Identification of DW532 as a novel anti-tumor agent targeting both kinases and tubulin

Article abstract of DOI:10.1038/aps.2014.33

Aim:7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (DW532) is one of simplified analogues of hematoxylin that has shown broad-spectrum inhibition on tyrosine kinases and in vitro anti-cancer activities. The aim of this study was to identify DW532 as a agent targeting both kinases and tubulin, and to investigate its anti-cancer and anti-angiogenesis activities.Methods:In vitro tyrosine kinases activity was examined with ELISA, and tyrosine kinases activity in cells was evaluated with Western blot analysis. Tubulin turbidity assay, surface plasmon resonance and immunofluorescence technique were used to characterize the tubulin inhibitory activity. Cell proliferation was examined with SRB assay, and cell apoptosis and cell cycle distribution were analyzed with Annexin-V/PI staining and flow cytometry. Tube formation, aortic ring and chick chorioallantoic membrane assays were used to evaluate the anti-angiogenesis efficacy.Results:DW532 inhibited EGFR and VEGFR2 in vitro kinase activity (the IC₅₀ values were 4.9 and 5.5 μmol/L, respectively), and suppressed their downstream signaling. DW532 dose-dependently inhibited tubulin polymerization via direct binding to tubulin, thus disrupting the mitotic spindle assembly and leading to abnormal cell division. In a panel of human cancer cells, DW532 (1 and 10 μmol/L) induced G₂/M phase arrest and cell apoptosis, which subsequently resulted in cytotoxicity. Knockdown of BubR1 or Mps1, the two core proteins of the spindle assembly checkpoint dramatically decreased DW532-induced cell cycle arrest in MDA-MB-468 cells. Moreover, treatment with DW532 potently and dose-dependently suppressed angiogenesis in vitro and in vivo.Conclusion:DW532 is a dual inhibitor against tubulin and tyrosine kinases, and deserves further development as a novel anti-cancer agent.

Full text of DOI:10.1038/aps.2014.33

Acta Pharmacologica Sinica (2014) 35: 916–928
© 2014 CPS and SIMM All rights reserved 1671-4083/14 $32.00
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Original Article
Identification of DW532 as a novel anti-tumor agent
targeting both kinases and tubulin
Ting PENG1, #, Jian-rui WU2, #, Lin-jiang TONG1, Meng-yuan LI1, Fang CHEN2, Yi-xin LENG2, Rong QU1, Kun HAN1, Yi SU1, Yi
1,
1,
CHEN1, Wen-hu DUAN2, , Hua XIE , Jian DING
*
*
*
1Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203, China; 2Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203, China
Aim: 7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (DW532) is one of simplified analogues of hematoxylin that has
shown broad-spectrum inhibition on tyrosine kinases and in vitro anti-cancer activities. The aim of this study was to identify DW532 as
a agent targeting both kinases and tubulin, and to investigate its anti-cancer and anti-angiogenesis activities.
Methods: In vitro tyrosine kinases activity was examined with ELISA, and tyrosine kinases activity in cells was evaluated with Western
blot analysis. Tubulin turbidity assay, surface plasmon resonance and immunofluorescence technique were used to characterize the
tubulin inhibitory activity. Cell proliferation was examined with SRB assay, and cell apoptosis and cell cycle distribution were analyzed
with Annexin-V/PI staining and flow cytometry. Tube formation, aortic ring and chick chorioallantoic membrane assays were used to
evaluate the anti-angiogenesis efficacy.
Results: DW532 inhibited EGFR and VEGFR2 in vitro kinase activity (the IC50 values were 4.9 and 5.5 μmol/L, respectively), and
suppressed their downstream signaling. DW532 dose-dependently inhibited tubulin polymerization via direct binding to tubulin, thus
disrupting the mitotic spindle assembly and leading to abnormal cell division. In a panel of human cancer cells, DW532 (1 and 10
μmol/L) induced G2/M phase arrest and cell apoptosis, which subsequently resulted in cytotoxicity. Knockdown of BubR1 or Mps1, the
two core proteins of the spindle assembly checkpoint dramatically decreased DW532-induced cell cycle arrest in MDA-MB-468 cells.
Moreover, treatment with DW532 potently and dose-dependently suppressed angiogenesis in vitro and in vivo.
Conclusion: DW532 is a dual inhibitor against tubulin and tyrosine kinases, and deserves further development as a novel anti-cancer
agent.
Keywords: anti-cancer agent; hematoxylin; combretastatin; tyrosine kinase; EGFR; VEGFR; tubulin; cell cycle; spindle assembly
checkpoint; anti-angiogenesis
Acta Pharmacologica Sinica (2014) 35: 916–928; doi: 10.1038/aps.2014.33; published online 26 May 2014
factor receptor (EGFR) and vascular endothelial growth factor
Introduction
Protein tyrosine kinases (PTKs) play crucial roles in signal receptor (VEGFR) have already been used in the clinic, and
transduction that regulate various cellular functions, such more are currently in different stages of preclinical or clinical
as cell proliferation, the cell cycle, differentiation and cell development[2, 3]
.
survival, and angiogenesis. Dysregulation of kinase activ-
Microtubules are components of the cytoskeleton with key
ity through overexpression or mutation is associated with roles in cellular functions, including intracellular transport,
the development of a number of different cancers[1]. Thus, cell shape maintenance, polarity, and mitosis. Microtubule
tyrosine kinases have been proven to be important targets targeting agents (MTAs) have also been recognized as a class
for cancer therapy. Several small molecule kinase inhibitors of the most effective medications in cancer treatment, and sev-
targeting different tyrosine kinases such as epidermal growth eral MTAs have been approved for the treatment of solid and
hematologic malignancies. MTAs are usually classified into
# These authors contributed equally to this work.
To whom correspondence should be addressed.
