Inhibition of Mitosis by Binding to the Colchicine ... - Cancer Research

Mechanism of Action of E7010, an Orally Active Sulfonamide
Antitumor Agent: Inhibition of Mitosis by Binding to the
Colchicine Site of Tubulin
Kentaro Yoshimatsu, Atsumi Yamaguchi, Hiroshi Yoshino, et al.
Cancer Res 1997;57:3208-3213.
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ICANCERRESEARCH57, 3208-3213. August 1, 19971
Mechanism of Action of E7010, an Orally Active Sulfonamide Antitumor Agent:
Inhibition of Mitosis by Binding to the Colchicine Site of Tubulin
Kentaro Yoshimatsu,' AtSUmi Yamaguchi, Hiroshi Yoshino, Nozomu Koyanagi, and Kyosuke Kitoh
Tsukuba Research Laboratories, Eisai Co. Ltd., 1-3 Tokodai 5-chome, Tsukuba-shi, Ibaraki 300-26, Japan
ABSTRACT
E7010 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyll-4-methoxybenze
nesulfonamide), an orally active sulfonamide antitumor agent that is
currently in a Phase I clinical trial, showed rather consistent growth
inhibitory activities against a panel of 26 human tumor cell lines
(IC@ 0.06-0.8 @ag/ml), in contrastto vlncristine(VCR;ICso 0.0002
0.04 @.tg/ml), 5-fluorouracil (IC@ = 0.2—30 gag/mI), Adriamycin
(IC50= 0.002-0.7 gag/ml),mitomycinC (ICso 0.0073 @Lg/Ifll),
arobinofuronoxylcytosine (ICse 0.005 to >30 gLg/ml), camptothecin
and tubulin-binding agent with different properties from those of
Taxol, VCR, or CLC.
MATERIALS AND METHODS
Drugs. E7010 and 14C-labeledE70l0 (specific activity, 56 mCi/mmol;
phenyl-U-RING-'4C; Fig. 3B) were synthesized at Tsukuba Research Labo
1-@3-D- ratories, Eisai Co. Ltd. Reference compounds were obtained from the follow
ing sources: VCR, vinblastine, CDDP, CPT, VP16, and melphalan were
@ (ICso 0.®2@0.4 andcisplatin(ICso 0.520 @LgJ1fll). It caused obtained from Sigma (St. Louis, MO); 5-FU was obtained from Tokyo Kasei
a dose-dependent increase in the percentage of mitotic cells in parallel (Tokyo, Japan); ADM, MMC, CLC, POD, and MTX were obtained from
with a decrease in cell proliferation, like VCR. It also showed a dose Wako Junyaku (Tokyo, Japan); and Ara-C (Cylocide) was obtained from
dependent inhibition of tubulin polymerization, which correlated well Nippon Sinyaku (Kyoto, Japan). Taxol was obtained from the Drug Synthesis
with the cell growth-Inhibitory activity. ‘4C-labeledE7010 bound to pu and Chemstry Branch, Division of Cancer Treatment, National Cancer Insti
rifled tubulin, and this binding was Inhibited by colchicine but not by tate. Trisnordolastatin was kindly provided by Dr. Shioiri (Nagoya City
VCR. However,its bindingpropertieswere differentfrom those of col University, Nagoya, Japan). [3H]VCR was purchased from Amersham, and
chicine, as well as those of VCR. E7010 was active against two kinds of [3HICLC was purchased from DuPont New England Nuclear.
VCR-resistant P388 cell lines, one of which showed multidrug resistance
due to the overexpressionof P-glycoproteln(resistantto Taxol),and the
Cell Lines. P388 murine leukemia and MX-l were supplied by Cancer
Chemotherapy Center, Japan Foundation for Cancer Research (Tokyo, Japan).
otherdidnotshowmultidrugresistance(sensitiveto Taxol).Furthermore,
four E7010-resistant P388 cell lines showed no cross-resistance to VCR, a
VCR-resistant P388 cell line (P388/VCR) with overexpression of P-glycopro
1cm(4)waskindlysuppliedbyDr.M.Inaba(CancerChemotherapy Center,
different pattern of resistance to podophyllotoxin, and collateral sensitiv Japan Foundation for Cancer Research). H460, H520, H596, HCT-!16,
ity to Taxol. Therefore, E7010 is a novel tubulin-binding agent that has a SW480, SW620, COLO2O5,DLD-!, HCF-l5, HT-29, MiApaca-Il, and HL-60
wider antitumor spectrum than VCR and has different properties from were obtained from the American Type Culture Collection (Rockville, MD).
those of VCR or Taxol.
