Design, synthesis, and biological evaluation of chalcone- containing shikonin derivatives as inhibitors of tubulin polymerization

Article abstract of DOI:10.1002/cmdc.201700001

The biological importance of microtubules in mitosis makes them an interesting target for the development of anticancer agents. In this study, a series of novel chalcone-containing shikonin derivatives was designed, synthesized, and evaluated for biologic

Full text of DOI:10.1002/cmdc.201700001

DOI: 10.1002/cmdc.201700001
Full Papers
Design, Synthesis, and Biological Evaluation of Chalcone-
Containing Shikonin Derivatives as Inhibitors of Tubulin
Polymerization
Han-Yue Qiu,[a, b] Fang Wang,[a, b] Xue Wang,[a, b] Wen-Xue Sun,[a, b] Jin-Liang Qi,[a, b] Yan-
Jun Pang,[a] Rong-Wu Yang,[a] Gui-Hua Lu,*[a, b] Xiao-Ming Wang,*[a, b] and Yong-Hua Yang*[a, b]
The biological importance of microtubules in mitosis makes
them an interesting target for the development of anticancer
agents. In this study, a series of novel chalcone-containing shi-
konin derivatives was designed, synthesized, and evaluated for
biological activities. Among them, derivative PMMB-259 [(R)-1-
(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methyl-
firmed that PMMB-259 can induce MCF-7 cell apoptosis,
reduce the mitochondrial transmembrane potential, and arrest
the cell cycle at the G2/M phase. Moreover, the morphological
variation of treated cells was visualized by confocal microsco-
py. The results, along with docking simulations, further indicat-
ed that PMMB-259 can bind well to tubulin at the colchicine
site. Overall, these studies may provide a new molecular scaf-
fold for the further development of antitumor agents that
target tubulin.
pent-3-en-1-yl
(E)-2-(4-(3-oxo-3-(3-(trifluoromethoxy)phenyl)-
prop-1-en-1-yl)phenoxy)acetate] was identified as a potent in-
hibitor of tubulin polymerization. Further investigation con-
Introduction
Mitosis is an elaborate and essential biological activity in so-
matic proliferating cells, resulting in the division of duplicated
sets of chromosomes into two genetically identical daughter
cells.[1] In the whole process of cell mitosis, the dynamic equi-
librium between microtubules and its heterodimers plays an
extremely important role.[2] When perturbed, the polymeri-
zation/depolymerization dynamics lead to cell-cycle arrest and
apoptosis.[3] Microtubule polymerization dynamics also affect
the cell’s capacity to maintain or change shape and undergo
critical processes such as intracellular transport, cell signaling,
and secretion.[4] Considering the significant role of microtu-
bules in actively proliferating and newly divided cells, it is not
surprising that microtubules are among the most important
molecular targets for the development of anticancer agents.[5,6]
To date, various classes of anti-tubulin agents have been ex-
plored and pre-clinically or clinically examined, with some al-
ready being used in cancer therapy.[7,8] However, the search for
new tubulin inhibitors with safer and more effective profiles re-
mains an important ongoing task.
Natural products are currently gaining significant status in
the drug design field. Strategies based on modifying natural
products have benefited from the innately complex scaffolds
of natural products, which are rarely covered in commercial
synthetic compound libraries.[9] In fact, the most prominent tu-
bulin inhibitors, paclitaxel and colchicine, were originally ex-
tracted from plants.[10,11] However, with natural products, there
remains considerable space for optimization in terms of oral
bioavailability, safety, efficacy, and other pharmacological prop-
erties.[12,13] As the main efficacious component isolated from
the root of the Chinese herbal medicine Lithospermum erythro-
rhizon, shikonin has been used in Europe and Asia since an-
cient times.[14] It is attracting considerable attention in the field
of natural product chemistry due to its manifold pharmacologi-
cal properties such as wound healing, antioxidant, antibacteri-
al, anti-inflammatory, and antitumor.[15–19] Unfortunately, shiko-
nin also exerts nonselective cytotoxic effects on normal cells,
which hinders its further development. Structural modifications
aimed at improving target selectivity help solve this dilemma.
