Synthesis, Evaluation, and Mechanism Study of Novel Indole-Chalcone Derivatives Exerting Effective Antitumor Activity Through Microtubule Destabilization in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.6b00021

Twenty-nine novel indole-chalcone derivatives were synthesized and evaluated for antiproliferative activity. Among them, 14k exhibited most potent activity, with IC₅₀ values of 3-9 nM against six cancer cells, which displayed a 3.8-8.7-fold increase in activity when compare with compound 2. Further investigation revealed 14k was a novel tubulin polymerization inhibitor binding to the colchicine site. Its low cytotoxicity toward normal human cells and nearly equally potent activity against drug-resistant cells revealed the possibility for cancer therapy. Cellular mechanism studies elucidated 14k arrests cell cycle at G₂/M phase and induces apoptosis along with the decrease of mitochondrial membrane potential. Furthermore, good metabolic stability of 14k was observed in mouse liver microsomes. Importantly, 14k and its phosphate salt 14k-P inhibited tumor growth in xenograft models in vivo without apparent toxicity, which was better than the reference compound CA-4P and 2. In summary, 14k deserves consideration for cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.6b00021

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Article
Synthesis, Evaluation, and Mechanism Study of Novel Indole-
chalcone Derivatives Exerting Effective Anti-tumor Activity
Through Microtubule Destabilization In vitro and In vivo
Jun Yan, Jie Chen, Shun Zhang, Jinhui Hu, Ling Huang, and Xingshu Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00021 • Publication Date (Web): 05 May 2016
Downloaded from http://pubs.acs.org on May 6, 2016
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Synthesis, Evaluation, and Mechanism Study of Novel
Indole-chalcone
Anti-tumor
Derivatives
Exerting
Effective
Activity Through
Microtubule
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Destabilization In vitro and In vivo
Jun Yan, Jie Chen, Shun Zhang, Jinhui Hu, Ling Huang*, and Xingshu Li*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006,
China
ABSTRACT
Twentyꢀnine novel indoleꢀchalcone derivatives were synthesized and evaluated for
antiproliferative activity. Among them, 14k exhibited most potent activity, with IC50
values of 3 ꢀ 9 nM against six cancer cells, which displayed a 3.8ꢀ8.7 – fold increase
in activity when compare with compound 2. Further investigation revealed 14k was a
novel tubulin polymerization inhibitor binding to the colchicine site. Its low
cytotoxicity towards normal human cells and nearly equally potent activity against
drugꢀresistant cells revealed the possibility for cancer therapy. Cellular mechanism
2
studies elucidated 14k arrests cell cycle at G /M phase, induces apoptosis along with
the decrease of mitochondrial membrane potential (MMP). Furthermore, good
metabolic stability of 14k was observed in mouse liver microsomes. Importantly, 14k
and its phosphate salt 14k-P inhibited tumor growth in xenograft models in vivo
without apparent toxicity, which was better than the reference compound CA-4P and 2.
In summary, 14k deserves consideration for cancer therapy.
INTRODUCTION
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Microtubules are dynamic polymers of two closely related 55 KDa proteins in cells
called αꢀ and βꢀtubulin and serve as key components of the cytoskeleton in eukaryotic
1ꢀ3
cells. Their pivotal role in a variety of essential cellular processes including the
movement of organelles, intracellular transportation, and the formation and
maintenance of cell shape makes microtubules an attractive target for cancer
0
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therapy. In recent decades, a large number of natural products have been identified
8
to interfere with the tubulinꢀmicrotubule system. Combretastatin Aꢀ4 (CAꢀ4), one of
the typical example, was first isolated from the bark of the African willow tree
Combretum caffrum in 1982. The discovery of CAꢀ4 motivated medicinal chemists to
9ꢀ13
develop extensive investigation of the structure modifications.
Natural products and their derivatives provide a diverse source of bioactive lead
14, 15
compounds for drug development.
Chalcones, bear an α,βꢀunsaturated ketone
moiety and can act as Michael acceptors which are considered as soft Lewis acids and
are able to alkylate multiple sulfhydryl residues of some critical protein such as
1
6, 17
thioredoxin reductases (TrxRs),
nuclear factor erythroid 2ꢀrelated factor 2
1
8, 19
(
Nrf2),
tubulinꢀmicrotubule system.
products such as curcumin, flavokawain, millepachine, and xanthohumol.
Apart from chalcones, the indole family also includes bioactive molecules that have
nuclear factor κB (NFꢀκB), and cysteine 239 or Glutamyl 198 residue of
20ꢀ22
Chalcones form a central core of various natural
1
6, 17, 23ꢀ25
2
6, 27
been widely used for the treatment of cancer.
In previous work, a class of benzylideneindanone derivatives were designed and
2
8
synthesized as tubulin polymerization inhibitors. Among them, compound 1 (Fig. 1)
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exerted moderate antiproliferative activity towards various human cancer cell lines.
Subsequently, considering the extensive pharmacological activities of indole
analogues, the benzene ring was replaced with an indole ring to provide a class of
indanoneꢀindole derivatives, and the most potent compound 2 in this series provided a
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nearly 10ꢀfold increase in antiproliferative activity in vitro.