E-mail jding@simm.ac.cn (Jian DING);
hxie@simm.ac.cn (Hua XIE);
whduan@simm.ac.cn (Wen-hu DUAN)
Received 2014-02-25 Accepted 2014-04-07
two main groups[4]. One group is known as the microtubule-
destabilizing agents, eg, vincristine (VCR), which inhibits
microtubule polymerization[5]. The other group is known as
the microtubule-stabilizing agents, eg, Taxol, which stimulates
microtubule polymerization. MTAs can arrest cells in mitosis,
*

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particularly rapidly dividing cancer cells, and apoptosis and broad-spectrum inhibition of tyrosine kinases and in vitro anti-
cell death likely follows[6].
tumor activity[12]. Hematoxylin has a tetracyclic compound
Cancer is a rather complex disease with robust and often structure with four hydroxyl groups, and it is barely soluble
redundant biological networks. Therefore, it is increasingly in water because the tetracyclic configuration often accounts
recognized that modulating more than one target may be a for the poor solubility of compounds. Although hematoxylin
better clinical approach than using single-targeting agents[7]. has interesting biological activity, its physical properties are
Clinical trials for cancer treatment using a combination of sub-optimal for clinical use. Moreover, from the structure
tubulin and kinase inhibitors were investigated with promis- of hematoxylin, we found that it contains the key pharmaco-
ing therapeutic effects. For example, combination of the EGFR phore combretastatin (CA-4) (Figure 1), a well-known tubulin
kinase inhibitor lapatinib and the microtubule-stabilizing inhibitor, which includes two phenyl groups with substituted
agent Taxol was found to significantly improve the progres- hydroxyls or methoxy groups. Therefore, we designed and
sion-free survival (FPS) and overall response (OR) of gastric synthesized a series of simplified analogues to achieve two
cancer patients in a phase III clinical trial[8]. Meanwhile, agents purposes: 1) target kinases and tubulin and 2) simultaneously
that target kinases and tubulin also have gained increasing decrease the complexity of the tetracyclic system of hema-
attention and achieved some progress. For example, Kadcyla, toxylin. One of the compounds that possess excellent bioac-
an antibody-drug conjugate that targets both HER2 and tubu- tivities is 7,8-dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-
lin, was approved by the FDA for the treatment of HER2-posi- chromen-2-one (DW532 in Figure 1). Biological results further
tive metastatic breast cancer. KX2-391, an oral inhibitor of Src confirmed that DW532 is an agent targeting both tubulin and
kinase and tubulin polymerization, is being tested in a Phase kinases. DW532 treatment resulted in growth inhibition, cell
II study of prostate cancer patients[9]. Tivantinib, the first MET cycle arrest and apoptosis in cancer cells, and it also exhibited
inhibitor in clinical research, was discovered to also affect the suppression of angiogenesis in vitro and in vivo.
tubulin polymerization[10]. Moreover, researchers are report-
edly attempting to modify the chemical structure of SU5416,
Materials and methods
an inhibitor of VEGFR, to obtain a dual inhibitor of VEGFR Drugs and chemicals
and tubulin[11]. All of these efforts highlight the importance of Synthesis of DW532 (Figure 1).
identifying and developing new inhibitors targeting kinases
and tubulin.
General
Hematoxylin, a natural product extracted from the heart- All chemicals obtained from Sigma-Aldrich (St Louis, MO,
wood of the the logwood tree (Haematoxylum campechianum) USA) and the Sinopharm Chemical Reagent Co, Ltd (Shang-
(Figure 1), was previously discovered by our group for its hai, China) were used without further purification. Melting
Figure 1. The structure of hematoxylin, combretastin, and DW532 Reagents and conditions for the synthesis of DW532: (a) Na, CO(OEt)2, reflux; (b)
AcOH, reflux; (c) (CF3SO2)2O, 0ºC; (d) 3-(benzyloxy)-4-methoxyphenylboronic acid, Pd(PPh3)4, CuI, K2CO3, DMF, 120ºC, 20 min; (e) CF3COOH.
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points (uncorrected) were measured with a Büchi B-510 melt- (d, J=9.1 Hz, 1H), 6.33 (s, 1H), 5.23 (s, 2H), 5.19 (s, 2H); MS (EI):
ing point apparatus. 1H NMR spectra were recorded with a 506 m/z [M+]; HRMS (EI) calcd for C24H17SF3O7 [M+]: 506.0647,
Varian Mercury 300 NMR spectrometer (Varian, Palo Alto, found: 506.0653.
CA, USA); tetramethylsilane and residual solvent signals were
used as internal standards. Chemical shifts are reported in 7,8-Bis(benzyloxy)-4-(3-(benzyloxy)-4-methoxyphenyl)-2H-
ppm (δ). Low-resolution mass spectra (MS) and high-reso- chromen-2-one (5)
lution mass spectra (HRMS) were obtained with an ionizing A mixture of 4 (80 mg; 0.16 mmol), tetrakis(triphenyl-
voltage of 70 eV using a Finnigan/MAT95 spectrometer. The phosphine) palladium (10 mg; 0.01 mmol), cuprous iodide
purity of the new derivatives was determined with an Agilent (34 mg; 0.18 mmol), sodium carbonate (118 mg; 1.20 mmol),
Technologies 1260 series HPLC system (Agilent Technology, and (3-(benzyloxy)-4-methoxyphenyl) boronic acid (82 mg;
Santa Clara, CA, USA) using a ZORBAX Eclipse Plus column 0.32 mmol) in 1,4-dioxane (15 mL) was degassed three times
(C18, 4.6×100 mm, 3.5 Micron). DW532 had a purity of greater with argon. The resulting mixture was heated in an argon
than 98%.