Our previous study also indicated that the toxicity of shikonin
against non-cancer cells can be decreased by modifying the
side chain hydroxy group.[20–22] Moreover, in our previous re-
search, various types of shikonin derivatives were designed
and found to have notable anti-tubulin activity with lower cy-
totoxicity.[23] Given these previous research results, in this study
we attempted to design a type of shikonin derivative that has
improved tumor targeting. We designed and synthesized
a series of chalcone-containing shikonin derivatives. Biological
evaluations indicated that some of these compounds can act
as potent inhibitors of tubulin polymerization. In addition, mo-
[a] H.-Y. Qiu, F. Wang, X. Wang, W.-X. Sun, Prof. Dr. J.-L. Qi, Dr. Y.-J. Pang,
Prof. Dr. R.-W. Yang, Dr. G.-H. Lu, Prof. Dr. X.-M. Wang, Prof. Dr. Y.-H. Yang
State Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint In-
stitute of Plant Molecular Biology, Nanjing University, Nanjing 210023
(China)
Fax: (86)25-8968-6305
E-mail: yangyh@nju.edu.cn
wangxm07@nju.edu.cn
guihua.lu@nju.edu.cn
[b] H.-Y. Qiu, F. Wang, X. Wang, W.-X. Sun, Prof. Dr. J.-L. Qi, Dr. G.-H. Lu,
Prof. Dr. X.-M. Wang, Prof. Dr. Y.-H. Yang
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing
Forestry University, Nanjing, 210037 (China)
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/cmdc.201700001.
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lecular docking simulations were performed in an attempt to
rationalize the biological results.
Table 1. The CDOCKER interaction energy (DGb) between target com-
pounds and 1SA0 and in vitro inhibition of tubulin polymerization.
Compound
R
DGb [ÀkcalmolÀ1
]
Tubulin IC50 [mm]
Results and Discussion
PMMB-248
PMMB-249
PMMB-250
PMMB-251
PMMB-252
PMMB-253
PMMB-254
PMMB-255
PMMB-256
PMMB-257
PMMB-258
PMMB-259
PMMB-260
PMMB-261
PMMB-262
PMMB-263
PMMB-264
PMMB-265
shikonin
H
57.2987
55.9599
63.6994
60.4691
58.2318
60.0841
66.6288
60.6049
63.2935
52.9641
64.3526
64.6074
53.5111
55.3211
57.4367
54.2322
51.1432
53.0311
16.14Æ0.65
17.29Æ0.51
6.26Æ0.29
15.36Æ0.46
18.35Æ0.36
13.65Æ0.22
3.85Æ0.39
16.99Æ0.25
10.23Æ0.46
28.35Æ0.27
4.63Æ0.11
4-OCH3
3,4,5-3OCH3
4-CF3
2,3,4-3Cl
2-CF3
2-OCH3
3-CF3
4-CH2CH3
2-F
3,5-2CF3
3-OCF3
3-OCH3
2-CH3
2-Cl
2-Br
Chemistry
The routes to synthesize the novel chalcone-containing shiko-
nin derivatives PMMB-248–PMMB-265 are outlined in
Scheme 1. All of these compounds are reported for the first
time (Table 1), and gave satisfactory analytical and spectro-
1
scopic data. H NMR and ESI-MS spectra are in full accordance
with the assigned structures, which are presented in the Sup-
porting Information (SI).
2.98Æ0.53
33.67Æ0.45
24.14Æ0.57
20.15Æ0.66
23.56Æ0.71
19.68Æ0.57
21.39Æ0.66
15.20Æ0.25
3.10Æ0.35
In vitro antiproliferative activity
3-CH3
4-CH3
Compounds PMMB-248–PMMB-265 were evaluated for their
antiproliferative activities by MTT assay against three cancer
cell lines: HeLa (human cervical cancer), MCF-7 (human breast
cancer), and A549 (human lung adenocarcinoma epithelial), as
well as two non-cancer cell lines: 293T (human kidney epithe-
lial) and L02 (human hepatic). The IC50 values were calculated
and are listed in Table 2. The data for intermediate compounds
B1–B18 were also collected and are provided in SI Table S1 for
comparison. We found that all compounds show moderate to
excellent antiproliferative effects on MCF-7 and A549 cancer
cell lines, but not HeLa. Especially against the MCF-7 cell line,
PMMB-259 showed the best antiproliferative activity, with
a lower IC50 value (2.36Æ0.32 mm) than that of shikonin (7.61Æ
0.16 mm). Subsequently, MTT assays against the two non-cancer
cell lines, 293T and L02, were performed to test the cytotoxicity
of the obtained compounds. As shown in Table 2, none of the
colchicine
compounds have any clear cytotoxic effects on the two non-
cancer cell lines, hinting at considerable selectivity.