PC1, PC9, and MKN28 were obtained from the Immuno Biology Laboratory
(Gunma, Japan). A549, WiDr, LS!74T, COLO32ODM, ZR-75-l, CCRF
INTRODUCTION
CEM, and K562 were obtained from Dainippon Pharmaceutical Co., Ltd.
(Osaka, Japan). PC1O,MKN45, and KATO-3 were obtained from the Japanese
As part of the search for clinically effective antitumor drugs to treat Cancer Research Resources Bank (Tokyo, Japan). All cell lines were main
refractory solid tumors, we have screened a number of sulfonamides,
because this class of compounds is well known to have a variety of
mined in RPMI 1640 containing 10% heat-inactivated fetal bovine serum,
penicillin (100 units/nil), streptomycin (100 @g/ml), 2-mercaptoethanol (50
biological activities, such as antibacterial, insulin-releasing, carbonic
anhydrase-inhibitory, and antithyroid activities (1). We found that
E7010 showed a wide therapeutic range of antitumor activity in the
colon 38 carcinoma model (2). It inhibited the growth of colon 38
cells inoculated s.c. by 60—99%when administered at doses of 25—
@LM), and sodium pyruvate (1 mM).
MTT Assay. Exponentially growing cells were plated into 96-well tissue
culture plates and precultured for 1 day. Various concentrations of test com
pounds were added, and the cells were incubated for an additional 3 days. The
antiproliferative activity was measured by MU assay, and the IC50 values
were determined.
100 mg/kg daily for 8 days (3). It was also active against mouse Mitotic Index. P388 cells were treated with the indicated concentrations of
tumors (s.c. inoculated M5076 fibrosarcoma, s.c. inoculated Lewis E70l0 or VCR for 12 h. Viable cell numbers were determined by the trypan
lung carcinoma, and i.p. inoculated P388 leukemia), a rat tumor (s.c. blue dye exclusion method, and cells were treated with 0.075 M KC1, fixed
inoculated SST-2 mammary carcinoma), and human xenografts (LC
376, LC-6, and LX- 1 lung cancer; H-8l , H-! 11, SC-2, and SC-6
gastric cancer; H-143, COLO32ODM, and WiDr colon cancer; and
H-31 and MX-l breast cancer) upon oral administration (3). More
over, E70l0 was effective against VCR2-resistant, CDDP-resistant,
and 5-Ri-resistant P388 cells in vivo (3).
The present study was designed to elucidate the mechanism of
(3:1, methanol:acetic acid), and stained with 0.1% crystal violet. The mitotic
index was determined by counting at least 500 cells.
Microtubule Polymerization Assay. Microtubule protein from bovine
brain was prepared by two cycles of assembly and disassembly in assembly
buffer [0.1 M morpholinoethanesulfonate, 1 mM EGTA, and 0.5 mM MgCl2
(pH 6.8); Ref. 5]. Protein concentration was determined using the Bio-Rad
protein assay kit. The reaction mixture contained 2 mg/mI mcrotubule protein
and the half-log step concentrations (0.89, 2.7, 8.9, and 27 ,.@M)of E70l0 in
action of E7010, and we report here that E70l0 is a novel antimitotic assembly buffer containing 1 mM GTP. Microtubule polymerization was
initiated by warming the sample from 0°Cto 37°Cin a 1-mi cuvette, and
Received 3/4/97; accepted 6/2/97.
turbidity (@A350) was recorded during the indicated time intervals with a
The costs of publication of this article were defrayed in part by the payment of page spectrophotometer (Fig. 3A). In the study of the relationship between antipro
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I To whom requests for reprints should be addressed. Phone: 81-298-47-5749; Fax:
81-298-47-2037.