atmosphere at 120°C for 20 min. After cooling to room tem-
perature, the reaction mixture was filtered to remove insoluble
substances. The filtration was evaporated to dryness, and
Ethyl 3-(3,4-bis(benzyloxy)phenyl)-3-oxopropanoate (2)
Sliced sodium (1.0 g; 43.06 mmol) was added to a mixture of the resulting residue was purified by flash chromatography
distilled diethyl carbonate (20 mL, 165.08 mmol) and 1 (1.5 g, (dichloromethane: methanol=40:1) to produce 5 as a brown
1
474.16 mmol), and the resulting mixture was stirred at 120°C solid (124 mg, 78.0%): mp: 163–165 °C; H NMR (300 MHz,
for 20 min. After the reaction mixture was cooled to room CDCl3) δ: 7.55 (dd, J=7.5, 2.4 Hz, 2H), 7.41–7.26 (m, 13H), 7.01
temperature and quenched with ethanol (10 mL), its pH value (s, 2H), 6.91 (d, J=8.4 Hz, 2H), 6.67 (d, J=9 Hz, 1H), 6.16 (s, 1H),
was adjusted to 6 with 6 mol/L hydrochloric acid. The result- 5.22 (s, 2H), 5.20 (s, 2H), 5.19 (s, 2H), 3.97 (s, 3H); MS (EI): 570
ing mixture was extracted with ethyl acetate (3×30 mL). The m/z [M+]; HRMS (EI) calcd for C37H30O6 [M+]: 570.2042, found:
combined extracts were dried over anhydrous sodium sulfate 570.2036.
and concentrated in a vacuum. The residue was purified by
flash chromatography (dichloromethane: methanol=40:1) to 7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one
1
produce 2 as a white solid (1.40 g, 80.1%): mp: 156–158°C; H (DW532)
NMR (300 MHz, DMSO-d6) δ: 11.83 (s, 1H), 77.63 (d, J=9.0 Hz, A mixture of 4 (30 mg; 0.06 mmol) in trifluoromethanesulfonic
1H), 7.43–7.27 (m, 10H), 6.81 (d, J=9.3 Hz, 1H), 5.24 (s, 2H), acid (2 mL) was stirred at 55°C for 2 h. The mixture was then
4.95 (s, 2H), 4.14 (s, 2H), 4.11 (d, J=6.0 Hz, 1H), 1.17 (t, J=7.1 evaporated to dryness, and the resulting residue was purified
Hz, 3H); MS (EI): 420 m/z [M+]; HRMS (EI) calcd for C25H24O6 by flash chromatography (dichloromethane: methanol=90:1)
[M+]: 420.1573, found: 420.1572.
to produce DW532 as a yellow solid (11 mg, 70.4%): mp:
118–120°C; 1H NMR (300 MHz, DMSO-d6) δ:10.16 (s, 1H), 9.39
(s, 1H), 9.37 (s, 1H), 7.05 (s, 1H), 6.89 (d, J=7.8 Hz, 3H), 6.79 (d,
7,8-Bis(benzyloxy)-4-hydroxy-2H-chromen-2-one (3)
A solution of 2 (1 g, 2.38 mmol) in acetic acid (5 mL) was J=8.9 Hz, 1H), 6.04 (s, 1H), 3.84 (s, 3H); MS (EI): 300 m/z [M+];
heated at reflux for 6 h. The reaction mixture was evapo- HRMS (EI) calcd for C16H12O6 [M+]: 300.0634, found 300.0637.
rated to dryness, and the resulting residue was purified by
Combretastatin, Taxol, ispinesib, SB743921 and vincristine
flash chromatography (dichloromethane: methanol=60:1) to (VCR) were purchased from Sigma-Aldrich (St Louis, MO,
produce 3 as a yellow solid (0.83 g, 92.7%): mp: 183–185 °C; USA). Aurora inhibitor II was purchased from Calbiochem
1H NMR (300 MHz, DMSO-d6) δ: 12.41–12.36 (m, 1H), 7.53 (d, (San Diego, CA, USA). All of the chemicals were prepared at
J=9.2 Hz, 1H), 7.50 – 7.28 (m, 12H), 7.19 (d, J=9.1 Hz, 1H), 5.47 10 mmol/L in 100% dimethyl sulfoxide (DMSO) as stock solu-
(s, 1H), 5.26 (s, 2H), 5.07 (s, 2H); MS (EI): 374 m/z [M+]; HRMS tions, and the aliquots were stored at -20°C.
(EI) calcd for C23H18O5 [M+]: 374.1154, found: 374.1159.
Cell culture
7,8-Bis(benzyloxy)-2-oxo-2H-chromen-4-yl trifluoromethane- The human cancer cell lines HT-29, K562, BT-474, T47D,
sulfonate (4)
MCF-7, PC-3, HCT-116, A549, A431, A375, KB, BxPC3, MDA-
A solution of trifluoromethanesulfonic anhydride (0.11 mL, MB-231, and MDA-MB-468 were obtained from the American
1.61 mmol) was added dropwise to a mixture of 3 (150 mg, Type Culture Collection (Manassas, VA, USA), SMMC-7721
0.41 mmol) and triethylamine (0.17 mL, 1.21 mmol) in dichlo- was obtained from the cell bank of the Chinese Academy of
romethane (12 mL). After addition, the mixture was stirred Sciences (Shanghai, China). All of the cell lines were cultured
at 0 °C for 12 h, and it was then quenched with brine and according to the suppliers’ instructions.
