IJC
International Journal of Cancer
MT119, a new planar-structured compound, targets the
colchicine site of tubulin arresting mitosis and inhibiting
tumor cell proliferation
Zhixiang Zhang1, Tao Meng2, Na Yang1, Wei Wang1, Bing Xiong2, Yi Chen1, Lanping Ma2,
Jingkang Shen2, Ze-Hong Miao1 and Jian Ding1
1
Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai, People’s Republic of China
2
Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai, People’s Republic of China
Cancer Therapy
Microtubule-targeted drugs are now indispensable for the therapy of various cancer types worldwide. In this article, we report MT119
[6-[2-(4-methoxyphenyl) -ethyl]-9-[(pyridine-3-ylmethyl)amino]pyrido[20 ,10 :2,3]imida-zo[4,5-c]isoquinolin-5(6H)-one] as a new
microtubule-targeted agent. MT119 inhibited tubulin polymerization significantly both in tumor cells and in cell-free systems, which
was followed by the disruption of mitotic spindle assembly. Surface plasmon resonance-based analyses showed that MT119 bound to
purified tubulin directly, with the KD value of 10.6 lM. The binding of MT119 in turn caused tubulin conformational changes as
evidenced by the quenched tryptophan fluorescence, the reduction of the bis-ANS reactivity and the decreased DTNB-sulfhydryl
reaction rate. Competitive binding assays further revealed that MT119 bound to tubulin at its colchicine site. Consequently, by
inhibiting tubulin polymerization, MT119 arrested different tumor cells at mitotic phase, which contributed to its potent antitumor
activity in vitro. MT119 was also similarly cytotoxic to vincristine-, adriamycin- or mitoxantrone-resistant cancer cells and to their
corresponding parental cells. Together, these data indicate that MT119 represents a new class of colchicine-site-targeted inhibitors
against tubulin polymerization, which might be a promising starting point for future cancer therapeutics.
Key words: mitosis, tubulin, the colchicine site, spindle assembly
Abbreviations: ADM: adriamycin; Bis-ANS: 4,40 -dianilino-1,10 binaphthyl -5,50 -disulfonic acid dipotassium salt; DMSO: dimethyl
sulfoxide; DTNB: 5,50 -dithiobis(2-nitrobenzoic acid); IC50: 50%
inhibitory concentration; MTX: mitoxantrone; MT119: 6-[2-(4methoxyphenyl)ethyl]-9-[(pyridine-3-ylmethyl)amino]pyrido[20 ,10 :2,
3]imidazo[4,5-c]isoquinolin-5(6H)-one; SPR: surface plasmon
resonance; SRB: sulforhodamine B; VCR: vincristine
Grant sponsor: The National Natural Science Foundation of China
(NSFC); Grant numbers: 30873092 and 30721005; Grant sponsor:
National Science & Technology Major Project ‘‘Key New Drug
Creation and Manufacturing Program’’ of China; Grant numbers:
No. 2009ZX09103-074 and No. 2009ZX09301-001; Grant sponsor:
The Science, Technology Commission of Shanghai Municipality
(STCSM); Grant number: No. 08PJ14113; Grant sponsor: National
Basic Research Program of China; Grant number: 2010CB934000
DOI: 10.1002/ijc.25661
History: Received 4 Apr 2010; Accepted 23 Aug 2010; Online 9 Sep
2010
Correspondence to: Drs. Jian Ding or Ze-Hong Miao, Division of
Antitumor Pharmacology, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, People’s Republic of China. Tel.:
+86-21-50805897, Fax: + 86-21-50806722, E-mail: [email protected].
ac.cn or [email protected]
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Int. J. Cancer: 129, 214–224 (2011) V
Microtubule is composed of a- and b-tubulin heterodimers,
whose dynamics is crucial for the proper function of spindles
and guarantees the mitotic progression.1 By interfering with
microtubule dynamics, tubulin inhibitors cause mitosis arrest
and ultimately lead to tumor cell death. Since the approval of
vinca alkaloids in 1960s and then taxol in 1990s for cancer
therapy, targeting microtubule has been recognized as the most
effective strategy against different types of malignancies,
including hematological, ovarian, mammary and lung cancers.2
According to the differential impacts on microtubule polymer
mass, tubulin inhibitors could be classified into microtubule
destabilizers, such as vinca alkaloids, colchicine and combretastatins and microtubule stabilizers, such as taxol and epothilones. Regardless of their distinct structural types, these inhibitors bind to microtubules mainly at one of the three sites: the
vinblastine site, the colchicine site or the taxol site. Despite of
their proven therapeutic success, some drawbacks such as limited natural sources, high neurotoxicity and poor solubility
have rendered serious questions onto the extensive use of current microtubule-targeted drugs in clinic.3,4 Therefore, it is
urgent to find more structurally-diverse compounds with tubulin inhibitory functions for future antitumor therapy.
