Combretastatin A-4 Analogue: A Dual-Targeting and Tubulin Inhibitor Containing Antitumor Pt(IV) Moiety with a Unique Mode of Action

Article abstract of DOI:10.1021/acs.bioconjchem.6b00353

Three new Pt(IV) complexes comprising a combretastatin A-4 analogue were designed and synthesized. The resulting antitumor Pt(IV) complexes could significantly improve the antiproliferative activity and overcome the drug resistance of cisplatin in vitro. Interestingly, these novel compounds not only can carry the DNA binding Pt(II) warhead into the cancer cells but also have a small molecule fragment that can inhibit tubulin polymerization. Among them, complex 13, which was attached to an inhibitor of tubulin at one axial position of Pt(IV) octahedral coordination sphere, could effectively enter cancer cells, arrest the cell cycle in HepG-2 cancer cells at G2/M phases, and induce activation of caspases triggering apoptotic signaling via the mitochondrial-dependent apoptosis pathways. Moreover, complex 13 has the ability to effectively inhibit the tumor growth in the HepG-2 xenograft model without causing significant loss of animal body weight in comparison with cisplatin.

Full text of DOI:10.1021/acs.bioconjchem.6b00353

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Article
Combretastatin A-4 Analogue: A Dual-targeting and Tubulin Inhibitor
Containing Antitumor Pt(IV) Moiety with a Unique Mode of Action
Xiaochao Huang, Rizhen Huang, Shaohua Gou, Zhi-xin Liao, Heng-Shan Wang, and Zhimei Wang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00353 • Publication Date (Web): 05 Aug 2016
Downloaded from http://pubs.acs.org on August 9, 2016
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Combretastatin Aꢀ4 Analogue: A Dualꢀtargeting and Tubulin
Inhibitor Containing Antitumor Pt(IV) Moiety with a Unique
Mode of Action
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1
†, ‡
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†, ‡
†, ‡*
†, ‡
Xiaochao Huang ,
Rizhen Huang ,
Shaohua Gou,
Zhimei Wang , Zhixin
†
, ‡
§, *
Liao, and Hengshan Wang
†
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, China
‡
Jiangsu Province HiꢀTech Key Laboratory for Biomedical Research, Southeast
University, Nanjing 211189, China
§
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources (Ministry of Education of China), School of Chemistry and Pharmaceutical
Sciences of Guangxi Normal University, Guilin 541004, China
Supporting Information
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Table of Contents Graphic
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H3CO
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OCH3
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in vivo antitumor activity
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Cl
Cl
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mitochondrial transmerabrane potential
DNA damage
p53
Bcl-2
Bax
Cyt c
p21
CDK1
Caspase-9
Cell cycle arrest G2
Caspase-3
Cell proliferation
Cell death
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ABSTRACT: Three new Pt(IV) complexes comprising a combretastatin Aꢀ4 analogue
were designed and synthesized. The resulting antitumor Pt(IV) complexes could
significantly improve the antiꢀproliferative activity and overcome the drug resistance of
cisplatin in vitro. Interestingly, these novel compounds can not only carry the DNA
binding Pt(II) warhead into the cancer cells but also have a small molecule fragment that
can inhibit tubulin polymerization. Among them, complex 13, which was attached to an
inhibitor of tubulin at one axial position of Pt(IV) octahedral coordination sphere, could
effectively enter cancer cells, arrest the cell cycle in HepGꢀ2 cancer cells at G2/M phases,
and induce activation of caspases triggering apoptotic signaling via the
mitochondrialꢀdependent apoptosis pathways. Moreover, complex 13 has the ability of
effectively inhibiting the tumor growth in the HepGꢀ2 xenograft model without causing
significant loss of animal body weight in comparison with cisplatin.
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INTRODUCTION
Platinum(II) based complexes, including cisplatin, carboplatin and oxaliplatin
(Figure 1), are the most frequently applied DNAꢀdamaging anticancer drugs, and among
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them cisplatin is the firstꢀline chemotherapeutic agent against certain types of
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malignancies. Nevertheless, the effectiveness of Pt(II) drugs has been heavily limited
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by high toxicity, severe side effects and inherent or acquired drug resistance. Therefore,
great efforts have been devoted to the design of novel platinum based drugs with
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improved therapeutic properties, clinical efficacy and without acquired resistance.
Recent studies have described the combination of platinum anticancer agents with
inhibitors of tubulin, primarily with paclitaxel and docetaxel, resulting in the
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improvement of therapeutic efficacy. The inhibitors of tubulin, such as the natural
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products vinblastine, vincristine, paclitaxel and docetaxel,
are forceful as antiꢀmitotic
agents that can induce tumor cells apoptosis and inhibit cancer cells proliferation and
angiogenesis. Microtubules are well known key components of the cytoskeleton which is
composed of α, βꢀtubulin heterodimers, playing a significant role in a variety of essential
cellular processes, notably mitotic spindle assembly during cell division, cell proliferation,