extracted with dichloromethane (3×10 mL). The combined
extracts were dried over anhydrous sodium sulfate and con- Sulforhodamine B (SRB) assays
centrated in a vacuum. The resulting residue was purified Cell proliferation was evaluated using the SRB (Sulforhoda-
by chromatography (petroleum ether: ethyl acetate=5:1) to mine B) assay as previously described[13]. Briefly, cells were
produce 4 as a white solid (153 mg, 75.4%): mp: 112–113°C; 1H seeded in 96-well plates and grown for 24 h. The cells were
NMR (300 MHz, CDCl3) δ: 7.38 (dd, J=28.7, 16.9 Hz, 11H), 7.00 then treated with various concentrations of test compounds
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with each concentration tested in triplicate and grown for an were as follows: 1250: 5’-CUGCACAACAGCCAGUUAUTT-3’
additional 72 h. The cells were then fixed with 10% pre-cooled (sense) and 5’-AUAACUGGCUGUUGUGCAGTT-3’ (anti-
trichloroacetic acid (TCA) for 1 h at 4°C and stained for 15 min sense) and 3133: 5’-GGGUCCUUCUGGAAACUUATT-3’
at room temperature with 100 μL of 4 mg/mL SRB solution (sense) and 5’-UAAGUUUCCAGAAGGACCCTT-3’ (anti-
(Sigma) in 1% acetic acid. The SRB solution was dissolved in sense). The Mps1 siRNA sequences were as follows:
150 μL of 10 mmol/L Tris base for 5 min and measured at 515 1813: 5’-CCGGAACGAAAUAGCUUAUTT-3’ (sense) and
nm using a multiwell spectrophotometer (VERSAmax, Molec- 5’-AUAAGCUAUUUCGUUCCGGTT-3’ (antisense) and
ular Devices). The inhibition rate for cell proliferation was cal- 2561: 5’-GGUCUGAAUUCUCCUAACUTT-3’ (sense) and
culated as [1–(A515 treated/A515 control)]×100%. The IC50 value 5’-AGUUAGGAGAAUUCAGACCTT-3’ (antisense). The
was obtained using the Logit method.
mock siRNA sequences were as follows: 5’-UUCUCCGA-
ACGUGUCACGUDTDT-3’ (sense) and 5’-ACGUGACACG-
UUCGGAGAADTDT-3’ (antisense). All of the RNA duplexes
Enzyme-linked immunosorbent assay (ELISA)
A total of 20 μg/mL Poly(Glu, Tyr)4:1 (Sigma, St Louis, MO, were synthesized by Genepharma Co (Shanghai, China).
USA) was precoated in 96-well ELISA plates as a substrate.
Cell cycle and apoptosis analysis
Active kinases were incubated with indicated drugs in 1×reac-
tion buffer (50 mmol/L HEPES pH 7.4, 20 mmol/L MgCl2, 0.1 Cells were treated with or without DW532 for the indicated
mmol/L MnCl2, 0.2 mmol/L Na3VO4, 1 mmol/L DTT) con- times. After treatment, cells were harvested and fixed in
taining 5 μmol/L ATP at 37°C for 1 h. After incubation, the 70% ethanol at -20 °C for 1 h followed by PBS washes. The
wells were washed with PBS and then incubated with an anti- cells were then incubated with propidium iodide (PI) and/or
phosphotyrosine (PY99) antibody (Santa Cruz Biotechnology, Annexin-V. After staining, the cell cycle distribution or apop-
Santa Cruz, CA) followed by a horseradish peroxidase (HRP)- tosis was determined using a FACSCalibur flow cytometer
conjugated secondary antibody. The wells were visualized (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with
using o-phenylenediamine (OPD) and read with a multiwell CELLQUEST software (BD Biosciences, Franklin Lakes, NJ,
spectrophotometer (VERSAmax™, Molecular Devices, Sunny- USA).
vale, CA, USA) at 492 nm.
Mitotic cell analysis
Western blot analysis
The percent of mitotic cells was quantified by flow cytometry
Cell samples were lysed in 1×SDS lysis buffer. Western blot as previously described[14]. Briefly, MDA-MB-468 cells were
analysis was subsequently performed using standard pro- treated with or without DW532 or siRNA for the indicated
cedures. Antibodies directed against the following proteins time. After treatment, the cells were harvested and fixed in
were used: p-KDR (Tyr1123), VEGFR, p-EGFR (Tyr1068), 90% methanol at -20°C for 1 h followed by PBS washes. Then,
EGFR, p-ERK, ERK, p-AKT (Ser473), AKT, p-H3 (Ser-10), H3, the cells were incubated with the p-H3 antibody (1:200, CST,
cyclin B1, and p-CDK1 (Tyr15), which were obtained from Cell Cambridge, MA, USA) for 1 h at 37°C. Afterward, the cells
Signaling Technologies (Cambridge, MA, USA), and GAPDH were labeled with an Alexa Fluor®488-conjugated goat anti-
and α-tubulin, which were obtained from Santa Cruz Biotech- mouse IgG antibody (Invitrogen, Carlsbad, CA, USA), and
nology (Santa Cruz, CA, USA).
their DNA was stained with propidium iodide (PI). Cytomet-
ric data were collected with a FACSCalibur flow cytometer
(BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using
Immunofluorescence Assays
Cells were seeded onto glass coverslips overnight and treated CELLQUEST software (BD Biosciences, Franklin Lakes, NJ,
with the indicated drugs for a certain period of time. After USA).
treatment, the cells were fixed for 15 min with 4% paraformal-
dehyde and then permeabilized for 15 min with 0.2% Triton Cellular microtubule stabilization assays
X-100 followed by blocking with 3% bovine serum albumin. After treatment with the indicated drugs, MDA-MB-468 cells
The cells were then incubated with the primary antibodies were harvested in lysis buffer (100 mmol/L PIPES pH 6.9, 1
α–tubulin (1:200) and α–pericentrin (1:100) for 1 h. The cells mmol/L EGTA, 1 mmol/L MgCl2, 30% glycerol, 5% DMSO,
were then stained with Alexa Fluor®488- or Alexa Fluor®633- 1% NP-40, 5 mmol/L GTP and protease inhibitors). The cell
conjugated secondary antibodies (Invitrogen, Carlsbad, CA, lysates were centrifuged at 180 000×g at 37 °C for 1 h, the
USA) for an additional hour. Finally, the cells were stained supernatants containing soluble tubulin were separated from
with DAPI, and the images were evaluated by microscopy.