We previously reported a combinatorial library of 6H-Pyrido[20 ,10 :2,3]imidazo [4,5-c]isoquinolin-5(6H)-ones that possessed potent antitumor activities in vitro.5 From this library,
Zhang et al.
we found a compound, designated as MT7 [6-(4-methoxybenzyl) pyrido[2’,1’:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one],
to destabilize cellular microtubules and arrest mitosis, suggesting that this library might represent a new class of microtubule-targeted scaffolds.6 However, the relatively weak activity of MT7 prevents us to further characterize how it acts on
microtubule. So we optimized MT7 and obtained a series of
new compounds. Among them, a compound designated as
MT119 [6-[2-(4-methoxyphenyl)ethyl]-9-[(pyridine-3-ylmethyl)amino]pyrido [20 ,10 :2,3]imidazo[4,5-c]isoquinolin-5(6H)one] showed a 10-fold increase in the in vitro anticancer activity, and was thus selected to elucidate the anti-microtubule
mechanism of this class of compounds.
In our study, we found that MT119 directly bound to tubulin at the colchicine site and resulted in the depolymerization
of cellular microtubules. Such disruption of microtubule dynamics further led to mitotic spindle abnormality and arrested
tumor cells at the mitotic phase. Persistent mitotic arrest finally
contributed to the growth inhibition of tumor cells induced by
MT119. Our data indicate that MT119 is a new tubulin inhibitor targeting the colchicine site. In contrast to the limited
resources of those reported naturally-originated tubulin inhibitors, its simple synthesis and its characteristic planar structure
make MT119 a promising starting point for the development
of new microtubule-targeted cancer therapeutics.
215
Chinese Academy of Sciences (Shanghai, China). Human
cancer MDA-MB-435, MKN45, SKOV3 and MCF7 cell lines
were obtained from the Japanese Foundation of Cancer
Research (Tokyo, Japan). The adriamycin-selected resistant
subline MCF7/ADM8,9 was purchased from the Institute of
Hematology, Chinese Academy of Medical Sciences (Tianjin,
China). The vincristine-selected resistant subline KB/VCR8–10
was purchased from Sun Yat-Sen University of Medical Sciences (Guangzhou, China). All the cell lines were cultured
according to the suppliers’ instructions.
Western blot analyses
All the antibodies were commercially available, including
those against histone H3, Ser10-phosphorylated histone H3
(Cell Signaling Technology, Danvers, MA), GAPDH (Kangchen Bio-tech, Shanghai, China), MPM-2 (Millipore, Billerica,
MA) and a-Tubulin (Invitrogen, Carlsbad, CA).
Immunofluorescence assays
After treatments with the indicated drugs, HeLa cells growing
on glass coverslips were fixed for 30 min with 4% paraformaldehyde and then permeabilized for 15 min with 0.2% TritonX-100. After subsequent saturation with 3% bovine serum
albumin, the cells were incubated with the primary antibody
against a-Tubulin (1:200, Invitrogen, Carlsbad, CA) for 1 hr.
Then, the cells were stained with Alexa FluorV 488-conjugated goat anti-mouse IgG (1:100, Invitrogen, Carlsbad, CA)
for another hour. Finally, after being counterstained with
DAPI, the cells were imaged under an Olympus BX51 fluorescence microscope system (Olympus, Tokyo, Japan) and a
Nikon Eclipse C1 Plus confocal microscope (Nikon, Tokyo,
Japan).
R
Drugs and chemicals
MT119 was readily prepared by three component condensation
of 2-amino-5-bromopyridine, phthaldehydic acid and 1-(2-isocyanoethyl)-4-methoxybenzene according to our previous publication,5 followed by the Ullmann condensation with 3-(aminomethyl)pyridine. Its purity (more than 99%) was determined
by RP-HPLC at two wavelengths of 214 nm and 254 nm. Paclitaxel (Taxol), colchicine, vincristine (VCR), adriamycin (ADM),
mitoxantrone (MTX), DTNB [5,50 - dithiobis(2-nitrobenzoic
acid)] and Bis-ANS (4,40 -dianilino-1,10 -binaphthyl-5,50 - disulfonic acid dipotassium salt) were purchased from SigmaAldrich (St. Louis, MO). MT119, Taxol, VCR, ADM and MTX
were dissolved at 10 mM in dimethyl sulfoxide (DMSO) as
stock solution, respectively. Colchicine was dissolved at 10 mM
in distilled water as stock solution. All the aliquots were stored
at 20 C. DTNB was dissolved at 4 mM in 100 mM K3PO4
pH6.8 prior to use. Bis-ANS was dissolved at 150 lM in distilled water prior to use. BODIPY FL-vinblastine was purchased
from Invitrogen (Carlsbad, CA). [3H]-colchicine was purchased
from PerkinElmer (Waltham, MA). DE81 cellulose paper was
purchased from Whatman (Maidstone, England).