2 The abbreviations used are: VCR, vincristine; 5-FU, S-fluorouracil; ADM, Adria
liferative activity and microtubule polymerization-inhibitory activity (Fig. 3B),
the wells of 96-well plates contained 1 mg/ml microtubule protein and log step
concentrations of E70l0 derivatives. The plates were incubated at 37°C,and
the turbidity was recorded with a mcroplate reader (ThermoMax; Molecular
mycin; MMC, mitomycin C; Ara-C, l-@-o-arabinofuranoxylcytosine; CPT, camptothecin;
CDDP,cisplatin;CLC, colchicine;POD, podophyllotoxin;NOC,nocodazole;MTX,
methotrexate; VP16, etoposide; MNNG, N-methyl-N'-nitro-N-mtrosoguanidine; MU,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MDR, multidrug-resistant;
Devices) for 20 mn kinetically. The turbidity at 20 mm (the polymerization in
the control reached a plateau level) was plotted on a linear scale versus the
concentration of the test compound on a log scale, and the concentration
CQS, chioroquinoxaline sulfonamide.
causing a 50% inhibition of polymerization was determined.
3208
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@
@
.@)
‘3
0
j
@iO@OO@ @-@(OOU)
@ .1.
c@
MECHANISM OF ACFION OF E70l0
Fig. 1. The effects of E70l0 and antitumor drugs on the proliferation of 26 human
tumor cell lines. Cells were treated with various concentrations of the compounds for 3
days.The antiproliferativeactivitywas determinedby MU assay,and IC50for each
compoundwascalculated.A:•,E7010;0, VCR;i@,5-Ri; D, ADM.B:0, MMC; @,
Ara-C; 0, CPT; X, CDDP. The cell lines used were non-small cell lung cancers PC1, PC9,
PC1O, A549, H520, H460, and H596; colon cancers WiDr, LS174T, COLO32ODM,
HCT-l 16, SW620, C0L0205, DLD-l, HCF-15, SW480, and HT29; stomach cancers
MKN28, MKN45, and KATO3; pancreatic cancer MiApaca-Il; breast cancers MX-l and
@-75-l;and leukemias CCRF-CEM, 1(562, and HL-60.
3209
E7010-resistant clones 0.5r-D, l.Or-H, and 4.Or-M were cloned, respectively.
E7010-resistant cell line 0.6r-8 was isolated by a four-step selection. MNNG
treatment was used at each step, and the dose of E7010 at each step was 0.1,
0.2, 0.3, and 0.6 @g/ml.After the four-step selection, 0.6r-8 was cloned.
RESULTS
The Effects of E7010 and Antitumor Drugs on the Proliferation
of Human Tumor Cell Lines. The antiproliferative activities of
E7010 and various clinically used antitumor drugs were tested against
a panel of 26 human tumor cell lines (7 lung cancers, 10 colon
cancers, 3 stomach cancers, 1 pancreatic cancer, 2 breast cancers, and
3 leukemias) by MU assay. E70l0 showed a dose-dependent and
broad-spectrum antiproliferative activity (Fig. 1). The IC50 values of
E7010 on various human tumors were rather similar (0.06—0.8 p.g/
ml), in contrast to those of VCR (0.0002—0.04 @.tg/ml),5-FU (0.2—30
j@g/ml), ADM (0.002-0.7 @tg/ml),MMC (0.007-3 @g/ml),Ara-C
(0.005->30 @.Lg/ml),CPT (0.002-0.4 @@g/ml), and CDDP (0.5-20
@@g/ml).
The Effect of E7010 on Cell Cycle Progression. Antitumor
agents are known to arrest cell cycle progression in S phase, 02, or M
phase (10, 11). Therefore, the effect of E7010 on the cell cycle
progression of P388 cells was examined by flow cytometry, and
E70l0 was found to arrest P388 cells in the G2-M phase. Next, we
examined whether E7010 increases the percentage of mitotic cells,
because agents that arrest cells in M phase, such as VCR, increase the
mitotic index, but agents that cause G2 arrest, such as VP16, decrease
it. E7010 caused a dose-dependent increase in the mitotic index, like
VCR (Fig. 2), and the rise in mitotic index was well correlated with
the decrease in cell proliferation. We also observed an increase in the
mitotic index of s.c. implanted M5076 tumor at 18—24h after the oral
administration of E7010 (data not shown).