Inhibition of tubulin polymerization
To determine whether the antiproliferative activity of com-
pounds PMMB-248–PMMB-265 result from their interaction
with tubulin, we evaluated their capacity to inhibit tubulin
polymerization in vitro. The results are listed in Table 1, indicat-
ing that the IC50 values for inhibiting tubulin polymerization
Scheme 1. Reagents and conditions: a) ethyl bromoacetate, K2CO3, acetone, 608C, 8 h; b) acetophenone, NaOH, 08C, 2 h; c) NaOH, EtOH, 908C, 6 h; d) DCC,
DMAP, CH2Cl2, 08C, 1 h.
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the monomeric form of JC-1, with low DYm in apop-
totic cells. Cells treated with PMMB-259 exhibited in-
creased green fluorescence and decreased red fluo-
rescence, suggesting the depolarization of mito-
chondria and reduced DYm. The ratio of cells with
high membrane potential exposed to PMMB-259 at
4 and 8 mm decreased from 86.6% to 78.9% and
53.3% after 4 h treatment. Moreover, the ratio of
cells with high membrane potential decreased from
81.8% to 78.1% and 66.9% after cells exposed to
PMMB-259 at 4 mm for 4 and 8 h treatment.
Table 2. Inhibition of proliferation of HeLa, MCF-7, A549, L02 and 293T cells by PMMB
(248–265).
Compound
IC50 [mm]
HeLa
MCF-7
A549
L02
293T
PMMB-248
PMMB-249
PMMB-250
PMMB-251
PMMB-252
PMMB-253
PMMB-254
PMMB-255
PMMB-256
PMMB-257
PMMB-258
PMMB-259
PMMB-260
PMMB-261
PMMB-262
PMMB-263
PMMB-264
PMMB-265
shikonin
11.70Æ0.01
14.82Æ0.08
8.91Æ0.07
11.59Æ0.13
13.9Æ0.12
8.56Æ0.05
5.29Æ0.04
11.11Æ0.42
9.13Æ0.17
8.42Æ0.21 12.49Æ0.10
9.68Æ0.05 17.68Æ0.27
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5.22Æ0.19
8.32Æ0.03
8.10Æ0.17
11.36Æ0.22
8.91Æ0.04 13.66Æ0.32
7.54Æ0.05
4.58Æ0.43
8.14Æ0.15
6.71Æ0.15
8.67Æ0.13
6.61Æ0.13
9.66Æ0.05
8.17Æ0.03
17.17Æ0.03 13.20Æ0.07 10.33Æ0.09
PMMB-259 induces cell-cycle arrest in MCF-7 cells
in a dose- and time-dependent manner
10.41Æ0.09
4.53Æ0.07
4.82Æ0.13
2.36Æ0.32
7.27Æ0.32
5.84Æ0.18
14.82Æ0.08 15.47Æ0.18 21.21Æ0.07
We further assessed the effect of PMMB-259 on the
cell cycle to ascertain whether MCF-7 cells are
blocked in mitosis. MCF-7 cells were treated with
PMMB-259 at increasing concentrations (0, 2, 4, and
8 mm) for 24 h and then incubated with propidium
iodide (PI) to analyze their genomic DNA content in
flow cytometry assays. As illustrated in Figure 3,
PMMB-259 induced a gradual accumulation of cells
in the G2/M phase in a dose-dependent manner, and
17.39Æ0.21 12.55Æ0.11
20.66Æ0.01
14.57Æ0.23
17.37Æ0.01
16.17Æ0.23
14.17Æ0.11
6.44Æ0.04
0.15Æ0.02
9.18Æ0.25 16.71Æ0.44
11.58Æ0.26
9.48Æ0.02
6.81Æ0.14
9.51Æ0.18
10.48Æ0.22 12.51Æ0.47
7.61Æ0.16
0.22Æ0.06
11.68Æ0.01 10.52Æ0.02 9.27Æ0.01
0.47Æ0.06 2.3Æ0.01 1.03Æ0.03
colchicine
show a similar trend with the respective IC50 values from the
cell antiproliferation assay. Among them, PMMB-259 showed
the most potent activity of anti-tubulin polymerization, with
an IC50 value of 2.98Æ0.53 mm. We therefore conclude that
most of the synthesized compounds can inhibit tubulin assem-
bly and that the antiproliferative effects might be produced in
part by their interaction with tubulin.