Cell culture
Human cancer HeLa, MDA-MB-468, HT29, HCT116, KB,
HL-60 and the mitoxantrone-resistant HL-60/MX27,8 cell
lines were obtained from the American Type Culture Collection (Manassas, VA). Human cancer SMMC-7721 cell line
was kept in the Shanghai institute of Materia Medica of the
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Int. J. Cancer: 129, 214–224 (2011) V
Tubulin turbidity assays
Tubulin was prepared from porcine brains by using the assembly-disassembly method as described by Shelanski et al.11
It was further purified by phosphocellulose chromatography
and stored at 80 C. Tubulin polymerization was assessed
by the turbidity assay. Indicated drugs were first mixed with
tubulin in a 96-well plate on ice, respectively. The plate was
put into a 37 C incubator and tubulin polymerization was
initiated and monitored by turbidity changes at 340 nm with
spectraMAX190 (Molecular Devices, Sunnyvale, CA). The
reaction buffer for this assay contained 80 mM PIPES pH6.9,
0.5 mM EGTA, 2 mM MgCl2 and 1 mM GTP.
Cellular microtubule stabilization assays
After treatments with the indicated drugs, HeLa cells were
harvested in the lysis buffer (100 mM PIPES pH6.9, 1 mM
EGTA, 1mM MgCl2, 30% glycerol, 5% DMSO, 1% NP-40, 5
mM GTP and protease inhibitors). After centrifugation at
180,000g 37 C for 1 hr, the polymerized tubulin fraction
(pellet) and the soluble tubulin fraction (supernatant) were
separated. These two fractions were adjusted to the same volume with SDS-PAGE loading buffer. Finally, the amount of
Cancer Therapy
Material and Methods
MT119 targets the colchicine site of tubulin
216
a-tubulin from equal aliquots of the polymerized tubulin
fraction and the soluble tubulin fraction was determined by
Western blot analyses.12
37 C, the bound [3H]-colchicine of each sample was determined using DE81 filter assays described by Gary G. Borisy14.
Vinblastine competitive binding assays
Surface plasmon resonance-based binding assays
Based on the surface plasmon resonance technology, the
binding affinity of MT119 to tubulin was determined with
the ProteOnTM XPR36 Protein Interaction Array System
(Bio-rad, Hercules, CA).13 For this purpose, tubulin was dissolved in 10 mM sodium acetate buffer (pH3.5) and immobilized to the ProteOnTM GLH sensor chip with the ProteOnTM
amine coupling kit. The final immobilization level was 8000
RU (Response unit). MT119 was two-fold diluted from 40
lM to 2.5 lM in HBS-T buffer (10 mM HEPES, 150 mM
NaCl, 3.4 mM EDTA, 0.005% Tween 20, pH7.4). MT119 of
different concentrations was then injected at a flow rate of 30
ll/min for 200 s, which was followed by a 300-s dissociation
phase. Data were analyzed with the ProteOnTM Manager
software, fitted to the 1:1 Langmuir model.
Determination of intrinsic tryptophan fluorescence
Tubulin (3 lM) was incubated with indicated drugs at 37 C
for 30 min. The samples were then excited at 280 nm and
the emission spectrum was monitored from 310 nm to 370
nm with spectraMAX2 (Molecular Devices, Sunnyvale, CA).
The reaction buffer for this assay contained 80 mM PIPES
pH 6.9, 0.5 mM EGTA and 2 mM MgCl2.
Determination of bis-ANS-tubulin fluorescence
Cancer Therapy
Tubulin (3 lM) was incubated with indicated drugs at 37 C
for 30 min, and then 15 lM Bis-ANS was added. After 15
min of reaction, the samples were excited at 400 nm and the
emission spectrum was monitored from 450 nm to 595 nm
with spectraMAX2 (Molecular Devices, Sunnyvale, CA). The
reaction buffer for this assay contained 80 mM PIPES pH6.9,
0.5 mM EGTA, 2 mM MgCl2.
Titration of tubulin sulfhydryl groups
Tubulin (3 lM) was preincubated with colchicine, MT119 or
vincristine at the indicated concentrations for 45 min, respectively, followed by the addition of BODIPY FL-vinblastine
(3 lM). After 20 min of incubation, the samples were then
excited at 490 nm and the emission spectrum was monitored
from 505 nm to 530 nm using Hitachi F-2500 spectrofluorometer (Hitachi, Tokyo, Japan). The reaction buffer for this assay
contained 80 mM PIPES pH6.9, 0.5 mM EGTA and 2 mM
MgCl2.
