 h at
37 °C in a humidified atmosphere containing 5% CO2 before Antiproliferative Activity of FTY720 It is known that
the WST-1 assay was conducted. FTY720 needs to be mono-phosphorylated by sphingosine
Effect of Sphingosine Kinase Inhibitor DMS on the
Western Blot MCF-7 cells on a 60-mm plate were culti- kinase in the cytosol in order to become biologically active
vated for 24 h in a serum-free medium prior to the addition on sphingosine-1-P-receptors, S1P-R1 and S1P-R3.9) We used
of the test compounds. The cells were incubated with a potent inhibitor of sphingosine kinase, DMS,16) to clarify
FTY720 (10 mM) or sphingosine (20 mM) in a PRMI-1640 the effects of the phosphorylation of FTY720 on the prolifer-
medium. After 0.5, 2, 4, 8, 12, or 24 h the medium was re- ation of a breast cancer cell line, MCF-7, on which S1P-R1
moved and the cells were washed twice with PBS. The cells and S1P-R3 are highly expressed.22) FTY720 in various con-
were lysed and protein extraction was performed. The com- centrations with or without 10 mM DMS was treated with
ponents of the lysis buffer were as follows: 1% Nonidet P-40 MCF-7 cells for 78 h and the cell viabilities were measured
(Roche Diagnostics GmbH, Penzberg, Germany), 0.1% SDS, (Fig. 3). The graph shows that the antiproliferative activity of
0.05% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 10% FTY720 was enhanced rather than inhibited by co-treatment
glycerol, 100 mM NaCl, 20 mM sodium pyrophosphate, 2 mM with DMS, suggesting that the endogenous phosphorylation
sodium orthovanadate, 1 mM NaF, 1.2 mg aprotinin, 0.2 mg of FTY720 is not required for its cytotoxicity. The increased
leupeptin, 0.02 mg pepstatin, and 1 mM PMSF in 10 mM activity may result from the increased amount of intact
Tris–HCl (pH 8.0). The samples were separated by SDS- FTY720 resulting from the inhibition of its phosphorylation.
PAGE (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan)
Effect of Phosphorylation of FTY720 on Cancer Cell
electrophoretically transferred to a polyvinylidene difluoride Proliferation In order to investigate the effects of exoge-
membrane (Pall Co., East Hills, NY, U.S.A.) and labeled nous phosphorylated FTY720, we synthesized FTY720-P
with rabbit polyclonal anti-ERK, anti-phospho-ERK, anti- and a phosphatase-tolerant phosphinane FTY720 derivative,
JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38 and cFTY720-P. The IC50 values of these agents against breast
anti-actin. They were visualized with peroxidase-conjugated cancer cell lines (MCF-7, MDA-MB-231, Sk-Br-3) and
second antibodies using a standard ECL kit (Amersham, Inc., colon cancer cell lines (HCT-116, SW620) were evaluated

1180
Vol. 31, No. 6
Fig. 3. Effect of Sphingosine Kinase Inhibitor DMS on Antiproliferative
Activity of FTY720 against Breast Cancer Cell Line MCF-7
Fig. 5. Effects of FTY720 and Sphingosine on Phosphorylation of
ERK1/ERK2, JNK1/JNK2 and p38 in MCF-7 Cells
Compounds with and without DMS (10 mM) were added to the wells and the plates
were incubated for 78 h (37 °C, 5% CO2) before a WST-1 assay was conducted. Bars
represent the S.D. of triplicate assays.
Cultured cells were incubated with 10 mM FTY720 for 0.5 h (lane 2), 2 h (lane 3), 4 h
(lane 4) and 8 h (lane 5) and with 20 mM sphingosine for 0.5 h (lane 6), 2 h (lane 7), 4 h
(lane 8) and 8 h (lane 9). The cell extracts were examined by Western blotting using the
respective antibodies as described in Materials and Methods. An actin antibody served
as the loading control.