the pellets containing polymerized tubulin. These two frac-
tions were adjusted to the same volume using SDS-PAGE
loading buffer. Finally, the amount of α-tubulin from equal
RNA interference (RNAi)
BubR1 and Mps1 protein expression were knocked down with aliquots of the polymerized tubulin fraction and free tubulin
small interfering RNA (siRNA) duplexes. The transfection fraction was determined by Western blotting.
of siRNAs was performed according to the manufacturer’s
instructions using the Oligofectamine transfection reagent Tubulin turbidity assays
(Invitrogen, Carlsbad, CA). The BubR1 siRNA sequences Tubulin turbidity was measured with the HTS-Tubulin
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Polymerization Assay Kit (Cytoskeleton, Inc, Denver, CO, further incubated for 48 h. The neighboring neo-vascular
USA). Briefly, tubulin proteins (>99% purity) were suspended zones were photographed with a stereomicroscope (MS5;
in G-PEM buffer containing 80 mmol/L PIPES, 2 mmol/L Leica, Heerbrugg, Switzerland).
MgCl2, 0.5 mmol/L EDTA, 1.0 mmol/L GTP (pH 6.9), and 5%
glycerol with or without the indicated drugs in a 96-well plate, Data analysis
and tubulin polymerization was initiated and monitored by Data are presented as the mean±SD of data from at least three
turbidity changes at 340 nm using a spectrometer (Spectra- independent experiments. Differences were considered sig-
MAX250, Molecular Devices, Sunnyvale, CA, USA).
nificant at P<0.05 as determined by Student’s t-test.
Surface plasmon resonance (SPR)-binding assay
Results
Tubulin binding assays were performed with an SPR tech- Chemistry
nology-based ProteOn TM XPR36 Protein Interaction Array Based on the structure of hematoxylin, a multi-kinase inhibi-
System (Bio-Rad, Hercules, CA, USA) as previously described. tor, we rationally designed and synthesized a series of com-
Briefly, tubulin was dissolved in 10 mmol/L sodium acetate pounds by decreasing the complexity of the hematoxylin
buffer (pH 3.5) and immobilized to a ProteOn TM CM5 sensor structure for the purpose of obtaining a dual inhibitor that
chip. The final immobilization level was 8000 RU (Response can target both kinases and tubulin. DW532 (Figure 1) was
unit). DW532 was diluted two-fold from 50 μmol/L to 3.6 a distinct derivative that was selected for further study.
μmol/L in HBS-T buffer (10 mmol/L HEPES, 150 mmol/L The synthesis of DW532 began with the starting material
NaCl, 3.4 mmol/L EDTA, 0.005% Tween 20, pH 7.4). Differ- 1-[3,4-bis(benzyloxy)-2-hydroxyphenyl]ethanone (1), which
ent concentrations of DW532 were then injected at a flow rate was prepared from benzene-1,2,3-triol using published proce-
of 300 μL/min for 200 s followed by a 300 s dissociation phase. dures with some modification (Figure 1). A condensation of
Data were analyzed with the ProteOnTM Manager software 1 with diethyl carbonate in the presence of sodium produced
and fitted to the 1:1 Langmuir model.
β-keto-phenylpropanoate 2, which was cyclized in refluxed
acetic acid to create Chromenone 3. Triflation of 3 followed by
Suzuki reaction with 3-(benzyloxy)-4-methoxyphenylboronic
Aortic ring assay
Aortic rings were obtained from 6-week-old Sprague-Dawley acid led to the precursor 5. Finally, debenzylation of 5 in tri-
rats. The Sprague-Dawley rats, weighing 280 to 320 g, were fluoroacetic acid led to DW532.
obtained from the Shanghai Institute of Materia Medica
(Shanghai, China). All experiments were approved by the DW532 inhibits the EGFR and VEGFR in vitro kinase activity
Institutional Animal Care and Use Committee (IACUC) and Because DW532 was designed and expected to be a dual
performed according to institutional ethical guidelines on inhibitor targeting kinases and tubulin, we first examined the
animal care. Each aorta was cut into 1 mm slices, which were activity of DW532 against different types of tyrosine kinases
embedded with 55 μL Matrigel, incubated with M199 medium using an in vitro kinase assay. The results showed that DW532
containing 10% FBS and various concentrations of DW532, effectively inhibited EGFR and VEGFR2 activities with IC50
and then photographed on day 7 with an inverted phase con- values of 4.9 and 5.5 μmol/L, respectively (Figure 2A and
trast microscope (IX-51, Olympus, Japan). The quantity of the 2B). This drug also exhibited inhibitory activity for c-Src
microvessels was evaluated by the relative area covered by kinase with less potency (IC50 at 8.6 μmol/L). In contrast, it
microvessels using Image-Pro Express.
showed no obvious inhibition of other kinases tested, includ-
ing PDGFR-α, PDGFR-β, Akt1, p70S6K, EPH-A2, and EPH-B2
even at 10 μmol/L (data not shown). The results showed that
Tube formation assay
Ninety six-well plates were coated with 55 μL Matrigel and as a simplified derivative of hematoxylin, DW532 maintained
then seeded with human umbilical vein endothelial cells kinase inhibitory activity.
(HUVECs) in M199 medium containing 20% FBS and the indi-
cated drugs. After adherence for 6 h, tubes were examined DW532 inhibits EGFR and VEGFR activation and downstream
with an inverted phase contrast microscope (DP70, Olympus, signal transduction in cells
Japan).