Molecular docking
The crystal structure of the tubulin in complex with colchicine
was retrieved from the Brookhaven Protein Data Bank (PDB
entry: 1SA0).15 Initially, the PDB structure was prepared by
removing the water atoms and colchicine. After that, the hydrogen atoms were added to protein with the AutoDock tool. Protein atom types and solvation parameters were assigned according to AutoDock 4.0 rules and the Amber Kollman atomic
charges were assigned to protein atoms. The ligand structure of
MT119 was optimized with the Cerius software16 with default
force field. The conjugated gradient method was used for energy
minimization with an energy convergence gradient value of 0.001
kcal/(molÅ). The advance docking program AutoDock 4.0 was
used to dock ligand to the colchicine binding site in tubulin.16,17
The Lamarckian genetic algorithm was applied to search for the
binding conformation of ligand. A Solis and Wets local search
was performed for energy minimization on a user-specified proportion of the population. The three-dimensional grid with 60 60 60 points and a spacing of 0.375 Å were created by the
AutoGrid algorithm to evaluate the binding energy between the
ligands and the proteins. In the docking phase, the number of
generations, energy evaluations, and docking runs were set to 3.7
104, 8 106 and 20, respectively. The lowest energy conformation was selected for further tubulin–ligand interaction analyses.
Sulfhydryl groups can act as probes to reflect the conformational changes of tubulin. Using the sulfhydryl-specific reagent DTNB, the kinetics of sulfhydryl group modification
was determined by measuring the absorbance at 412 nm with
spectraMAX190 (Molecular Devices, Sunnyvale, CA). Tubulin
(3 lM) was incubated with indicated drugs at 37 C for 30
min, and then 400 lM DTNB was added. After 1 hr of reaction, the number of modified sulfhydryl groups was determined by using a molar extinction coefficient of 12,000. The
reaction buffer for this assay contained 80 mM PIPES pH6.9,
0.5 mM EGTA and 2 mM MgCl2.
Cells were seeded into 6-well plates, cultured overnight, and
treated with MT119 for the indicated time. Cells were then
harvested and washed with PBS, fixed with pre-cooled 70%
ethanol at 4 C. Staining went along in PBS containing 40 lg/
ml RNase A and 10 lg/ml propidium iodide in the dark for
30 min. For each sample, at least 1104 cells were collected
with FACS Calibur (BD Biosciences, Franklin Lakes, NJ) and
analyzed by using the CELLQUEST software (BD Biosciences, Franklin Lakes, NJ).
Colchicine competitive binding assays
Sulforhodamine B (SRB) assays
Tubulin (3 lM) was preincubated with colchicine, MT119 or
vincristine at the indicated concentrations for 1 hr, respectively, followed by the addition of [3H]-colchicine (5 lM, final
specific activity ¼ 0.05 lCi). After 30 min of incubation at
Cells were seeded into 96-well plates, cultured overnight and
treated with MT119 for 72 hr. Cells were then fixed with
10% pre-cooled trichloroacetic acid (TCA), washed with distilled water, and stained with SRB (Sigma-Aldrich, St. Louis,
Flow cytometry
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Zhang et al.
217
MO) in 1% acetic acid. SRB in the cells was dissolved in 10
mM Tris-HCl and was measured at 515 nm with spectraMAX190 (Molecular Devices, Sunnyvale, CA). The cell proliferation inhibition rate was calculated as: proliferation inhibition (%) ¼ [1-(A515 treated/A515 control)] 100%. The average
IC50 values were determined with the Logit method from at
least three independent tests.18–20
Results
We previously identified a new antimitotic compound, MT7,
from the library of 6H-Pyrido[20 ,10 :2,3]imidazo[4,5-c]isoquinolin-5(6H)-ones. Although MT7 shares similar gene expression
profiles in tumor cells with the known tubulin inhibitors and
disrupts cellular microtubule networks, whether it changes,
directly or indirectly, tubulin polymerization remains to be
clarified due to its relatively poor solubility and weak bioactivity.6 Since then, great efforts have been put into the structural
optimization based on MT7 and yielded a new derivative,
MT119 (Fig. 1a), with 10-fold increase in the in vitro anticancer activity. To assess the possible effect of MT119 on in
vitro polymerization of tubulin, tubulin turbidity assays were
performed. The polymerization of purified tubulin was monitored in the absence or presence of different drugs at the indicated concentrations. MT119 was shown to inhibit tubulin polymerization in a concentration-dependent manner, similar to
the classic microtubule destabilizer, vincristine. At the concentration of 10 lM, MT119 suppressed tubulin polymerization by
50%, while at 20 lM, it almost completely inhibited tubulin
polymerization (Fig. 1b). The data suggest that MT119 may
directly act on tubulin and thus interfere with its polymerization in this cell-free system.