However, this
compound displayed only an approximately 21.7% antiꢀtumor inhibition rate in vivo
evaluation. To continue the efforts in discovering and developing novel small
molecule compounds that target the tubulinꢀmicrotubule system as potential cancer
therapeutic agents, the indanone moiety of compound 2 or 3 was modified to afford
indoleꢀchalcone derivatives, which greatly improved the antiꢀtumor activity in vitro
and in vivo. Herein, we reported the synthesis, evaluation of their antiꢀtumor activities
in vitro and in vivo, selectivity against human normal cells, tolerance against
drugꢀresistance cells and their underlying cytotoxic mechanisms were also elucidated.
Figure 1. Design strategy of indoleꢀchalcone derivatives.
RESULTS AND DISCUSSION
Chemistry. The main purpose in this work was to investigate the structure ꢀ activity
relationships (SARs) of these indoleꢀchalcone derivatives. We aimed to discuss the
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effects of the location of methoxyl group, the introduction of a methyl group at the
Nꢀ1 position, the activity differences between the acetophenone and propiophenone
derivatives. We started the synthesis with the commercially available 4, via the aldol
condensation reaction with commercially available 5a or 6a, to obtain compounds 7a
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or 7c. The Nꢀmethylation of 5a or 6a gave 5b or 6b. Similarly, compound 5b or 6b
3
1
reacted with 4 via the same reaction to provide 7b or 7d.(scheme 1).
Scheme 1. Reagents and conditions: (a) EtOH, piperidine, reflux.
Propiophenone analogues 1ꢀ(3,4,5ꢀtrimethoxyphenyl)propanꢀ1ꢀone (10) and
substituted benzaldehydes 12 or 13 were synthesized according to a method
32, 33
previously reported in the literature.
The aldol reaction of 10 with substituted
benzaldehydes 12 or 13 in the presence of piperidine provided the compounds 14a-14l
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(scheme 2).
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Scheme 2. Reagents and conditions: (b) CH CH MgBr, anhydrous THF; (c) PCC, DCM, rt. (d) POCl , DMF, rt; (e)
3
2
3
CH
3
I, NaH, THF; (f) EtOH, piperidine, reflux.
Similarly,
as
shown
in
scheme
3,
1ꢀ(5ꢀmethoxyꢀ2,
2
ꢀdimethylꢀ2Hꢀchromenꢀ8ꢀyl)ethanꢀ1ꢀone (17) was prepared according to the method
3
4
previously described in the literature, then reacted with different substituted
different substituted indoleꢀ3ꢀaldehydes via an aldol reaction to afford compounds
18a-18f.
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Scheme 3. Reagents and conditions: (g) DBU, CuCl
2
, CH
3
CN; (h) pyridine, reflux; (i) EtOH, piperidine, reflux
.
Finally, considering the excellent antiꢀproliferative activity of compound 14k, a series
of analogues, 19a-19f, were synthesized with the different substitutions at the
Nꢀposition of compound 14k via alkylation, hydroxymethylation, hydroxyethylation
and so on (scheme 4).
+
ꢀ
Scheme 4. Reagents and conditions: (j) 19a:HCHO, Bu N F , K CO , 80℃ ; 19b, 19c, 19f: CH CH Br or
4
2
3
3
2
HOCH
2
CH
2
Br or CH
3
CH
2
CH
2
Br, NaH, DMF; 19d: BrCH
2
CH
2
Br, K
2
CO
3
2 2
, DMF, reflux; 19e: BrCH CH Br,
2 3
K CO , DMF, rt.
In addition, to improve the solubility of the target compound, phosphoramidate was
substituted at the Nꢀposition of compound 14k, and the general synthetic procedure
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was listed in scheme 5. The preparation of dibenzylphosphoryl chloride (22) was
35
according to the literature method. Firstly, a mixture of dimethylaniline and benzyl
alcohol was added slowly with stirring and cooling to phosphorus trichloride
dissolved in benzene solution to prepare dibenzyl phosphite (21). Subquently,
compound 21 were reacted with Nꢀchlorosuccinimide to obtain intermidate
dibenzylphosphoryl chloride (DBPCl, 22). Then, compound 14k was phosphorylated
with 22 in the presence of NaHMDS to prepare the Nꢀphosphorylated derivative (23).
The deprotection of 23 and neutralization with NaOH afforded the targeted compound
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1
4k-P.
Scheme 5. Synthesis of target compound 14k-P.
Biology
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Anti-proliferative activities and the SARs. Starting from the leadꢀcompound 2,
7
aꢀ7d were synthesized and their antiꢀproliferative activities were evaluated in
various human cancer cell lines by MTT assay. The results listed in Table 1 indicated
that this series of compounds exhibited decent antiproliferative activity and retained
the same magnitude of activity as compound 2.