We further evaluated whether DW532 could inhibit the ligand-
induced activation of kinases and downstream signaling
transduction in A431 human epithelial cancer cells and human
Chick chorioallantoic membrane (CAM) assay
Fertilized chicken eggs were incubated (7 day) in a humidified umbilical vein endothelial cells (HUVEC), which express high
Roll-X base egg incubator (Lyon Electric Company, CA, USA). levels of EGFR or VEGFR2, respectively[15, 16]. As demonstrated
Gentle suction was applied to the hole located at the broad in Figure 2C, DW532 dose-dependently inhibited the VEGF-
end of the egg to create a false air sac directly over the chicken induced phosphorylation of VEGFR2 and its downstream
chorioallantoic membrane, and a 1 cm2 window was immedi- signaling, which was indicated by phosphorylated Akt, in
ately removed from the eggshell. Glass coverslips (0.5 cm×0.5 HUVEC cells. Similar inhibition potency on the EGF-induced
cm) saturated with compounds or normal saline were placed phosphorylation of EGFR and its downstream signaling was
on areas between pre-existing vessels, and the embryos were also observed in A431 cells treated with DW532 (Figure 2D).
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Figure 2. DW532 inhibited EGFR and VEGFR2 kinase activity. (A and B) DW532 inhibited EGFR and VEGFR2 in vitro kinase activity. The kinase
profiling assay was conducted as described in the Experimental section by using ELISA kinases assay. IC50 values were obtained by Logit method
based on the data obtained from three independent experiments and expressed as mean±SD. (C and D) Effects of DW532 on VEGF-induced VEGFR2
phosphorylation in HUVEC cells (C) and EGF-induced EGFR phosphorylation in A431 cell line (D). Cells were starved for 12 h and treated with indicated
concentration of DW532 for 2 h and then stimulated by 50 ng/mL EGF and 100 ng/mL VEGF for 15 min. Cells were harvested and subjected to
Western blot analysis.
These results demonstrated that DW532 possesses VEGFR2 When fitted to the 1:1 Langmuir model, DW532 displayed
and EGFR inhibitory activity at the cellular level.
high binding affinity with an equilibrium dissociation con-
stant (KD value) of 3.6 μmol/L (Figure 3B). Therefore, these
DW532 inhibits tubulin polymerization in vitro by directly binding data indicate that DW532 could directly bind tubulin and thus
with tubulin
inhibit tubulin polymerization.
We then examined whether DW532 also affects tubulin polym-
erization as expected by using an in vitro turbidity assay with DW532 inhibits tubulin polymerization in cancer cells and
recombinant tubulin protein in the presence of various con- causes disordered mitosis
centrations of DW532. DW532 was shown to inhibit tubulin Tubulin inhibitors could change the dynamics of cellular
polymerization in a concentration-dependent manner, which microtubules by promoting (ie, microtubule stabilizer) or
is similar to the activity of the classic microtubule destabiliz- inhibiting (ie, microtubule destabilizer) tubulin polymeriza-
ers VCR and combretastatin (CA4) but quite different than the tion[4]. We then evaluated whether DW532 affected tubulin
microtubule-stabilizing agent Taxol (Figure 3A). At a concen- polymerization in cancer cells using an ultracentrifugation
tration of 20 μmol/L, DW532 suppressed tubulin polymeriza- method (Figure 3C). The treatment of MDA-MB-468 cells with
tion by 30%, and at 40 μmol/L, tubulin polymerization was DW532 substantially decreased polymerized tubulin (P) in
almost completely inhibited. The results demonstrated that pellets in a dose-dependent manner, which was in accordance
DW532 could inhibit tubulin polymerization as a microtubule with upregulation of soluble tubulin (S) in the supernatant,
destabilizer but not as a microtubule stabilizer.
indicating that DW532 promotes the transition of cellular
To identify how DW532 interacts with tubulin protein, we tubulin from the polymerized state to the free state (Figure
investigated its potential binding affinity using an in vitro SPR 3C). Treatment with the positive control microtubule desta-
assay. The results revealed that DW532 treatment led to RU bilizer VCR resulted in similar results as DW532, whereas the
increase in a concentration-dependent manner (Figure 3B). microtubule stabilizer Taxol led to the upregulation of polym-
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Figure 3. DW532 inhibited tubulin polymerization through directly binding with tubulin protein and disrupted mitotic spindle assembly. (A)
DW532 inhibited tubulin polymerization in vitro. DW532 and reference compounds were incubated with purified tubulin protein at 37ºC, and then
polymerization were kinetic monitored by SpectraMAX250 at 340 nm every 1 min for 30 min at 37ºC. (B) DW532 bound to tubulin directly. The
tubulin binding affinity was assessed by SPR assay. Different concentrations of DW532 were prepared as indication and then injected to Biacore
3000 instrument with tubulin coated chip, and the data were analyzed with the ProteOnTM Manager software. (C and D) DW532 inhibited tubulin
polymerization in cancer cells. MDA-MB-468 cells were treated with indicated concentrations for 12 h and then harvested by lysis buffer. After
centrifuged 180000×g 37ºC for 1 h, the free and polymerized tubulin [soluble (S) and polymerized (P)] were separated and detected by Western
blotting (C). A549 cells were treated with indicated concentration of drugs for 6 h. The cells were then processed for immunofluorescence staining and
confocal microscopy (×600) (D). (E) DW532 disrupted mitotic spindle assembly. A549 cells were treated with indicated concentration of drugs for 12 h.
The cells were then processed for immunofluorescence staining and confocal microscopy (×600).
erized tubulin, which is consistent with previous reports and stretched network, whereas DW532 treatment resulted
(Figure 3C).
in dispersed tubulin staining, which is similar to that found
Immunofluorescence staining was then performed to detect in VCR-treated cells. In contrast, Taxol treatment caused a
microtubule networks in cancer cells treated with DW532. As significant increase in microtubule bundles. The above results
demonstrated in Figure 3D, untreated cells exhibited an intact further illustrated that DW532 disrupted the microtubule net-
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923
works in cancer cells by destabilizing microtubules.
play a major role in the antitumor activity of DW532.