To further evaluate whether MT119 affects cellular tubulin
polymerization, we employed two independent assays. Firstly,
the microtubule networks of interphase HeLa cells treated
with MT119 were examined by immunofluorescence staining.
As indicated by a-tubulin, the MT119-treated cells exhibited
dispersed tubulin staining and seldom filamentous microtubule structures could be detected, which shared the same feature with the vincristine-treated cells. By contrast, the
untreated cells typically showed an intact and stretched network, while the taxol-treated cells produced a significant
increase in microtubule bundles. The data revealed that
MT119 disrupted the cellular microtubule networks (Fig. 1c).
Secondly, we analyzed the MT119-mediated quantitative
changes in free and polymerized tubulin fractions in HeLa
cells based on ultracentrifugation. As indicated by the soluble
form, the free fraction of cellular tubulin was increased in
response to the treatment with MT119, in concert with a
steady reduction of the polymerized fraction, which was
marked by the pellet form. At 1 lM, MT119 led to the complete transition of cellular tubulin from the polymerized state
to the free state (Fig. 1d). By contrast, MT7 inhibited the cellular tubulin polymerization to a similar extent requiring a
concentration as high as 10 lM.6 Obviously, MT119 exhibits
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Int. J. Cancer: 129, 214–224 (2011) V
Figure 1. MT119 inhibited tubulin polymerization. (a) Chemical
structure of MT119 and MT7. (b) MT119 inhibited tubulin
polymerization in vitro. MT119 at the indicated concentrations was
incubated with purified porcine tubulin at 37 C. Its effect on tubulin
polymerization was examined by turbidity changes at the wavelength of
340 nm. (c) MT119 disrupted intracellular microtubule networks at
interphase. HeLa cells were treated with MT119 (0.5 lM), Taxol (0.1
lM) or VCR (0.1 lM) for 6 hr, respectively. The cells were then
processed for immunofluoscence staining and confocal microscopy
(600). (d) MT119 promoted the transition from polymerized tubulin to
free tubulin within HeLa cells. HeLa cells were treated with MT119 at
the indicated concentrations for 12 hr. The polymerized fraction and
the free fraction of tubulin were separated by ultracentrifugation and
processed for Western blotting. All the data shown were representative
of three independent experiments with similar results. [Color figure can
be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Cancer Therapy
MT119 inhibits tubulin polymerization
MT119 targets the colchicine site of tubulin
218
Figure 2. MT119 directly bound tubulin. a and b, Binding kinetics of MT119 (a) or colchicine (b) to tubulin determined by the SPR
technology. Tubulin was immobilized on the chip surface, followed by the injection of the compounds at the indicated concentrations. (c)
MT119 quenched intrinsic tryptophan fluorescence of tubulin. MT119 at the indicated concentrations were incubated with tubulin for 30
min at 37 C and then the emission spectrum was monitored from 310 nm to 370 nm with the excitation at 280 nm. (d) MT119 decreased
bis-ANS-tubulin fluorescence. MT119 at the indicated concentrations was incubated with tubulin for 30 min at 37 C and then 15 lM Bis-
Cancer Therapy
ANS was added. After 15 min of reaction, the emission spectrum was monitored from 450 nm to 595 nm with the excitation at 400 nm. (e)
MT119 affected tubulin sulfhydryl reactivity. MT119 at the indicated concentrations were incubated with tubulin for 30 min at 37 C and
then 400 lM DTNB was added. After 1 hr of reaction, the reactivity of sulfhydryls was determined by measuring the absorbance at 412 nm.
All the data above were representative of three independent experiments with similar results.
10-fold higher potency in inhibiting tubulin polymerization
than its parent compound MT7.
Together, the independent evidence from the three different assays collectively suggests that MT119 may directly act
on tubulin and thus disrupt its polymerization.
MT119 directly binds to tubulin
To clarify how MT119 interacts with tubulin, the surface
plasmon resonance (SPR) technology was used to determine
the possible binding affinity.21–23 Different concentrations of
MT119 were injected to allow the interaction with the tubulin protein immobilized on the chip surface. The result
showed that when interacting with tubulin, MT119 displayed
a similar association-dissociation kinetic process to colchicine
(Figs. 2a and 2b). The response unit (RU) increased in the
MT119-concentration-dependent manner. When fitted to the
1:1 Langmuir model, MT119 displayed modest binding affinity with the equilibrium dissociation constant (KD value) of
10.6 lM (Fig. 2a), which was a bit higher than that for colchicine (Fig. 2b). Detailed kinetic analyses revealed that
MT119, in comparison with colchicine, exhibited relatively
fast association-dissociation kinetics with relatively high Ka
and Kd values. These data indicate that like the classical
tubulin inhibitor colchicine, MT119 can directly bind tubulin.