The Effects of E7010 and Its Derivatives on Microtubule
Polymerization. The results described above and the immunofluo
rescence observation that E70l0 caused the disappearance of cyto
plasmic microtubules in colon 38 cells (data not shown) led us to
examine E7010 for effects on microtubule polymerization. E70l0
caused dose-dependent inhibition of microtubule polymerization (Fig
ure 3A). Furthermore, a linear relationship between the antiprolifera
Binding Assay. Tubulin was purified from microtubule protein on a phos
60
8
phocellulose column (6). The binding assay was performed using the DEAE
cellulose filter method of Borisy (7). The reaction mixture (0.2 ml) contained
tubulin and ‘4C-labeled E70l0 (2 aiM;0.0224 pCi/reaction), [3H]VCR (0.25
50
,.tM; 0.05 MCi/reaction), or [3H]CLC (1 ,.LM;0.1 pCi/reaction) in assembly
buffer. The reaction was stopped by rapid cooling to 4°C, and then the mixture
40
was diluted with 5 ml ofO.lX assembly buffer, filtered through a stack of three
Whatman DE81 ion exchange paper discs using a BRANDEL cell harvester
@
(Biomedical Research and Development Laboratories, MD), and washed with
10 ml of 0.lX assembly buffer.
Isolation ofNon-MDR VCR-reslstant P388. VCR-resistant cell lines that
did not show the MDR phenotype were isolated from MNNG (Aldrich)-treated
P388 cells by selection with both increasing doses of VCR (0.5, 1.0, 2.0, and
4.0 ng/ml) and 10 @Mverapamil to avoid the development of the mdr
phenotype (8, 9). When the relative resistance of VCR-resistant cells to VCR
reached about 10 times, the VCR-resistant clone P3881V4—4 was cloned in
x
S
.u
.E
U
0
30
20
10
1@
VCR and verapamil-containing medium using the limiting dilution method.
The relative resistance was determined in the absence of verapamil by MU
0
.001 .01 .1 1 10
assay.
Isolation of E7010-resistant P388. E70l0-resistant cell lines 0.5r-D,
Concentration (@sg/mI)
1.Or-H,and 4.Or-M were isolated by MNNG-treated P388 cells by selection Fig. 2. The effects of E70l0 and VCR on proliferation and the mitotic index. P388 cells
with increasing doses of E70l0 (0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2,
2.0, 3.0, and 4.0 p@g/ml).When the relative resistance of E7010-resistant cells
were treated with the indicated concentrations of E70l0 (•, ) or VCR (0, []) for 12 h.
The number of viable cells (, El) and the mitotic index (•,0) were determined as
described in “Materials and Methods.â€Values represent the mean ±SE (bars) of three
to E70l0 reached about 5 (13 weeks), 10 (18 weeks), and 40 times (21 weeks), independent experiments.
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0.3
@ J//,,,,,,,—,—.--..,—------'—--———--.
Control
0
A
i
B
@-U,
00
C
.2a)
.0@
C
0.2
0.1
0.0
0
4
3
5 10 15 20 25
Time (mm)
l4@
R@H@Q
@1fl2
14
MECHANISM OF ACTION OF E7010
0.89 sM
2.7 @tM
8.9 sM
27 pM
R= 0.92
R2=H
R3=OH A
2 3 4 5
InhibitIon of tubulin polymerization
log [1C50(nM)]
6
Fig. 3. The effect of E70l0 on microtubule polymerization (A) and the correlation
between antiproliferative activities and inhibitory activities against microtubule poly
merization in E7010 derivatives (B). A, the reaction mixture contained 2 mg/mI micro
tubule protein and the indicated concentration of E70l0 in assembly buffer. Microtubule
polymerization was recorded as described in “Materials and Methods.â€B, in this study, the
effects of E7010 derivatives on microtubule polymerization were determined using 1
mg/mI microtubule protein. Each point, the antiproliferative activity and antipolymeriza
tion activity of an E7010 derivative. 1, R' CH3O, R2 = OH, and R3 = H; 2,
R' = CH3O,R2= H,andR3= H;3, R' = CH1O,R2= H,andR3= F;4, R' CH3O,
R2 = CH@,and R3 = OH; 5, R' = C2H5, R2 = H, and R3 = H; 6, R' = CH3O, R2 = H,
and R3 = Cl; 7, R' CH1O, R2 = H, and R3 = OH; 8, R' CH3, R2 = H, and R3 = H;
9, R' = C2H50, R2 = H, and R3 = H; 10, R' = CH3O, R2 = H, and R3 = C2H5O; 11,
R@= CH1O,R2= H,andR3= phenyl;12,R' = CH1O,R2= H,andR3= COOC2H5;
13, R' = CH3O, R2 = H, and R3 = CH3; 14, R' = CH3O, R2 = H, and R3 = CH3O;
15, R' = CH3O, R2 = H, and R3 = SCH3; 16, R' = CH3CO, R2 = H, and R3 = H; 17,
R' = CN,R2= H,andR3= H; 18,R' = NO2,R2= H,andR3= H; 19:R' = CH3O,
R2 CH3O,andR3= CH3O.