Because microtubules are required for assembly of the
Therefore, our results demonstrated that DW532 potently
mitotic spindle, which is essential for accurate chromosome inhibited the in vitro proliferation of tumor cells via cell cycle
segregation[17], we further investigated changes in the cen- arrest and apoptosis induction.
trosomes and spindles of cancer cells treated with or with-
out DW532 as indicated by staining with pericentrin and DW532-induced abnormal division depends on the spindle
α-tubulin, respectively. As shown in Figure 3E, the cellular assembly checkpoint
chromosomes were scattered in the cytoplasm, and multipolar The above mentioned findings showed that tubulin inhibi-
spindles were formed with DW532 treatment, which is con- tion might play a major role in the cell cycle arrest induced by
sistent with other traditional microtubule destabilizers. Inter- DW532. Generally, disruption of the mitotic spindle through
estingly, a small proportion of monopolar spindles was also tubulin inhibition reduces the microtubule tension or attach-
observed in DW532-treated but not in control cells, which is ment on kinetochores, which then activates the spindle assem-
rarely caused by tubulin inhibitors and deserves further inves- bly checkpoint (SAC) and delays the onset of anaphase[17]
.
tigation.
To identify the role of DW532-mediated SAC activation in
cell cycle arrest, we further evaluated the role of BubR1 and
DW532 potently inhibits the proliferation of tumor cells via cell Mps1[21–23], two core SAC proteins, in the cell cycle arrest activ-
cycle arrest and apoptosis induction
ity mediated by DW532 using gene knockdown assays. The
The presence of both tyrosine kinase and tubulin inhibition efficiency of siRNA knockdown was first examined (Figure
could result in cell-cycle arrest, apoptosis and growth inhi- 5A, 5B), and it was confirmed that siRNA transfection had no
bition. Therefore, we further examined these activities in obvious effects on the cell cycle (upper panel of Figure 5C,
DW532-treated cancer cells. The in vitro antitumor activity of 5D). As expected, the percentage of mitotic cells induced by
DW532 was evaluated in a panel of 16 cancer cell lines with DW532 was dramatically decreased in cells transfected with
different tissue origins. As illustrated in Figure 4A, DW532 siRNAs directed against BubR1 or Mps1 compared with con-
inhibited the proliferation of the tested cancer cells with an trol cells (Figure 5C, 5D, lower panel). These results indicate
average IC50 of 1.82 μmol/L (range: 0.35 to 7.32 μmol/L). that tubulin inhibition and the subsequent SAC activation con-
DW532 displayed non-selective in vitro anti-proliferative activ- tribute to the mitotic arrest induced by DW532 treatment.
ity against different types of tumor cells.
We then evaluated whether DW532 caused apoptosis in DW532 exhibits anti-angiogenesis activity in vitro and in vivo
cancer cells by the Annexin V/PI staining method. The results As VEGFR2 and tubulin play key roles in the process of
revealed that treatment with DW532 led to a dose-dependent angiogenesis[24, 25], we further evaluated the anti-angiogenesis
increase in apoptosis in the different types of cancer cells activity of DW532 in vitro and in vivo. The formation of func-
tested, including KB mouth epidermal carcinoma cells, MDA- tional tubules in HUVECs plated on Matrigel was first deter-
MB-468 human breast carcinoma cells and A431 human mined. As shown in Figure 6A, HUVEC cells form a complete
epithelial carcinoma cells (Figure 4B). For example, approxi- network structure within 6 h with serum stimulation. After
mately 59.7% of KB cells underwent apoptosis following DW532 treatment, the tubule formation was suppressed in a
treatment with DW532 at 1 μmol/L for 48 h. Approximately dose-dependent manner. At a concentration of 0.1 μmol/L
70% of the cells underwent apoptosis when the concentration DW532, the inhibition was approximately 41%, and at 10
given was 10 μmol/L. We further examined the mechanisms μmol/L, there was nearly complete suppression (Figure 6B).
involved in the apoptosis induced by DW532, and the results We further tested the angiogenesis activity of DW532 using
showed that with increasing DW532 concentrations, the cleav- the aortic ring assay, which is a unique ex vivo method that
age of PARP, caspase-3 and caspase-9 concomitantly increased recapitulates the key steps in the angiogenesis process[26]. As
(Figure 4C), demonstrating that DW532 induced apoptosis illustrated in Figure 6C, DW532 showed potent inhibition of
through a caspase-related mechanism.
the microvessel formation of the aortic ring with 18.7% inhibi-
To clarify the effects of DW532 on the cell cycle, six types of tion observed at 0.1 μmol/L and 60.9% and 84.1% inhibition at
sensitive cell lines were selected for treatment to test their cell 1 and 10 μmol/L, respectively (Figure 6D). Moreover, we also
cycle distribution. The results showed that 1 μmol/L DW532 examined the effects of DW532 on the new blood vessel for-
treatment nearly arrested all six cell lines at the G2/M phase mation process using the CAM assay, a model used to study
(Figure 4D). In agreement with these results, the level of two in vivo neovascularization since the early 1970s when it was
mitotic markers, cyclin B1 and phosphorylated histone H3, adapted by Folkmann et al[27] The results showed that vascular-
was significantly elevated, and the negative regulatory phos- ization in chick embryos was significantly inhibited when they
phorylation site in CDK1 was also distinctly decreased (Figure were treated with DW532 (0.1 or 1 nmol per egg) compared
4E). From previous reports, it is known that tubulin inhibitors with a no treatment control (Figure 6E). Therefore, these
generally cause cell cycle arrest in G2/M phase[18, 19], while pro- results suggest that as an agent targeting both tubulin and
tein kinase inhibitors induce G1 phase arrest[20]. It is evident VEGFR2, DW532 potently inhibits angiogenesis in vitro and in
that DW532 can arrest different tumor cell lines at the G2/M vivo.