To investigate what consequences result from the binding
of MT119 to tubulin, we detected the conformational changes
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Zhang et al.
Figure 3. MT119 directly bound tubulin at the colchicine site. (a). MT119 competed with colchicine for the binding site on tubulin. MT119,
colchicine or VCR at the indicated concentrations was incubated with tubulin for 1 hr at 37 C and then 0.05 lCi [3H]-colchicine was added. After
30 min of reaction, the bound [3H]-colchicine was determined by DE81 filter assays. (b). MT119 did not compete for the vinblastine site. MT119,
colchicine or VCR at the indicated concentrations was incubated with tubulin for 45 min at 37 C and then 2 lM BODIPY FL-vinblastine was
added. After 20 min of reaction, the emission spectrum was monitored from 505 nm to 530 nm with the excitation at 490 nm. All the results
were expressed as mean 6 SD of three independent experiments. (c) Binding models of MT119 and colchicine. a-tubulin and b-tubulin were
shown with solid ribbons colored blue and orange, respectively. All the carbon, nitrogen and oxygen atoms of MT119 were colored yellow while
those atoms of colchicine were colored white. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
of tubulin using different methods.24 Tubulin contains eight
tryptophan residues, the intrinsic fluorescence of which can
be used as a marker to monitor the conformational changes
of tubulin.25 The result showed that MT119 quenched the
intrinsic fluorescence of tryptophan in a concentration-deC 2010 UICC
Int. J. Cancer: 129, 214–224 (2011) V
pendent manner (Fig. 2c). Bis-ANS binds the hydrophobic
surface of tubulin and inhibit its polymerization and thus has
been used as a useful agent to probe possible tubulin conformational changes.26 Just like colchicine and vincristine,
MT119 elicited a steady reduction of the bis-ANS-tubulin
Cancer Therapy
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220
MT119 targets the colchicine site of tubulin
fluorescence (Fig. 2d). In addition, tubulin sulfhydryls distribute globally through its primary sequence. The structural
changes of tubulin upon ligand binding can be detected with
the sulfhydryl-specific reagent DTNB.27–29 MT119, like colchicine, decreased the DTNB-sulfhydryls reaction rate, but
different from vincristine, it did not affect the total number
of reactive sulfhydryls apparently (Fig. 2e). All the data indicate that MT119 can induce the conformational changes of
tubulin, which, vice versa, further strengthens the experimental evidence of the binding of MT119 to tubulin.
Cancer Therapy
MT119 binds to tubulin at its colchicine site
Generally, microtubule-destabilizing agents bind to tubulin at
either the colchicine site or the vinblastine site.1 To identify
the binding site of MT119, we performed competitive binding assays to test whether MT119 shared the same binding
site with either of them.30–32
The colchicine site is located at b-tubulin, near its interface with a-tubulin.1 MT119 of different concentrations was
incubated with tubulin, followed by the addition of [3H]-colchicine. Both MT119 and colchicine inhibited the binding of
[3H]-colchicine to tubulin significantly. At 20 lM, MT119
led to a inhibition rate of up to 52.2% (Fig. 3a). In contrast,
vincristine did not interfere with the binding of [3H]-colchicine to tubulin. Further evaluations showed that even at 20
lM, MT119, like colchicine, did not significantly change the
binding of BODIPY FL-vinblastine to tubulin that was apparently suppressed by vincristine, a vinblastine analogue (Fig.
3b). The data indicate that MT119 binds to tubulin at the
colchicine site rather than at the vinblastine site.
We then applied molecular docking to analyze the binding
mode of MT119 to the colchicine site. MT119 was revealed
to bind to tubulin in a colchicine-like manner (Fig. 3c). First,
the methoxyl group of MT119 overlapped with the one of
ring C of colchicine. The methoxyl group of colchicine (at
position 10) is crucial for its binding to tubulin as a hydrophobic center33 and somehow determines its inhibitory
capacity against tubulin polymerization.34 Similarly, the
methoxyl group of MT119 is also unchangeable and even
minor modifications at this group could lead to the complete
loss of its antimitotic activity (unpublished data). On the
other hand, the (pyridine-3-ylmethyl)amino group of MT119
at position 9 is similar in chemical space to the acetamide
group of ring B of colchicine. The acetamide of colchicine is
not considered as a pharmacophore of colchicine analogues33
and can be substituted or even eliminated without any detectable loss of its potency.35 Similarly, the substituent of
MT119 at position 9 has no impacts on its antimitotic activity (unpublished data). Thus, these docking data clarify the
structure-activity relationship of MT119 that is helpful to
understand the binding mode of MT119.