Table 2. Effect of Phosphorylated FTY720 Derivatives, FTY720-P and
cFTY720-P on Cancer Cell Proliferation
IC50 (mM)
MCF-7. Treatment of MCF-7 cells with FTY720 for 0.5 h
caused a strong down-regulation of phosphorylated ERK1/
ERK2 (p-ERK1/p-ERK2), whereas long term incubation
caused a recovery of p-ERK1/p-ERK2 expression level. The
phosphorylation of ERK was also inhibited by treatment of
the cells with sphingosine for 0.5 h. On the other hand, the
phosphorylation of JNK1/JNK2 was induced 2—8 h after the
addition of FTY720 or sphingosine. Both FTY720 and
sphingosine led to the activation of JNK1/JNK2, but not of
p38 (Fig. 5).
Cell line
FTY720-P
cFTY720-P
MCF-7
MDA-MB-231
Sk-Br-3
HCT-116
SW620
79.1ꢄ2.0
59.9ꢄ9.2
72.9ꢄ7.7
ꢅ100
ꢅ100
ꢅ100
ꢅ100
ꢅ100
ꢅ100
40.0ꢄ12.1
IC50 values were determined by linear interpolation between the values immediately
above and below the 50% inhibition. The relative growth ratio of the cells was deter-
mined by WST-1 assay 48 h after the treatment of the test compounds. Values are
meansꢄS.D. from at least three independent experiments.
DISCUSSION
We evaluated the antiproliferative effects of FTY720,
sphingosine and their analogues on human breast cancer cell
lines MCF-7, MDA-MB-231, and Sk-Br-3 as well as human
colorectal cancer cell lines HCT-116 and SW620. FTY720,
sphingosine and ISP-I-55 inhibited the growth of these cells.
FTY720 showed the highest cytotoxicity among the tested
compounds. ISP-I-55 has a structure lacking a benzene ring
found in FTY720. Judging from the mouse allogeneic mixed
lymphocyte reaction,17) the immunosuppressive effect of ISP-
I-55 is less active than that of FTY720. A similar structure–
activity relationship has been shown in the cytotoxicity
between these two compounds. Western blot analysis of the
cytosolic fraction of MCF-7 cells demonstrated that 10 mM
FTY720 or 20 mM sphingosine temporally deactivated the
phosphorylation of a survival MAPK, ERK, and activated
Fig. 4. Concentration Dependence of Cell Viabilities of MCF-7 Cells
Treated with FTY720-P and Sphingosine-1-P
The relative growth ratio of the cells was determined by a WST-1 assay conducted
48 h after the treatment of the test compounds. Values are meansꢄS.D. from at least
three independent experiments.
using a WST-1 assay (Table 2). Both compounds showed the phosphorylation of death kinase, JNK without modula-
considerably high IC50 values in comparison to FTY720. The tion of p-p38. The recovery of p-ERK expression after the
low concentration range of FTY720-P induced cell growth temporal down-regulation would be associated with the regu-
slightly in MCF-7. This tendency was stronger for sphingo- lation of apoptosis or survival signaling in the cells.23,24) The
sine-1-P, although this compound was more cytotoxic in a results suggested that the antiproliferation of FTY720 and
higher concentration range (Fig. 4).
sphingosine proceeded in a similar manner including the in-
The Effect of FTY720 and Sphingosine on the Activa- duction of apoptosis. Although FTY720-P and cFTY720-P
tion of Major MAPKs in MCF-7 Cells We evaluated the are active mediators in the immunomodulation and endothe-
effect of FTY720 (10 mM) and sphingosine (20 mM) on the lial cell functions, these compounds affected little to the cell
phosphorylation of major MAPKs, survival MAPK/extracel- growth. Observation of the low cytotoxicity of FTY720-P
lular signal-regulated kinase, ERK1/ERK2, the stress-acti- may be caused by endogenous dephosphorylation, since
vated MAPK c-Jun NH2-terminal kinase, JNK1/JNK2 and phosphatase-tolerant cFTY720-P has much less of an effect.
the death kinase p38 in a human breast cancer cell line, Furthermore, the pretreatment of DMS did not inhibit the ef-

June 2008
1181
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the cytotoxicity of the compound.
Acknowledgments This work was supported by a Grant-
in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan and
Kansai University’s Overseas Research Program 2005.
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