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a
Table 1. Antiꢀproliferative activities of 7a - 7d and 2 against human cancer cell lines .
b
IC50 (µM)
compound
A549
Hela
Belꢀ7402
MCFꢀ7
A2780
HCTꢀ8
7a
0.036±0.006
0.038±0.002
0.063±0.014
0.050±0.004
0.041±0.04
0.057±0.02
7
b
0.030±0.009
0.031±0.023
0.032±0.01
0.038±0.02
0.023±0.02
0.041±0.07
7
c
0.033±0.03
0.053±0.011
0.026±0.003
0.022±0.017
0.057±0.021
0.027±0.010
0.034±0.015
0.077±0.013
0.035±0.040
0.028±0.027
0.061±0.007
0.047±0.019
0.028±0.01
0.045±0.039
0.035±0.027
0.034±0.04
0.067±0.031
0.038±0.015
7d
2
CA-4
0.005±0.001
0.012±0.001
0.003±0.002 0.013±0.001
0.009±0.002
0.0017±0.002
a
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the
b
Experimental Section. IC50 values are indicated as the mean ± SD (standard error) of at least three independent
experiments.
In searching for potential compounds that display better activity, the reaction of
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acetophenone or propiophenone analogues with different substituted
indoleꢀ3ꢀaldehydes was performed to obtain compounds 14aꢀ14l. In this series of
analogues, we mainly investigated the effects of the location of methoxyl group, the
introduction of a methyl group at the Nꢀ1 position, the activity differences between
the acetophenone and propiophenone analogues, and obtained some obvious SARs.
The results listed in Table 2 indicated that the position of the methoxyl group on
indole moiety was crucial for the antiꢀproliferative activity. Compounds 14a-14d,
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which contain ꢀOCH
3 1
(R ) at the Cꢀ4 position of indole moiety, displayed relatively
lower antiꢀproliferative activity (IC > 1 ꢁM) toward six cancer cell lines regardless
50
of whether the R
from the Cꢀ4 position to the Cꢀ5 or Cꢀ6 position significantly increased the
antiꢀproliferative activity. Compounds 14g and 14h, which contained ꢀOCH at the
Cꢀ5 (R ) position, gave the IC50 values of 0.032 ꢀ 0.096 ꢁM and 0.235 ꢀ 0.726 ꢁM
toward six cancer cell lines, respectively. Interestingly, when the ꢀOCH was moved
4 5 3
and R groups were methyl or hydrogen. Moving the ꢀOCH group
3
2
3
to the Cꢀ6 position (R
3
) and the R
4
3
was ꢀCH group, the obtained compound 14k
displayed the best activity (IC50 values of 0.003 ꢀ 0.009 ꢁM toward six cancer cell
lines), which was better than the reference compound CAꢀ4. So, the relationships
between the location of ꢀOCH
3
group and the antiꢀproliferative activities were
6ꢀOCH > 5ꢀOCH > 4ꢀOCH . On the other hand, the introduction of a methyl group
3
3
3
at the Nꢀ1 position displayed contrary effects on the acetophenone and propiophenone
derivatives. Interestingly, Nꢀmethylation was favorable for the acetophenone
analogues but unfavorable for their corresponding propiophenone analogues. For
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example, compounds 14f and 14j, with a methyl group at the Nꢀ1 position (R )
derived from 14e and 14i, respectively, showed significant increases in
antiproliferative activity. However, compound 14l, with a methyl group at the Nꢀ1
0
1
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position (R ) derived from 14k, showed a 13.7ꢀfold decrease in antiproliferative
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activity toward A549 cells. Finally, the target compounds derived from propiophenone
(
14g, 14h, 14k, and 14l) displayed better activity than their corresponding
acetophenone analogues (14e, 14f, 14i, and 14j) when comparing the results of 14e vs
4g, 14f vs 14h, 14i vs 14k, and 14j vs 14l. The phosphate disodium salt of 14k
1
caused 3.44ꢀ8.67ꢀ fold decrease in antiproliferative activity, which might be resulted
from the relatively low cell permeation of 14kꢀP.
a
Table 2. Antiꢀproliferative activities of 14a–14l and 14k-P against human cancer cell lines .
b
IC50 (µM)
compound
A549
>1
Hela
>1
Belꢀ7402
>1
MCFꢀ7
>1
A2780
>1
HCTꢀ8
>1
1
4a
4b
1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
14c
14d
1
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4f
0.164 ±0.013 0.531±0.025
0.256±0.014
0.231±0.039
0.283±0.017
0.032±0.012
0.315±0.021
0.048±0.002
1
4g
4h
0.074±0.022
0.078±0.011
0.096±0.037 0.033
0.356±0.018 0.548
±
0.005
0.027
1
0.331±0.012
0.224±0.023
0.023±0.016
0.235±0.035
0.261±0.012
0.062±0.024
±
0.427±0.124
0.118±0.017
0.025±0.017
0.726±0.037
0.274±0.138
0.047±0.005
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14i
0.351±0.024
0.061±0.015
0.246±0.014
0.087±0.019
14j
1
4k
0.003±0.001
0.041±0.011
0.056±0.023
0.026±0.003
0.005±0.001
0.007±0.004
0.031±0.017
0.009±0.002 0.006
±
0.003
0.024
0.008±0.001
0.034±0.003
0.043±0.017
0.035±0.027
0.009±0.002
0.006±0.002
0.061±0.022
0.092±0.028
0.038±0.015
0.0017±0.002
1
4l
0.028±0.021 0.042
±
14k-P
2
0.078±0.021 0.061±0.035
0.038±0.012
0.027±0.010
0.012±0.001
0.035±0.040 0.047±0.019
0.003±0.002 0.013±0.001
CA-4
a
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the
b
Experimental Section. IC50 values are indicated as the mean ± SD (standard error) of at least three independent
experiments.