43.76% of cells were arrested in G2/M phase upon exposure to
8 mm PMMB-259 for 24 h. We then treated MCF-7 cells with
4 mm PMMB-259 for 0, 12, 24, and 36 h. The results show that
PMMB-259 could block the cell cycle in G2/M phase in a time-
dependent manner, and the maximum accumulation of cells in
G2/M phase was observed after treatment with PMMB-259 at
4 mm for 36 h. Furthermore, we performed western blotting on
PMMB-259 with cycle-related proteins to confirm its G2/M
arrest effect. As research has revealed that cyclin B1 and CDK1
play crucial roles in regulating the transition from G2 to M
phases, their expression was examined in the test. The western
blot results show that PMMB-259 can increase the expression
of cyclin B1 and CDK1 in a dose-dependent manner, which are
in accordance with the above results.
PMMB-259 causes apoptosis in MCF-7 cells in a dose- and
time-dependent manner
We next performed an Annexin V–FITC/PI double staining
assay to validate the apoptosis-stimulating effects of PMMB-
259 on MCF-7 cells. We treated MCF-7 cells with varying con-
centrations (0, 2, 4, and 8 mm) of PMMB-259 for 24 h, and ana-
lyzed cells for changes in apoptotic markers with a flow cytom-
eter. As shown in Figure 1, the results indicate that increasing
concentrations of PMMB-259 yield an increased apoptotic rate
in MCF-7 cells. Meanwhile, we treated MCF-7 cells with PMMB-
259 at 4 mm for 0, 12, 24, and 36 h, and found that the per-
centage of apoptotic cells is markedly elevated in a time-de-
pendent manner. In conclusion, it is clear from the aforemen-
tioned results that PMMB-259 can induce apoptosis in MCF-7
cells in a dose- and time-dependent manner.
PMMB-259 inhibits tubulin polymerization and disrupts the
microtubule system
To further investigate the effect of PMMB-259 on the cytoskele-
tal network of tubulin, MCF-7 cells were immunostained and
analyzed by confocal microscopy. As illustrated in Figure 4, the
microtubule network of control cells displayed a normal ar-
rangement and organization, which is slim and fibrous. Treat-
ment with paclitaxel at 1 mm dramatically enhanced microtu-
bule polymerization, with an increase in the density of cellular
microtubules and formation of long thick microtubule bundles.
In contrast, the cell membrane microtubules of cells treated
with 1 mm colchicine showed depolymerization and solubility.
With reference to these two groups and an untreated control
group, we found that a significant decrease in the density of
microtubules and the radial organization of microtubules was
perturbed after treatment with PMMB-259 at 2 mm. We there-
fore conclude that PMMB-259 can inhibit tubulin polymeri-
PMMB-259 reduces mitochondrial transmembrane potential
in MCF-7 cells
The mitochondrial transmembrane potential (DYm) was next
monitored by JC-1 staining. Figure 2 shows representative JC-
1 fluorescence in both FL-1 and FL-2 channels. Red fluores-
cence represents the mitochondrial aggregate form of JC-1,
with high DYm in normal cells. Green fluorescence represents
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Figure 1. Apoptosis study of PMMB-259 tested on MCF-7 cells. A) Cells treated with various concentrations (0, 2, 4, and 8 mm) of PMMB-259 for 24 h were col-
lected and analyzed. B) Cells treated with 4 mm PMMB-259 at different time points (0, 12, 24, and 36 h) were collected and analyzed. The percentage of early
apoptotic cells is shown in the lower right quadrant (Annexin V–FITC-positive/PI-negative cells), and late apoptotic cells are located in the upper right quad-
rant (Annexin V–FITC-positive/PI-positive cells). Images are representative of three independent experiments. Data are the meanÆSD of three independent
experiments; *p<0.05, **p<0.01.
zation just like colchicine, and these results further confirm
that the observed effects are due to its interaction with tubu-
lin, resulting in a prolonged mitotic arrest that ultimately leads
to cell death.