MT119 disrupts mitotic spindle assembly
Microtubule is required for the assembly of mitotic spindle,
which is essential for accurate chromosome segregation.36–38
Figure 4. MT119 disrupted mitotic spindle assembly. (a) MT119
induced aberrant mitotic spindle formation. HeLa cells were treated
with MT119 (0.5 lM) for 12 hr. Then, the cells were processed for
immunofluoscence staining and confocal microscopy (600). The
images from the two confocal sections (z-spacing, 2.2 lM) at the
same location of the same MT119-treated cell were presented at the
middle panel and the bottom panel, respectively. (b) The
quantitative analysis of MT119-induced spindle abnormality. The
number of spindle poles of each cell was counted and the
percentage of mitotic cells with or without two centrosomes was
quantified. Five microscope fields were randomly selected and at
least 100 cells were examined in each experiment. All the results
were expressed as mean 6 SD of three independent experiments.
[Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
To demonstrate whether the binding of MT119 to microtubule
is translated into the disruption of mitotic spindle assembly,
we examined centrosomes and spindles formed as indicated by
pericentrin and a-tubulin immunofluorescence staining in
HeLa cells. In contrast to the untreated HeLa cells, whose mitotic spindles were bipolar, those MT119-treated cells contained at least three centrosomes as evidenced by pericentrin
staining (two in the first confocal section and one in the other
section), indicating the multipolar spindle assembly. Consistently, DAPI-stained chromosomes did not align well at the
metaphase plate, but scattered in the cytoplasm (Fig. 4a). Statistically, upon MT119 treatment, the percentage of mitotic cells
with normal bipolar spindles dropped from 92.90% to 10.60%,
whereas the percentage of mitotic cells with aberrant spindles
increased from 7.10% to 89.40% (Fig. 4b). The data indicate
that MT119 disrupts normal mitotic spindle assembly.
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Zhang et al.
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(Fig. 5a). Consistently, the levels of two mitotic markers,
MPM-2 epitopes and phosphorylated histone H3, were both
elevated significantly by MT119 (Fig. 5b). Therefore, MT119
arrested different tumor cell lines at mitosis.
Persistent mitotic arrest always gives rise to cell death,
which accounts for the anti-proliferation effects of most
tubulin inhibitors. To determine the tumor cell fate after the
prolonged treatments with MT119, SRB assays were carried
out in 10 tumor cell lines from 8 different tissue origins. The
results revealed that MT119 inhibited the proliferation of the
tested tumor cells with an averaged IC50 of 0.34 lM ranging
from 0.06 lM to 0.53 lM (Fig. 5c), about 10-fold more
potent than its parent MT7 (with an averaged IC50 of 2.58
lM). More importantly, it was shown that MT119 could
effectively overcome multiple drug resistance, which was now
an intractable obstacle of many conventional chemotherapeutics against human cancers (Table 1). In three resistant cell
lines (KB/VCR, MCF-7/ADM and HL-60/MX2), the resistance factor values (RFs) of MT119 were significantly lower
than those of the corresponding reference drugs vincristine
(2.7 vs. 155.0), adriamycin (2.2 vs. 876.3) and mitoxantrone
(0.6 vs. 46.6).
Figure 5. MT119 induced mitosis arrest and inhibited proliferation
in various tumor cell lines. (a) MT119 induced mitosis arrest. Four
tumor cell lines were treated with MT119 (0.5 lM) for 12 hr, and
then the cells were stained with DAPI and processed for flow
cytometry analyses. (b) MT119 up-regulated molecular markers of
mitosis. The cells treated as above were lysed and immunoblotted
with the indicated antibodies. (c) MT119 inhibited tumor cell
proliferation. Proliferation of tumor cell lines was assessed by SRB
assays after 72-hr treatments with MT119 at a range of
concentrations. All the results were expressed as mean 6 SD of
three independent experiments.
MT119 arrests mitosis and inhibits proliferation in various
tumor cell lines
Microtubule abnormality is always monitored by the spindle
assembly checkpoint, which subsequently leads to cell cycle
arrest at mitosis.36,39,40 Considering that MT119 could depolymerize microtubule by binding the colchicine site as
stated above, it was of interest whether this compound interfered with cellular mitosis as MT7 did.6 To test this possibility, we used 4 different tumor cell lines to monitor the cell
cycle progression by flow cytometry in response to the treatments with MT119. The result showed that MT119 increased
the accumulation of all these tumor cells at the G2-M phase
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Int. J. Cancer: 129, 214–224 (2011) V
We have generated a library of 6H-Pyrido[20 ,10 :2,3]imidazo[4,5c]isoquinolin- 5(6H)-ones with antitumor activities,5 in which
MT7 has been found to destabilize cellular microtubules.6 However, its relatively low solubility and weak bioactivity limits the
detailed elucidation of its underlying mechanism. Based on MT7
through chemical modifications, a more bioactive compound
with improved solubility, MT119, was synthesized. In our study,
we clearly clarify that MT119, as a representative compound of
this chemical skeleton, directly binds tubulin at the colchicine
site, disrupts its normal dynamics and function of tubulin and
thus results in mitosis arrest and anticancer activity.