phase, supporting the notion that tubulin inhibition might
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Figure 4A–4C. DW532 inhibited the proliferation of tumor cell lines and induced cell mitosis disorder. (A) DW532 inhibited the proliferation of a panel
of human cancer cells. Proliferation of the tumor cells were assessed by SRB assays after 72 h treatments with DW532. All the Results were expressed
as mean±SD of three independent experiments. (B and C) DW532 induced cell apoptosis. Cells were treated with indicated concentration of DW532
for 48 h and harvested for flow cytometry assays (B) and Western blot (C), respectively.
and evaluated their in vitro antitumor activities. One of the
Discussion
The aim of this study was to design and identify novel representative derivatives, DW532, showed distinct antitumor
inhibitors targeting both kinases and tubulin. Based on the activity against cancer cells in preliminary screening (data not
chemical structure of hematoxylin, a natural product that we shown) and thus was selected for further investigation. This
previously reported to have kinase inhibitory activity, we compound has moderate inhibitory activity toward the EGFR
rationally designed and synthesized a series of derivatives and VEGFR2 kinases in vitro and in cancer cells. In addition,
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Figure 4D–4E. (D) DW532 induced mitosis arrest at G2/M phase. Six cell lines were treated with DW532 at 1 and 10 μmol/L for 24 h followed by flow
cytometry analysis. (E) DW532 affected mitotic associated protein. Six cell lines were treated with DW532 at 1 and 10 μmol/L for 24 h followed by
Western blot to examine the expression of the proteins.
Figure 5. Knockdown of the spindle assembly checkpoint component decreased the DW532 induced mitotic arrest. (A and B) Efficiency of BubR1 and
MPS1 siRNA in MDA-MB-468 cells. (C and D) Percentage of mitotic cells after knockdown of spindle assembly checkpoint component. After transfected
with BubR1 siRNA or Mps1 siRNA (50 nmol/L) or control siRNA for 24 h, MDA-MB-468 cells were treated with DW532 (1 μmol/L) for another 24 h.
Mitotic cells were analyzed by p-H3 and PI labeled flow cytometry assays.
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Figure 6. DW532 had anti-angiogenesis activity. (A) DW532 inhibited the in vitro tube formation of HUVECs. HUVECs containing indicated drugs
were seeded in 96-well plates which was coated with Matrigel. After 6 h align, the tube was examined with an inverted phase contrast microscope.
(B) Relative tube formation was quantitated and plotted. (C) DW532 inhibited microvessel outgrowth arising from rat aortic rings. Aortic rings were
embedded in Matrigel in 96-well plates, and then fed with medium containing various concentrations of DW532 for 7 d. (D) The area of microvessels
was quantified and normalized to untreated controls. (E) DW532 showed anti-angiogenesis in a chorioallantoic membrane model. Glasscover-slip
saturated with DW532 or normal saline was placed areas between pre-existing vessels in the fertilized chicken eggs and incubated for 48 h. The
coverglass saturated with or without DW532 was placed on the right side of per field. Arrows indicated the edge line of the coverglass. cP<0.01 vs
control.
DW532 exhibited more potent inhibitory activity on tubu- Further experimental results revealed that this molecule dis-
lin polymerization by directly binding with tubulin protein. rupted the microtubule networks in cancer cells. This effect
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927
is quite similar to VCR, demonstrating that it belongs to the in vitro and in vivo. These characteristics, combined with bet-
microtubule-destabilizing group of MTAs[4]. Therefore, our ter solubility and a convenient approach to synthesis, make
results demonstrated that DW532 could inhibit kinase activ- DW532 a promising anti-cancer agent in cancer therapy.
ity and tubulin polymerization concurrently, which indicates
the success of our original concept of the chemical designing
Acknowledgements
of a dual-functional agent for tumor therapy. Until now, only This work was supported by the National Natural Science
a few agents have been reported to possess dual inhibitory Foundation of China (No 81173080, 81273365, and 81321092)
effects on both kinases and tubulin[8–10, 28, 29]. These reported and the National Science and Technology Major Project “Key
agents are structurally different from DW532. Therefore, New Drug Creation and Manufacturing Program” of China
DW532 is a dual inhibitor of kinases and tubulin with a new (No 2012ZX09103101-024).
scaffold, and it deserves further investigation.
Inhibition of tubulin or kinases can result in many effects
Author contribution
on tumor cells, such as cell-cycle arrest, apoptosis and growth Ting PENG, Wen-hu DUAN, Hua XIE, and Jian DING partici-
inhibition[6]. Our results show that DW532 treatment causes pated in the research design; Ting PENG, Jian-rui WU, Lin-
cell cycle arrest, apoptosis and subsequently anti-proliferation jiang TONG, Meng-yuan LI, Kun HAN, Rong QU, Yi SU, and
activity in a panel of cancer cells. The results from flow Yi CHEN conducted experiments; Ting PENG, Jian-rui WU,
cytometry revealed that DW532 treatment led to G2/M phase Fang CHEN, and Yi-xin LENG contributed new reagents or
cell cycle arrest in different type of cancer cells with concomi- analytic tools; Ting PENG, Jian-rui WU, Wen-hu DUAN, and
tant alterations in the mitotic cell cycle-associated proteins Hua XIE performed the data analysis; Ting PENG, Jian-rui
cyclin B1, p-CDK1, and p-H3[30]. Generally, tyrosine kinase WU, Wen-hu DUAN, and Hua XIE wrote or contributed to the
inhibitors trigger G1 phase arrest, while tubulin inhibitors writing of the manuscript.
induce G2/M phase arrest, and our results support the idea
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DW532 exhibited potent inhibition of angiogenesis, which
contributes to the antitumor activity of the compound.
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