Molecular docking
To gain a better understanding into the binding mode of the
synthesized compounds, we proceeded to examine the inter-
action of all compounds with the crystal structure of tubulin
(PDB ID: 1SA0) using the Discovery Studio 3.5 software pack-
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Figure 2. Mitochondrial transmembrane potential was analyzed in MCF-7 cells by JC-1 staining.
age (Accelrys, Co. Ltd). Molecular docking was performed by
inserting the compounds into the colchicine binding site of tu-
bulin, and the general workflow is described in the Experimen-
tal Section below. The CDOCKER_INTERACTION_ENERGY values
of molecular docking are listed in Table 1. It is clear that
PMMB-259 shows the best interaction energy of all the synthe-
sized compounds, with a calculated interaction energy value of
À64.6074 kcalmolÀ1. The model of PMMB-259 docked with tu-
bulin is depicted in Figure 5. In the binding model, PMMB-259
is nicely bound to the colchicine binding site of tubulin by
three hydrogen bonds with Ser178, Tyr202, and Lys254. These
molecular docking results, along with the biological assay data,
suggest that PMMB-259 is a potential inhibitor of tubulin poly-
merization.
colchicine. According to the analysis of the binding model of
PMMB-259 with tubulin, three hydrogen bonds were observed
with residues in the colchicine binding site, which may contrib-
ute to its anti-tubulin polymerization activity. Our findings indi-
cate that PMMB-259 could be a lead molecule for further re-
search into tubulin polymerization inhibitors.
Experimental Section
Materials and measurements: All chemicals (reagent grade) used
were purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing,
China). Paclitaxel and colchicine were purchased from Sigma–Al-
drich (St. Louis, MO, USA). All 1H NMR spectra were recorded on
a Bruker DPX 500 model spectrometer in CDCl3, and chemical
shifts (d) are reported as parts per million (ppm). ESI-MS spectra
were recorded a Mariner System 5304 mass spectrometer. Melting
points were determined on an XT4 MP apparatus (Taike Corp., Bei-
jing, China). Thin-layer chromatography (TLC) was performed on
silica gel plates (silica gel 60 GF254) and visualized by UV light
(l 254 nm). Column chromatography was performed using silica
gel (200–300 mesh) eluting with ethyl acetate and petroleum ether
(bp: 30–608C). The Annexin V–FITC Apoptosis Detection Kit (A211-
01/02) was purchased from Vazyme Biotech Co. Ltd. (Nanjing,
China); the JC-1 Mitochondrial Membrane Potential Assay Kit
(C2006) was purchased from Beyotime Biotech (Shanghai, China);
the Cell-Cycle Assay Kit (A411-01/02) was purchased from Vazyme
Biotech Co. Ltd. (Nanjing, China).
Conclusions
In conclusion, a series of novel chalcone-containing shikonin
derivatives, PMMB-248–PMMB-265, were designed, synthe-
sized, and evaluated as potential inhibitors of tubulin polymeri-
zation. Most of them showed potent antiproliferative activity,
and PMMB-259 displayed the most potent activity against tu-
bulin polymerization (IC50: 2.98Æ0.53 mm) and MCF-7 cell pro-
liferation (IC50: 2.36Æ0.32 mm). Furthermore, mechanism of
action studies proved that PMMB-259 can induce apoptosis in
MCF-7 cells, reduce the mitochondrial membrane potential,
cause an accumulation of cells in the G2/M phase of the cell
cycle, and effect severe disruption of the microtubule system
in a manner similar to that of the positive control compound,
General procedure for the synthesis of compounds PMMB-248–
PMMB-265: A mixture of 4-hydroxybenzaldehyde (10 mmol) and
anhydrous potassium carbonate (15 mmol) were dissolved in ace-
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Figure 3. Effect of PMMB-259 on the cell-cycle distribution of MCF-7 cells. A) Cells treated with 4 mm PMMB-259 at different time points (0, 12, 24, and 36 h).