Millepachine, a chalcone derivative with a 2,2ꢀdimethylbenzopyran feature, was first
isolated from the Millettia pachycarpa. Z. Yang, et al. synthesized a series of
millepachine analogues as tubulin polymerization inhibitors that exhibited potent
2
3
antiꢀtumor activity. By replaceing the pꢀmethoxyphenyl moiety of millepachine with
an indole moiety, a new series of chalconeꢀindole analogues were obtained; their
antiꢀproliferative activities are listed in Table 3. The SARs above were also valid in
this series; namely, the methoxyl group at the Cꢀ6 position was required, and the Nꢀ
methylation did not show significant effect for the antiꢀproliferative activity.
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a
Table 3. Antiꢀproliferative activities of 18a-18f against human cancer cell lines .
0
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b
IC50 (µM)
compound
A549
>1
Hela
>1
Belꢀ7402
>1
MCFꢀ7
>1
A2780
>1
HCTꢀ8
>1
18a
1
8b
>1
>1
>1
>1
>1
>1
1
8c
0.272±0.006
0.452±0.008
0.327±0.004
0.415±0.003
0.456±0.114
0.389±0.087
0.782±0.006
0.567±0.013
0.398±0.024
0.497±0.097
0.557±0.217
0.675±0.124
1
8d
1
8e
0.055±0.006
0.058±0.009
0.026±0.003
0.049±0.001
0.093±0.007
0.027±0.010
0.078±0.013
0.097±0.042
0.056±0.013
0.045±0.023
0.041±0.014
0.061±0.032
0.035±0.027
0.077±0.023
0.087±0.014
0.038±0.015
18f
2
0.035±0.040 0.047±0.019
0.003±0.002 0.013±0.001
CA-4
0.005±0.001
0.012±0.001
0.009±0.002
0.0017±0.002
a
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the
b
Experimental Section. IC50 values are indicated as the mean ± SD (standard error) of at least three independent
experiments.
Among the three series of compounds above, 14k displayed the best activity toward
six cancer cell lines, leading to the further development of more potent agents with
various modifications of 14k. Then, starting from 14k, different substitutions were
introduced, including ethyl, propyl, ethylene, hydroxymethyl, hydroxyethyl, and
bromoethyl at the Nꢀ1 position of the indole ring to provide compounds 19a-19f.
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Most of compounds in this series displayed good antiproliferative activities, of which
the IC50 values were all below 100 nM, except for the values of 19e and 19f.
Compared with 14k, all compounds in this series displayed a decrease in
antiproliferative activities (Table 4), which showed that the alkylation in the Nꢀ1
position was indeed unfavorable for this activity. Similar results were obtained for the
extension of the alkyl chain length in the Nꢀ1 position. However, hydroxymethylation
in the Nꢀ1 position (19a) maintained relatively excellent activity, with the IC50 values
of 0.008 ꢀ 0.021 nM towards six cancer cell lines. The SARs of all the synthesised
compounds were summarized in Fig. 2.
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a
Table 4. Antiꢀproliferative activities of 19a-19f against human cancer cell lines .
b
IC50 (µM)
compound
A549
Hela
Belꢀ7402
MCFꢀ7
A2780
HCTꢀ8
19a
0.008±0.002
0.013±0.005
0.009±0.007
0.019±0.010
0.021±0.009
0.017±0.003
1
9b
0.039±0.005
0.025±0.022
0.089±0.026
0.065±0.012
0.035±0.014
0.067±0.024
0.015±0.004
0.021±0.004
0.091±0.017
0.032±0.014
0.014±0.003
0.064±0.019
0.021±0.009
0.031±0.014
0.049±0.013
0.056±0.014
0.020±0.004
0.079±0.039
1
9c
1
9d
1
9e
0.126±0.014
0.514±0.256
0.146±0.047
0.531±0.123
0.259±0.027
0.614±0.014
0.389±0.017
0.497±0.036
0.274±0.017
0.597±0.065
0.169±0.023
0.679±0.017
19f
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2
0.026±0.003
0.005±0.001
0.027±0.010
0.012±0.001
0.035±0.040 0.047±0.019
0.003±0.002 0.013±0.001
0.035±0.027
0.009±0.002
0.038±0.015
0.0017±0.002
CA-4
a
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the
0
1
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0
b
Experimental Section. IC values are indicated as the mean ± SD (standard error) of at least three independent
5
0
experiments.
Figure 2. The summarized SARs of synthesised compounds.
In vitro tubulin polymerization inhibition activity. To elucidate whether the
synthesized compounds target the tubulinꢀmicrotubule system, the in vitro tubulin
polymerization inhibition activities of 14k, 14g, 14j, 18e, and 19a were evaluated in
regard to their relatively better antiꢀproliferative activities in the initial cytotoxicity
screening among all the tested compounds. Purified and unpolymerized tubulin was
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incubated with the tested compounds at the indicated concentrations, and
polymerization was measured by the method originally described by Bonne, D. et al.