As the first step to investigate the impact of MT119 on
tubulin, it was found to inhibit tubulin polymerization in
both cell and cell-free systems. Then, the SPR analyses
revealed that MT119 directly bound tubulin and subsequently
led to apparent conformational changes in tubulin. Next, we
used competitive binding assays and molecular docking to
clearly define that the binding site of MT119 on tubulin was
the colchicine and that the methoxyl group of MT119 was
critical for this binding. After that, MT119 was further shown
to disrupt mitotic spindle assembly and thus obviously
increased supernumerary centrosomes and multipolar spindles. Finally, MT119 was revealed to induce mitotic arrest
and growth inhibition in different tumor cell lines. All these
lines of evidence from the interactions between MT119 and
its target (tubulin), the changes in tubulin conformation and
dynamics, the biological consequences and the potential therapeutic values, collectively indicate that MT119 is a new
tubulin destabilizer targeting the colchicine site.
As a colchicine-like anti-tubulin agent, MT119 is different
from colchicine in several aspects. The two compounds
Cancer Therapy
Discussion
MT119 targets the colchicine site of tubulin
222
Table 1. Effects of MT119 on vincristine (VCR)-, adriamycin (ADM)- or mitoxantrone (MTX)-resistant cancer cells
IC50(lM)
IC50(lM)
IC50(lM)
Compounds
KB
KB/VCR
RF
MCF-7
MCF-7/ADM
RF
HL-60
HL-60/MX2
RF
MT119
0.15660.003
0.42260.267
2.7
0.55860.409
1.23160.035
2.2
0.27560.059
0.17060.020
0.6
VCR
0.00260.000
0.31060.052
155.0
NA
NA
NA
NA
NA
NA
ADM
NA
NA
NA
0.04060.012
35.05263.000
876.3
NA
NA
NA
MTX
NA
NA
NA
NA
NA
NA
0.00860.002
0.37360.006
46.6
IC50s were expressed as mean 6 SD (lM), which came from three separate experiments. The resistance factor (RF) was calculated as the ratio of
the IC50 value of the drug-resistant cells to that corresponding parental cells.
possess different chemical skeletons as shown in Figure 3.
MT119 is especially distinct due to its typical planar structure
(which has been demonstrated not to interfere with cellular
DNA-related activities). The structural differences could also
been evidenced from the molecular docking studies. For
example, the three methoxyl groups of ring A of colchicine
function as the indispensable tubulin-drug complex stabilizing anchor. In contrast, at the corresponding chemical space
of MT119, no methoxyl or methoxyl-like group(s) existed,
suggesting that MT119 might be anchored into the colchicine
site of tubulin via a distinct mechanism. These differences
may further contribute to their differential binding affinity,
polymerization inhibition and potential anticancer activity.
On the other hand, MT119 inhibits the polymerization of
purified tubulin in vitro with an IC50 value of about 10 lM,
nearly equal to the KD value (10.6 lM) determined by SPR
assays. It means that about 50% of the soluble tubulin could
be bound by MT119 at the concentration that causes halfmaximal inhibition of microtubule polymerization. This is in
striking contrast to colchicine, which has been reported to
totally suppress tubulin polymerization in vitro even at only
4% of its occupancy.41 Therefore, MT119 may inhibit tubulin
polymerization in a distinct mode, rather than in the endpoisoning manner like colchicines.1,42,43 Those differences lay
an excellent basis on future optimization of this series of
compounds. Moreover, the much easier synthesis of MT119
via a multiple-component reaction ensures its rich resource
as a tubulin probe and an anticancer drug lead, as compared
to those reported colchicine-site-targeted inhibitors with natural origins.
Up to now, the colchicine-site-targeted tubulin inhibitors
including colchicine have not been formally approved for anticancer therapeutics because of its severe or even life-threatening toxicity.44,45 However, there are several colchicine-site
inhibitors under investigations in clinical trials, such as
ZD6126, ABT-751 and CA4P, which show reduced toxicity
and improved tolerance,46–50 suggesting that the toxicity is not
inherent in the colchicine-site binders. On the other hand, the
colchicine-site inhibitors may circumvent the drug resistance
arising from the uses of taxanes and vinca alkaloids due to their
different binding sites and acting mode. Thus it is reasonable
to make MT119 an important lead for the development of new
anticancer therapeutics tageting the colchicine site of tubulin.
Acknowledgements
We sincerely thank Mrs. Li-Juan Lu, Mr. Yong Xi and Miss Yan-Yan Shen
for their technical supports. The authors have no conflicting financial
interests.
Cancer Therapy
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