B) Cells treated with various concentrations (0, 2, 4, and 8 mm) of PMMB-259 for 24 h were collected and disposed for detection. C) The expression of cyclin B1
and CDK1 were detected by western blot analysis. D) Relative expression levels were analyzed by ImageJ software.
tone, and ethyl bromoacetate (12 mmol) was thereafter added
dropwise. The reaction mixture was held at reflux for 8 h, and the
precipitate was filtered before vacuum distillation. The solvent was
then concentrated, water (150 mL) was added, and the solution
was adjusted to pH 5.0 with dilute HCl. Product extraction was car-
ried out with ethyl acetate, dried over Na2SO4, and the organic
layer was then concentrated in vacuo to afford compound A1.
Shikonin, C1–C18, 4-dimethyaminopyridine (DMAP), and N,N’-dicy-
clohexylcarbodiimide (DCC) were dissolved in dichloromethane.
The reaction was stirred on an ice bath for 1 h and monitored by
TLC. After completion of the reaction, the products PMMB-248–
PMMB-265 were obtained and purified by column chromatogra-
phy.
Cell culture: Human cervical cancer (HeLa), human breast cancer
(MCF-7), human lung adenocarcinoma epithelial (A549), human
hepatic (L02) and human kidney epithelial (293T) cell lines were
purchased from Nanjing Keygen Technology (Nanjing, China). Cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Hyclone; high glucose) with l-glutamine supplemented with 10%
fetal bovine serum (FBS, BI), 100 UmLÀ1 penicillin and 100 mgmLÀ1
streptomycin (Hyclone), and incubated at 378C in a humidified at-
mosphere containing 5% CO2.
A mixture of A1 (5 mmol), substituted acetophenones (6 mmol)
and sodium hydroxide (10 mmol) in ethanol (40 mL) was stirred at
08C for 2 h. After completion of the reaction, the precipitate was
filtered and washed with ethanol three times to furnish com-
pounds B1–B18. Without further purification, a mixture of B1–B18
(1 mmol), sodium hydroxide (1.5 mmol) in ethanol (20 mL) and
water (10 mL) was stirred at 908C for 6 h. The solution was cooled,
and the solution was adjusted to pH 3.0 by diluted HCl. The pre-
cipitated solid was collected by filtration and recrystallized form
ethanol to afford pure C1–C18.
Antiproliferation assays: The antiproliferative activities of the pre-
pared compounds against HeLa, MCF-7, A549, L02, and 293T cell
lines were evaluated using a standard 3-(4,5-dimethylthiazol-2-yl)-
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positive control reference drugs (20 mm) in DMSO were prepared.
Cells were then treated with the target compounds at 0, 1, 10, and
100 mm in the presence of 10% FBS for 24 h. Cell viability was then
assessed by conventional MTT reduction assay, carried out strictly
according to the manufacturer’s instructions (Sigma). The absorb-
ance (OD570) was read on an ELISA reader (Tecan, Austria). In all ex-
periments, three replicate wells were used for each drug concen-
tration. Each assay was carried out at least three times.
Tubulin polymerization assays: Tubulin was purified from bovine
brain as described previously. To characterize the effect of the com-
pounds on tubulin assembly in vitro, compounds (ranging from 0.1
to 50 mm) were incubated with bovine brain tubulin (10 mm) in glu-
tamate (0.8 m, pH 6.6 with HCl) at 308C and then cooled to 08C.
After GTP (0.4 mm) was added, the mixtures were transferred to
cuvettes at 08C in a recording spectrophotometer and warmed up
to 308C. Finally, the assembly of tubulin was observed turbidimetri-
cally at l 350 nm. The IC50 value is defined as the compound con-
centration required to inhibit the extent of assembly by 50% after
20 min incubation.