3
6, 37
with some modifications.
The increased fluorescence intensity of the control
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samples with time at 37 ℃ indicated that tubulin polymerization had occurred. When
tubulin was incubated with the tested compounds at indicated concentrations, the
increased tendency of the fluorescence intensity was obviously slowed down. The
inhibitory concentrations that reduced the polymerized tubulin by 50% (IC50) of
compounds 14k, 14g, 14j, 18e, and 19a were 2.68 ± 0.15 ꢁM, 5.06 ± 0.02 ꢁM, 3.48 ±
0
.14 ꢁM, 4.39 ± 0.04 ꢁM, and 3.59 ± 0.27 ꢁM, respectively (Fig. 3).
Figure 3. Tubulin polymerization inhibition activities of 14k, 14g, 14j, 18e, and 19a. Purified tubulin protein at 10
ꢁ
M in a reaction buffer was incubated at 37 °C in the absence (control) or presence of 14k, 14g, 14j, 18e, and 19a
at the indicated concentrations. Polymerizations are followed by an increase in fluorescence emission at 410 nM
over a 60 min period at 37 °C (excitation wavelength was 340 nM).
Molecular modeling study. Compound 14k displayed the best antiꢀproliferative
activity and effective tubulin polymerization inhibition in the initial screening and we
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5
6
then selected it as the optimized compound for the following studies. To investigate
the potential binding site of 14k with tubulinꢀmicrotubule system, molecular
modeling studies were performed. When we reꢀdocking colchicine, Fig. 4A showed
that colchicine could be docked in the colchicine binding site in a similar
conformation to that of colchicine in the crystal structure (PDB code: 1SA0).
Reꢀdocking colchicine indicated the feasibility of our docking method. The docking
results of 14k displayed that the trimethoxyphenyl moiety of 14k was positioned in
the binding cavity buried in tubulin. The hydrophobic residues Leu248 and Lys254
acted as thumb and index finger in a left hand firmly grasped the head of 14k, which
was critical for the binding of 14k with tubulin (Fig. 4B). And this interaction has
0
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28
been previously observed for colchicine. Moreover, the Met259 and Thr314 residues
hold the tail of 14k (the indole ring part) like the third finger and litter finger. In order
to elucidate the SARs we concluded from the antiꢀproliferative activities above, the
molecular docking were also performed to study the binding mode of some
representative compounds. Comparing the docking results of 14k and 14i (the
representative compounds of propiophenone series and acetophenone series,
respectively), compound 14i without the methyl located in the αꢀposition alleviated
the hydrophobic interaction with the Lys254 (Fig. 4C), which play a vital role in
binding with tubulin. In comparison with 14i, the increase in the antiꢀproliferative
activity of 14j might due to the increase of hydrophobic inteaction of Nꢀmethylation
with the Met259 and Thr314 residues (Fig. 4D), which might explain that Nꢀ
methylation is favorable for acetophenone series. Comparing to 14k, the methoxyl
3 3
group of 14c (4ꢀOCH ) and 14g (5ꢀOCH ) project outwards of the pocket and thus
unfavorable for the conformation stability (Fig. 4E). This is in good agreement with
the high impact on the activity of the methoxy group’s position on the cytotoxic
potency. Similarly, we found the activity decline of 14l because the indole ring more
easily exposed to the solvent environment although its Nꢀmethylation contributes
some interaction with the Met259 and Thr314 residues (Fig. 4F).
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Figure 4. The molecular docking results of the superimposed conformation for the optimized
compounds (A) colchicine (shown in green) (B) 14k (shown in yellow) and colchicine (C) 14k
and 14i (shown in cyan) (D) 14j (shown in carmine) and 14i (E) 14k, 14c (shown in gray), and
14g (shown in hyacinth) (F) 14k and 14l (shown in pink) with the tubulinꢀcolchicine complex
1SA0.pdb.
The molecular modeling study indicates an acceptable binding mode for 14k in the
3
colchicine binding site of tubulin, we further performed the [ H]colchicine
competitive binding assay to confirm our hypothesis. As shown in Table 5, 82% and
96% binding inhibition occurred with 14k at 1 ꢁM and 5 ꢁM, respectively, which
displayed similar inhibition activity with the reference compound CAꢀ4. Along with
the molecular modeling study, we have reasons to believe that the binding of the
indole –chalcone compounds occurs at the colcchicine binding site of tubulin.
3
a
Table 5. Compound 14k competes with [ H]colchicine for the same binding site on tubulin
b
inhibition of colchicine binding (%) inhibition ± SD
compound
1
ꢁM
5 ꢁM
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5
5
5
5
5
5
5
6
1
4k
82 ± 9
78 ±11
96 ± 6
92 ± 5
CAꢀ4
a
The tubulin competitionꢀbinding assay was performed as indicated in the Experiment section. Tubulin, 1ꢁM;
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
3
b
[
H]Colchicine, 5ꢁM; 14k and CAꢀ4, 5 or 1 ꢁM. Values are indicated as the mean ± SD (standard error) of at least
three independent experiments.