tive activity of a compound and its activity to inhibit microtubule
polymerization was found for various E70l0 derivatives that had been
synthesized during the development of E70l0 (Fig. 3B). These results
strongly suggest that the inhibition of microtubule polymerization is
responsible for the antiproliferative action of E7010.
The Binding of ‘4C-labeledE7010 to Tubulin. Compounds that
arrest the cells in mitosis and inhibit tubulin polymerization are
known to bind to tubulin at the Vinca site or the CLC site (12—15).
The binding of E7010 to tubulin was examined and compared with
the binding of VCR and CLC, which are typical tubulin-binding
agents (Fig. 4). The binding of E70l0 to tubulin was rapid and did
not show temperature dependence. On the other hand, the binding
of VCR was slow at 4°C,whereas CLC could not bind to tubulin
at 4°Cas reported previously (12), and its binding was slow even
at 37°C.
3210
Inhibition of ‘4C-labeledE7010, [3H]VCR, or [3H]CLC Binding
to Tubulin. To determine the binding site of E7010, inhibition of the
binding of ‘4C-labeledE70l0, [3H]VCR, or [3H]CLC to tubulin by
each unlabeled compound was examined. The binding of E70l0 to
tubulin was inhibited by CLC and enhanced by VCR (Fig. 5A). The
binding of CLC was inhibited by E7010 and was also enhanced by
VCR (Fig. SB). The enhancement of the binding by VCR was caused
by stabilization of the CLC-binding activity of tubulin (12, 16). The
binding of VCR was not inhibited by E7010 or CLC (Fig. SC).
Lineweaver-Burk analysis demonstrated that E7010 is a competitive
inhibitor of the binding of CLC and binds at the CLC site on tubulin
(Fig. SD). The K1value for E70l0 of [3H]CLC was 3.3 @M(Fig. SD),
and the K1 values for CLC, POD, and NOC, reported to bind at the
CLC site, were 2.7, 0.94, and 2.3 @M, respectively (data not shown).
Cross-Resistance in VCR-resistant P388 Cells. Antimitotic
drugs that are widely used in cancer chemotherapy, such as VCR,
vinblastine, and Taxol, are good substrates for transport by P-glyco
protein (classical MDR phenotype; Refs. 17—19).Moreover, other
types of resistance to Vinca alkaloids have been reported (20—22),and
we isolated VCR-resistant cells (P388/V4—4) that did not show the
classical MDR phenotype. Therefore, the effects of VCR, Taxol,
ADM, CLC, and E70!0 on two MDR P388 cells (P388IVCR and
P388/V0.256) and P3881V4—4cells were examined. The results are
summarized in Table 1. P388/VCR and P388N0.256 cells, which
overexpress P-glycoprotein, were significantly resistant to VCR,
Taxol, CLC, and ADM but were sensitive to E7010. P3881V4—4cells,
0
E
0.
•0
C
0
B
0
E
0.
•0
C
0
0 30 60 90 120
Time (mm)
Fig. 4. The binding of ‘4C-labeledE70l0, [3H]VCR, and [3H]CLC to tubulin at 4°C
and 37°C.Purified tubulin (0.45 mg/ml) was incubated with ‘4C-labeledE70l0 (•),
[3H]VCR (0), or [3H]CLC (@) at 4°Cor 37°Cfor the time indicated. The binding to
tubulin was determined by the DEAE-cellulose filter method as described in “Materials
and Methodsâ€and expressed as the mean for duplicate samples in a single experiment,
which was repeated with similar results.