Apoptosis assays: Approximately 105 cells per well were plated in
a 24-well plate and allowed to adhere. The medium was then re-
placed with fresh culture medium containing PMMB-259. Untreat-
ed wells received an equivalent volume of ethanol (<0.1%). After
treatment, they were trypsinized, washed in phosphate-buffered
saline (PBS) and centrifuged at 2000 rpm (300ꢁg) for 5 min. The
pellet was then resuspended in 500 mL staining solution (contain-
ing 5 mL Annexin V–FITC and 5 mL PI in binding buffer), mixed
gently and incubated for 15 min at room temperature in the dark.
The samples were then analyzed by a FACSCalibur flow cytometer
(Becton Dickinson, San Jose, CA, USA).
Figure 4. Effect of PMMB-259 on the tubulin network of MCF-7 cells. Micro-
tubules labelled with rhodamine (red) and nuclei tagged with DAPI (blue)
were observed by confocal microscopy.
Mitochondrial membrane potential evaluations: Mitochondrial
transmembrane potential (DYm) was detected using a JC-1 mito-
chondrial membrane potential assay kit (Beyotime Biotech), follow-
ing the manufacturer’s protocol. After treatment, the cells were in-
cubated at 378C for 20 min with 5 mgmLÀ1 JC-1 (5,5,6,6-tetra-
chloro-1,1,3,3- tetraethylbenzimidazolylcarbocyanine iodide), then
washed twice with PBS and placed in fresh medium without
serum. The samples were analyzed using a FACSCalibur cytometer
(Becton Dickinson) equipped with a 488 nm argon laser.
2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay
with some modification. Cell lines were grown to log phase in
DMEM supplemented with 10% FBS. Cell suspensions were pre-
pared, and 100 mL per well were dispensed into 96-well plates,
giving 104 cells per well. The subsequent incubation was carried
out at 378C under 5% CO2 atmosphere for 24 h to allow the cells
to reattach. Stock solutions of the synthesized compounds and
Figure 5. Molecular docking 2D and 3D modeling of PMMB-259 with the colchicine binding site of tubulin.
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Cell-cycle analysis: Cells were plated in six-well plates (106 cells
per well) and incubated at 378C for 24 h. Exponentially growing
cells were then incubated with PMMB-259. Cells were centrifuged
at 2000 rpm (300ꢁg) at 48C for 5 min after treatment, and then
fixed in 70% ethanol at 48C for at least 12 h and subsequently re-
suspended in PBS containing 0.1 mgmLÀ1 RNase A and 5 mgmLÀ1
PI. The cellular DNA content, for cell-cycle distribution analysis was
measured by flow cytometry, plotting at least 10000 events per
sample. The percentage of cells in the G0/G1, S, and G2/M phases of
the cell cycle were determined using Flowjo 7.6.1 software.
Keywords: antitumor agents · docking studies · shikonin ·
tubulin polymerization
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Acknowledgements
The authors are grateful to the Program for Changjiang Scholars
and Innovative Research Team in University (IRT_14R27), the fund
for University PhD Program from the Ministry of Education of
China (20120091110037), and the National Natural Science Foun-
dation of China (NSFC; Nos. 31470384 and 31171161).
Manuscript received: January 1, 2017
Revised: February 13, 2017
Accepted Article published: February 13, 2017
Final Article published: && &&, 0000
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FULL PAPERS
Combined effect: A series of chalcone-
containing shikonin derivatives were de-
signed, synthesized, and evaluated as
potential inhibitors of tubulin polymeri-
zation. Most showed potent antiprolifer-
ative activity, and studies into the mech-
anism of action revealed induction of
apoptosis, reduction of mitochondrial
membrane potential, accumulation of
cells in the G2/M phase, and severe dis-
ruption of the microtubule system to an
extent similar to that of the reference
compound colchicine.
H.-Y. Qiu, F. Wang, X. Wang, W.-X. Sun,
J.-L. Qi, Y.-J. Pang, R.-W. Yang, G.-H. Lu,*
X.-M. Wang,* Y.-H. Yang*
&& – &&
Design, Synthesis, and Biological
Evaluation of Chalcone-Containing
Shikonin Derivatives as Inhibitors of
Tubulin Polymerization
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These are not the final page numbers! ÞÞ