As tubulinꢀmicrotubule system plays a vital role in the maintenance of cell shape and
basically cellular functions, an immunofluorescence assay was performed to study
whether compound 14k could disrupt the microtubule dynamics in living cells. As
shown in Fig. 5A, in the control group, the microtubule network in the A549 cells
exhibited normal arrangement and organization, characterized by regularly assembled,
normal filiform microtubules wrapped around the uncondensed cell nucleus. In
contrast, after exposure to 14k for 24 h, the spindle formation demonstrated distinct
abnormalities and was heavily disrupted. Furthermore, when the concentration
increased to 10 nM, the microtubule spindles significantly shrunk around the center of
the cells, and a number of dotted disorder formations were easily observed. The
changes of the mitotic spindles were observed more clearly by increasing the
magnification of the laser confocal microscope. As shown in Fig. 5B, wellꢀorganized
and bipolar mitotic divisions were present in the DMSOꢀtreated cells. However, the
multiꢀpolarization of the spindle and multiꢀnucleation phenomena was nonꢀnegligible
in the drugꢀtreated group, especially in the 10 nM of 14kꢀtreated group. These
morphological microtubules changes indicated that 14k dramatically disrupted the
microtubule organization, induced the multiꢀpolarization of the mitotic spindle, and
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interfered with the mitosis of A549 at much lower effective concentrations, which
might eventually lead to cell cycle disorder.
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Figure 5. 14k disrupted the organization of the cellular microtubule network (A) and induced multipolar mitosis
(
B) at nanomolar concentrations. A549 cells were plated in confocal dishes and incubated with 14k at the indicated
concentrations for 24 h, followed by direct microscopy. The detection of the fixed and stained cells was performed
with an LSM 570 laser confocal microscope (Carl Zeiss, Germany). The experiments were performed five times,
and the results of the representative experiments were shown.
Selectivity of 14k towards normal cells. In contrast, four types of nonꢀtumorigenic
cell lines from different origins, including human umbilical vein endothelial cells
(HUVECs), MCFꢀ10A cells (human mammary epithelial cell line), BJ (human dermal
fibroblasts), and HLF cells (human embryonic lung fibroblast) were used to test the
selectivity of 14k toward cancer cells and normal cells. As shown in Table 6, 14k was
associated with high cytotoxicity toward A549 cells and no apparent toxicity toward
those four nonꢀtumorigenic cell lines, which exhibited 54.3ꢀ, 891ꢀ, 2973ꢀ, and
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1
1
2
2
2
2
2
2
2
2
2
2
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3
3
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3
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3
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5
5
5
5
5
5
6
1
891ꢀfold selectivity ratios for HUVECs, MCFꢀ10A, BJ, and HLF cells, respectively.
Table 6. Antiproliferative activity of compound 14k against human normal cells.
a
c
IC50, mean ± SE (ꢁM)
Selectivity ratio
0
1
2
3
4
5
6
7
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9
0
1
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0
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9
0
1
2
3
4
5
6
7
8
9
0
A549
Quiescent HUVECs
MCFꢀ10A
BJ
0.003 ± 0.001
0.163 ± 0.003
2.672 ± 0.56
8.921±0.24
b
54.3
891
2973
1891
HLF
5.674±0.38
a
b
Data are presented as the mean ± SE from the doseꢀresponse cures of at least three independent experiments. For
quiescent growth conditions, HUVECs were cultured in endothelial cell medium containing 0.5% fetal calf serum
c
and in absence of growth factor. Selectivity ratio = (IC50 human normal cells) / (IC50 A549).
Anti-proliferative activity of 14k against drug-resistant cancer cell lines. Rapidly
acquired drug resistance, which frequently results in clinical chemotherapy failure,
has recently caused little improvement in longꢀterm cancer therapy in the past few
38ꢀ40
decades.
Therefore, it is important to develop new antiꢀproliferative agents against
drugꢀresistant cancer cell lines. To evaluate the selectivity of compound 14k against a
panel of parental and drugꢀresistant cancer cell lines, commercially available human
ovarian carcinoma cell line A2780, human ileocecal carcinoma cell line HCTꢀ8, and
the cell line A2780/TAX (paclitaxelꢀresistant cell line) and HCTꢀ8/VCT
(vincristineꢀresistant cell lines) were selected. Furthermore, the wellꢀknown
cisplatinꢀresistant cell line A549/CDDP and the adriamycinꢀresistant cell line
MCFꢀ7/ADM were also chosen as two other representative cell lines. Although
exhibited a high activity in parental cancer cell lines, the positive drugs paclitaxel,
vincristine, cisplatin, and adriamycin exhibited 1136ꢀ, 22.5ꢀ, 10.4ꢀ, and 49.4ꢀfold
resistances to the drugꢀresistant cell lines, respectively. Comparatively, except for in
A2780 and A2780/TAX, the resistance index (RI) of 14k was in the range of 0.42 to
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.50, which showed that compound 14k exhibited practically equally potent activities
in the parental cells and the drugꢀresistant cells. The drugꢀresistant index of the
positive compounds in Table 7 indicated the drugꢀresistant cell lines were also
resistant to all of the positive compounds and displayed the multiꢀdrug resistant
property.