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Fig. 5. A comparison of the effects of E7010, CLC, and VCR on the
binding of ‘4C-labeledEbb, [3HICLC, and [3H]VCR to tubulin.
Microtubule protein (0.4 mg/mI) was incubated with ‘4C-labeled E70l0
(A),[3HICLC(B andD) or [3H]VCR(C)andthe indicatedconcentra
tions of unlabeled compounds for 2 h at 37°C.A, B, and C: •,E70l0;
& CLC;0, VCR.In the Lineweaver-Burkanalysis(D),thefollowing
concentrations of [3H]CLC were used: 0.56, 0.83, 1.25, 2, 4, and 8 @sM.
The binding was determinedby the DEAE-cellulose filter methodas
described in “Materials and Methods.â€Points, the mean for duplicate
samples in a single experiment, which was repeated with similar results.
MECHANISMOF ACI1ON OF E70l0
0
1@
•1
C
0
U
which were isolated by exposure to VCR in the presence of verapamil,
showed cross-resistance to vinblastine, vindesine, and trisnordolasta
tin (a dolastatin analogue; Ref. 23) with resistance factors of 4.7, 9.4,
and 8.7, respectively, but they had a collateral sensitivity to Taxol.
P388/V4—4 cells were sensitive to E7010 and slightly resistant to
CLC.
Cross-Resistance in E7010-resistant P388 Cell Lines. For the
clinical application of a new drug, understanding the mechanisms by
which resistance is developed is important. Therefore, we isolated
four E7010-resistant cell lines and examined the sensitivity of E70l0resistant
cell lines to clinically used antitumor drugs (VP16, 5-FU,
CDDP, ADM, MTX, MMC, Ara-C, and melphalan; Fig. 6A) and
antimitotic agents (VCR, Taxol, CLC, NOC, and POD; Fig. 6B).
None of E7010-resistant cell lines (0.6r-8, 0.5r-D, l.Or-H, and 4.Or-M)
showed cross-resistance to any of the antitumor drugs of which the
target is not tubulin. On clinically used antimitotic drugs, E70l0resistant
cell lines exhibited no significant resistance to VCR but had
increased sensitivity to Taxol. Among antimitotic agents that bind to
the CLC site, NOC showed a similar pattern of resistance across the
spectrum of E70l0-resistant cell lines (resistance factor, 4.Or
M 1.Or-H > 0.5r-D 0.6r-8). On the other hand, POD exhibited
a different pattern of resistance (resistance factor, O.5r-D and l.Or
H > 0.6r-8 and 4.Or-M). The pattern of resistance to CLC was similar
to that of POD, although the resistance to CLC was minimal.
•0
C
0
.0
0
0
p..
Lu
a
0
I-
C
0
U
0
•0
C
0
.0
0
> I
C')
@10@@ io6 io@ i@-@ io@ io6 io@ io@
Concentration (M) Concentration (M)
3211
1o6 io@ io@
Concentration (M)
DISCUSSION
-1 0 1
1/Coichicine (1/pM)
E70l0 was found by screening a number of sulfonamides with
widely different chemical structures (2) and shows a broad spectrum
of activity against mouse and human tumors (3). In a Phase I study of
E70l0 using a single-dose schedule, some responses were observed
(24), and a 5-day continuous administration study is in progress. We
have demonstrated here that E7010 is an antimitotic agent, and the
target of its antiproliferative action is tubulin. CQS was reported to
have antiproliferative activity in vitro (25, 26), but the mechanism of
its action has not yet been elucidated. Therefore, we examined the
effect of CQS on cell cycle progression and tubulin polymerization.
CQS did not arrest P388 cells in the G2-M phase and did not affect
tubulin polymerization. Therefore, E70l0 seems to be a unique sul
fonamide antitumor agent.