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Multidrug resistance is a major obstacle for successful treatment of cancer, which was
mainly mediated by Pꢀglycoprotein (Pꢀgp) or multidrugꢀresistanceꢀassociated protein
(MRP). As compound 14k does not seem too hydrophobic, it is not expected to be a
substrate of Pꢀgp which is mainly responsible for the excellent efficacy towards
several kinds of drugꢀresistant cancer cell lines. The specific and detailed
drugꢀresistant mechanism of 14k and its in vivo antiꢀtumor efficiay on the
drugꢀresistant xenograft tumour models will be elucidated in the further study.
Table 7. Drug tolerances against different drugꢀ resistant cancer cells.
a
IC50, mean ± SE (ꢁM)
Compd.
A2780/
TAX
HCTꢀ8
/VCT
A549/
CDPP
0.004±
0.002
0.014 ±
0.007
0.023
±0.017
0.561±
0.02
MCFꢀ
7
MCFꢀ7
/DOX
b
A2780
DRI
HCTꢀ8
DRI
0.42
2.94
6.57
22.5
A549
DRI
1.33
2.80
2.56
9.20
10.4
DRI
1.50
2.07
11.3
12.9
0
.01±
.001
0.124 ±
0.02
0.012 ±
0.002
0.005 ±
0.001
0.003±
0.001
0.005 ±
0.001
0.009
±0.002
0.061±
0.013
9.15 ±
0.03
0.006± 0.009 ±
1
4k
12.4
6.44
1136
6.46
0
0.003
0.013±
0.001
0.012
±0.009
0.098±
0.012
10.5 ±
1.31
0.006
0.027±
0.023
0.135 ±
0.13
0
.009±
.002
0.058±
0.013
5.68 ±
0.21
0.0017 ± 0.005 ±
CA-4
TAX
VCT
0
0.002
0.019 ±
0.013
0.35 ±
0.03
0.002
0.125 ±
0.52
0.005 ±
0.02
0.026±0 0.168±0
7.86 ±
0.15
1.261±
0.135
21.4 ±
2.13
.
003
2.5±
.31
0.98±
.02
ND
1
21.5±
3.56
92.4±
9.83
95.5 ±
0.14
CDDP
DOX
ND
4.30
1.29
2.03
49.4
1
3.29±
3.35
1.17±
1.51±
2.17±
3.14±
1.44 1.02 ±
50.4
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.13
Data are presented as the mean ± SE from the doseꢀresponse cures of at least three independent experiments.
DRI: drugꢀresistant index, (IC50 of drug resistant cancer cell)/(IC50 of parental cancer cell).
1.02
1.14
1.36
1.02
1.17
0.013
±1.05
a
b
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4k induces G
2
/M phase arrest and regulates the expression of G
2
/M-related
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proteins. As most tubulinꢀdestabilizing agents display the ability to disrupt the
41
regulated cell cycle distribution, a flow cytometry analysis was performed to
2
determine the arrest effect of 14k on the G /M transition, a rigorously regulated
process in the cell cycle. As shown in Fig. 6, 14k induced cell cycle arrest at the G /M
2
phase in a doseꢀ and timeꢀdependent manner. When A549 cells were treated with 14k
from 1 nM to 10 nM for 24 h, the percentage of cells at the G
2
/M phase increased
sharply, along with concomitant losses in the G phase (Fig. 6A and 6D); this trend
1
was more obvious after 48 h incubation. After 48 h treatment, most cells were arrested
at G /M phase at relatively high concentration (3 nM or 10 nM) (Fig. 6B and 6E).
2
2
While, after the incubation time was extended to 72 h, the G /M cell cycle arrest
effect was still obvious at 1 nM. But at relatively high concentration (3 nM or 10 nM),
the subꢀG1 peak (a characteristic hypodiploid peak) was the main peak, which
indicated most cells underwent DNA fragment (Fig. 6C and 6F). What’s more, the
hypodiploid peak (subꢀG1) which indicated apoptotic cells, was a very small amount
at 24 h, displayed a modest increase at 48 h and significantly accumulated at 72 h.
Entry into mitosis of eukaryotic cell is regulated by activation of Cdc2 kinase, which
is controlled by several steps including cyclin B1 binding and cdc25C
42, 43
phosphorylation.
Therefore, the expression of cell division regulated proteins was
investigated. As shown in Fig. 6G and Fig. 6H, 14k caused a doseꢀ and
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timeꢀdependent decrease in Cdc2, cyclin B1, and Cdc25C. These results, which were
consistent with the cell cycle analysis and the intracellular microtubule
immunofluorescence assay, further illustrated the mechanism of the cell cycle arrest
effect.
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Figure 6. Cell cycle arrest effect of 14k. A549 cells were treated with 14k at the indicated concentrations for 24 h,
48 h, and 72 h. Then, the cells were trypsinized and harvested for PIꢀstained DNA content by flow cytometry (A-C)
or lysed and the total proteins were harvested for the western blot analysis to detect the expression of Cyclin B1,
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Cdc25c, and Cdc2 (G and H). Quantitative analysis of the percentage of cells in each cell cycle phase by EXPO32
ADC analysis software (D-F). The experiments were performed at least three times, and the results of the
representative experiments are shown.
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1
4k induces apoptosis via the regulation of apoptosis-related protein expression.