Most of the tumor cell lines tested were almost equally sensitive to
E7010 (IC50 = 0.06—0.8 @sg/m1).This is consistent with the idea that
the target of E7010 is tubulin, because microtubules in the mitotic
spindle are important for cell proliferation of all cells. In contrast, a
wider range of IC50 values (0.002—0.04 p.g/ml) was observed with
VCR. This might be due to the differential drug accumulation of VCR
among these tumor cells, which express P-glycoprotein to various
extents. Indeed, HCT-15 and DLD-l, which were sensitive to E70l0
(IC50 values of 0.08 and 0.14 p@g/ml,respectively) but very insensitive
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2

,
linesP388
Table 1 The effects of VCR, Taxol, ADM, CLC, and E7010 on three VCR-resistant P388 cell
cells and VCR-resistant P388 cell lines were treated with various concentrations of the compounds for 3 days. Antiproliferative activity was determined as indicated in
“Materialsand Methods,â€and IC50 for each compound was calculated. Resistance factors were calculated line.Resistance as IC50 for the resistant cell line: IC50 for the sensitive cell
factorCell
ADMP388/VCR line VCR Taxol E7010 CLC
11.0 ±2.4'@ 6.3 ±0.9 1.2 ±0.2 — —
P388N@,56 82.8 ±7.7 16.9 ±0.3 1.5 ±0.3 64.5 29.9 ±2.9
0.08a P388/V4-4 8.4 ±0.7 0.18 ±0.05 1.3 ±0.2 2.7 ±0.2 0.58 ±
Values representthe mean ±SE.
to VCR, were reported to express P-glycoprotein and to show differ
ential sensitivity to non-MDR versus MDR drugs (27). These obser
vations are consistent with the finding that P388IVCR and P388/
V0.256 were resistant to VCR but sensitive to E7010. The patterns of
antiproliferative activity against tumor cell lines except HCT-l5 and
DLD-l were similar for VCR and E7010, although the ranges of IC50
values were different.
To examine the mechanism of action of E70l0, we first examined
the effect on cell cycle progression, because this is related to the
mechanism of action (10, 11). Antimetabolites, such as MTX and
Ara-C, cause cells to accumulate in the S phase. Topoisomerase
inhibitors, VP16 and CPT, ADM, and MMC inhibit cell cycle pro
gression in the 02 phase. Vinca alkaloids and Taxol arrest cells in the
M phase. By flow cytometric analysis, we found that E70l0 accumu
lated P388 cells in G2-M phase. E70l0 caused dose-dependent in
creases in the mitotic index in parallel with an inhibition of cell
proliferation, like VCR.
Because the antimitotic effects of antimitotic agents may be medi
ated by disruption (VCR) or stabilization (Taxol) of microtubules in
mitotic spindles, the effects of E70l0 on microtubule structure in
colon 38 cells were examined, and E7010 was shown to cause the
disappearance of cytoplasmic microtubules and mitotic spindles (data
not shown). Therefore, the effects of E7010 and E70l0 derivatives on
microtubule assembly in a cell-free system were examined and corn
pared with the growth-inhibitory activities. These experiments clearly
demonstrated that the growth-inhibitory activity of E70l0 is caused
by the inhibition of rnicrotubule assembly. Next, we examined
whether E70l0 can bind to tubulin and compared the binding prop
erties of E70l0 with those of VCR and CLC, although the structure of
E70l0 is quite different from those of the known tubulin binders,
Fig. 6. Effects of antitumor agents and antimitotic agents on E7OIO
resistant P388 cell lines. P388 cells and E7OIO-resistant cell lines
(0.6r-8, 0.5r-D, I Or-H, and 4.Or-M) were treated with various concen
trations of the compounds for 3 days. Antiproliferative activity was
determined as indicated in “Materialsand Methods,â€and IC,0 for each
compound was calculated. Resistant factors were calculated as IC50 for
the resistant cell line:IC@0 for the sensitive cell line and shown graph
ically as the mean value ±SE (bars; n = 3—10).
MECHANISM OF ACtiON OF E7010
L-PAM
Ara-C
MMC
which bind to the CLC site or Vinca site on tubulin, such as CLC,
NOC, Vinca alkaloids, rhizoxin, halichondrin, and so on. We found
that E7010 binds to purified tubulin and that the binding site is the
CLC site, not the Vinca site, although the binding properties of E7010
to tubulin were quite different from those of CLC. Therefore, E7010
is quite distinct in its action from clinically used Vinca alkaloids or
Taxol. The preliminary structure-activity study (Fig. 3B) reveals that
the addition of methoxyl groups to the lower phenyl ring decreases the
activity (compounds 2, 14, and 19), which is intriguing in relation to
the structural factors of other CLC site ligands such as CLC, and
suggests that the ligand recognition on tubulin may be different
between E7010 and CLC.
The antimitotic drugs, Vinca alkaloids and Taxol, are key drugs in
cancer chemotherapy, but drug resistance mediated by P-glycoprotein
limits their usefulness against MDR tumors that have acquired resist
ance in the course of chemotherapy or naturally (17—19).In this study
and in the previous one (3), we found that MDR cells selected by
exposure to VCR did not show cross-resistance to E7010. In the in
vitro tumor panel study, E7010 seemed to be effective against natu
rally resistant MDR cell lines such as HCT15, DLD-1, and
OVCAR-4. There was also no cross-resistance with other MDR cell
lines (KB 8—5,KB C-!, and KB C-2; data not shown). Because other
types of drug resistance to VinCa alkaloids have been reported (20—
22), we selected VCR-resistant P388/V4—4 cells, which did not show
the classical MDR phenotype, by exposure to VCR and verapamil.
Interestingly, P388/V4—4 cells showed resistance to Vinca alkaloids
(VCR, vinblastine, and vindesine) and trisnordolastatin, an analogue
of dolastatin 10 that binds to the Vinca site on tubulin (13) but had
collateral sensitivity to Taxol. Surprisingly, P3881V4—4cells were not
cross-resistant to E70l0. Furthermore, to clarify the resistance to
III
d A
U
III 4.Or-M
J.4 U 1.Or-H
0 O.5r-D
0 O.6r-8
@ t I I I I I t I I I t I I I I I I I I
MDC
ADM
CDDP
5-FU
VP16
E7010
p
0.1 1 10
Resistance factor
3212
NOC
POD
CLC
Taxoi
VCR
E7010
llllllllllllllllllP
1nhT@.I
@-I El 4.Or-M
U 1.Or-H
0 O.5r-D
@l 0 O.6r-8
100 0.1 1 10 100
Resistance factor
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Research.
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E7010, four E70l0-resistant P388 cell lines were selected by exposure
to E7010. E7010-resistant P388 cell lines were not cross-resistant to
VCR, but collateral sensitivity to Taxol was observed. The pattern of
the cross-resistance to tubulin binders that bind at the CLC binding
site was very interesting. The pattern of the cross-resistance to NOC
across the spectrum of four E70l0-resistant P388 cell lines was
similar to that of E70l0, but the pattern of the cross-resistance to POD
was quite different. The pattern ofcross-resistance to CLC was similar
to that of POD, although the resistance to CLC was minimum. These
differences in the resistance pattern seem to be consistent with the
differences of the binding properties (binding site, temperature de
pendency, rate of binding, and reversibility of binding) among tubulin
binders. These suggest the involvement of alterations of the target,
tubulin itself or the polymerization state of tubulin, that specifically
affect the binding of the E70l0-type agent to tubulin in E70l0resistant
cell lines, because the accumulation of 14C-labeled E70l0
was similar in sensitive P388 cells and in E70l0-resistant cell lines
(data not shown). CDDP-, 5-FU-, VP16-, and CPT-resistant cell lines
also lacked cross-resistance to E70l0 (data not shown).
Few CLC site binding agents are active in in vivo solid tumor models,
although many compounds have been reported to bind to the CLC site on
tubulin, such as CLC analogues (28), steganacin (29), POD analogues
(30),combrestatin analogues (31),benzimidazole derivatives (32),1,2hydropyrido[3,4-b]pyrazine
derivatives (33), 2-styrylquinazolin-4(3H)one
derivatives (34), 2,3@ihydro-2-(aiyl)4(lfI)-quinazolinone deriva
tives (35), and curacin A (36). We have shown here that E70l0 is a
unique antimitotic agent with a broad spectrum ofantitumor activity. This
drug is considered to be a candidate for clinical use, either alone or in
combination chemotherapy.
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Research.