According to the cell cycle analysis results above, it was hypothesized that
4kꢀtreatment might induce the apoptosis of A549 cells, which was evidenced by the
appearance of the characteristic hypodiploid DNA content peak (subꢀG1) (Fig. 6A –
C). To confirm this property, A549 cells were treated with various indicated
1
4
concentrations of 14k for 24 h or 48 h; then, the cells were harvested, stained with
Annexin VꢀFITC and PI and analyzed by flow cytometry (Fig. 7A - 7B). As shown in
Fig. 7, 14k caused cell apoptosis in a doseꢀ and timeꢀdependent manner. When the
cells were incubated with 14k at 1 nM, 3 nM, and 10 nM for 24 h, the percentages of
early and late apoptosis cells were 14.9%, 32.2%, and 37.7%, respectively (Fig. 7A).
While, after 48 h incubation at 1 nM, 3 nM, and 10 nM, the percentages of early and
late apoptotic cells were strikingly increased to 33.5%, 60.2%, and 78.6%,
respectively (Fig. 7B). Bclꢀ2 family proteins are crucial components of mitochondrial
44
stressꢀinduced cellular apoptosis. Therefore, the expression of apoptosisꢀrelated
proteins was also determined. Further evidence from the Western blot assay
confirmed that 14k upꢀregulated the expression of proꢀapoptotic proteins (e.g., Bad
and Bax) and correspondingly downꢀregulated the expression of antiꢀapoptotic
proteins (e.g., Bclꢀ2 and Bclꢀxl) in a doseꢀ and timeꢀdependent manner (Fig. 7C and
7
D).
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Figure 7. 14k induced the apoptosis of A549 cells. A549 cells were treated with 14k at the indicated
concentrations for 24 h or 48 h. Then, the cells were trypsinized, harvested and stained with Annexin VꢀFITC and
PI solution for flow cytometry (A and B) or lysed to harvest the total proteins for the Western blot analysis to
detect the levels of the apoptosisꢀrelated proteins Bax, Bad, Bclꢀ2, and Bclꢀxl (C and D). The percentages of cells
in each stage of apoptosis were quantitated by flow cytometry. (B1, upper left quadrant) necrotic cells; (B2, upper
right quadrant) late apoptotic cells; (B3, bottom left quadrant) live cells; and (B4, bottom right quadrant) early
apoptotic cells. (AꢀB). Representative images from five independent experiments were shown.
14k caused mitochondrial dysfunction through decreasing the mitochondrial
membrane potential. Increasing evidence has indicated that mitochondria play an
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vital role in the progression of apoptosis. Decreased mitochondrial membrane
potential (MMP, Ψm) has been implicated as an early event in apoptotic cells. Flow
△
cytometry analysis and a laser scanning confocal microscope were used in this study
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to detect the MMP. The results shown in Fig. 8A indicated that after cells exposed to
1
4k from 1 nM to 10 nM, the red fluorescence intensity (JCꢀ1 aggregates) decreased
from 97.2% to 40.7%, which was consistent with the green fluorescence intensity
JCꢀ1 monomers) correspondingly increase from 1.78% to 57.2%. The same
(
phenomena were also observed by confocal microscopy, which was evidenced by a
shift toward the form of the monomers and decrease of the aggregates (Fig. 8B).
These results demonstrated that compound 14k induced MMP collapse and
mitochondrial dysfunction, altered the expression of apoptosisꢀrelated proteins, and
eventually triggered apoptotic cell death.
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Figure 8 Compound 14k decreased the mitochondrial membrane potential of A549 cells. The A549 cells were
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treated with 14k at the indicated concentrations for 24 h followed by incubation with the fluorescence probe JCꢀ1
for 30 min. Then, the cells were analyzed by flow cytometry (A) or visualized by LSM 570 laser confocal
microscope (Carl Zeiss, Germany) (B). The experiments were performed five times, and the results of the
representative experiments were shown.
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Metabolic stability of 14k. Metabolic stability of 14k. To evaluate the metabolic
stability of 14k which might provide some guidence of drugꢀlike properties, the assay
in vitro with rat liver microsomes was performed. Compound 14k was in vitro
incubated with rat liver microsomes at 37
℃ for indicated time in buffer solution
containing NADPH regenerating system and the residue was determined by HPLC.
CAꢀ4 was used as a reference compound (Table 8). The results indicated that 14k was
relatively quite stable. There still remained 42.4% of 14k after 3 h incubation under
the applied conditions (see the Experimental Section); however, only 28.6% of CAꢀ4
remained after incubation for 1 h, indicating that CAꢀ4 displayed a relatively faster
metabolic profile than 14k. As we all known, a short halfꢀlife of pharmaceutical
agents might result in low bioavailability in vivo and required for frequently
administrated with a high dose, which might result in drug accumulation and produce
related toxicity. In comparison to the reference compound CAꢀ4 which had shorter
halfꢀlife time (t1/2< 60 min), the t1/2 displayed by 14k (t1/2 = 157.5 min) had greatly
improved, which encoruaging us to carry out further detailed study in vivo.
Table 8. The metabolic stability of 14k and CAꢀ4.
Remaining%
Compd.
Time/min
t
1/2
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