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intracellular transport, cell signaling, and migration.
Meanwhile, inhibition of the
assembly of tubulin into microtubules, or inversely, the depolymerization of microtubules
leading to the arrest of cell division and eventually to apoptosis, makes the microtubule
cytoskeleton an effective and attractive molecular target for cancer chemotherapeutic
agents. CombretastatinꢀA4 (CAꢀ4, Figure 2), as a natural cisꢀstilbene product, isolated
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from the bark of African willow tree Combretum caffrum, is one of the well known
tubulinꢀbinding molecules that strongly inhibit tubulin polymerization by binding to the
colchicine binding site, causing rapidly vascular shutdown and cell death in tumor cells.
CAꢀ4 has shown strong cytotoxicity against a large number of human cancer cells,
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including those that are multidrug resistant ones.
Particularly, Pt(II) complexes
comprising a CAꢀ4 analogous chalcone seemed to primarily target the tubulin at a
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physiologically meaningful concentrations. Hence, a combination of cytotoxic DNA
damaging agents, such as platinum drugs, with inhibitor of tubulin can be a fascinating
strategy for targeting microtubules, and at least in theory, can enhance the efficacy and
overcome the resistance of the platinum drugs. However, the activities of these
compounds, which hinge on the intracellular aquation of the Pt(II) complexes to release
the inhibitor of tubulin, were not as potent as expected. Furthermore, it is extremely
difficult to control major obstacles in administering freeꢀdrug formulations, such as the
definitive exposure to the targets of interest, individual pharmacokinetics, or
bioꢀdistribution parameters, when drugs are individually administered.
Rather than depend on aquation for the synchronous release of two antiꢀtumor drugs
inside the cancer cells, it might be an alternative way to construct a single prodrug which
takes advantage of the favorable chemical properties of Pt(IV) complexes towards that
end. Recently, there have been several reports on Pt(IV) prodrugs derived from cisplatin
or oxaliplatin with chalcone derivatives or phenylbutyrate as the axial ligands in the
octahedral geometry of Pt(IV) complexes, in which the Pt(IV) compounds were reduced
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to Pt(II) equivalents in the tumor cells and released other molecular fragments to activate
p53, suppress efficacy of histone deacetylases (HDACs) and induce cell cycle arrest at S
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phase and cell death through apoptosis, respectively.
However, hitherto, no examples
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of a prodrug containing both cisplatin and combretastatinꢀA4 (CAꢀ4) has been reported as
a inhibitor of tubulin polymerization. In recent years, the work of Hsieh and Nam showed
that 3,4,5ꢀtrimethoxy substitution on the Aꢀring and the cisꢀorientation were essential for
efficient CAꢀ4 inhibition of tubulin polymerization and antitumor activities.31,32 In this
mind, we designed and tried to obtain a novel dualꢀtargeting platinum(IV) anticancer
prodrug with the ability to release cisplatin and CAꢀ4 analogue for their respective
biological actions that can not only carry the DNA binding platinum warhead into the
tumor cells but also have a small molecule fragment that can inhibit tubulin
polymerization (Figure 3.).
Figure 1. FDA approved platinum(II) anticancer agents, platinum(IV) prodrug in clinical
trials, satraplatin, and platinum(IV) compound commonly used as precursor for acylation
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reactions, oxoplatin.
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Figure 2. Structures of natural products and clinical trial agent (CAꢀ4P) that bind at the
colchicineꢀbinding site of tubulin.
Figure 3. Design of a novel platinum(IV) prodrug as a potential antiꢀtubulin and
antiꢀtumor agent.
RESULTS AND DISCUSSION
Synthesis and Characterization. The CAꢀ4 analogue were synthesized according to
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the reported procedures.
Platinum(IV) precursor complexes were synthesized
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according to the reported procedure. Target compounds purity was determined by
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microanalysis and their structures were characterized by HRꢀMS, H and C NMR
spectrometry. The synthetic profiles of the compounds and their chemical structures are
listed in Schemes 1 and 2.
Scheme 1. Synthetic Pathway to Target Compounds 10ꢀ12. Reagents and Conditions:
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Scheme 2. Synthetic Pathway to Target Compounds 13ꢀ15. Reagents and Conditions:
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(a) NaBH , CH OH, 0 C; (b) PBr , CH Cl , 0 C; (c) PPh , toluene, 110 C (d) NaH,
4 3 3 2 2 3
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rt.; (h) TBTU, Et N, DMF, rt
.
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H CO
CHO
H3CO
H3CO
3
H CO
3
Br
OH
a
b
H CO
3
H CO
3
OCH3
OCH3
OCH3
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H CO
3
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PPh Br
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c
OHC
OTBDMS
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H CO
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OCH3
OCH3
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OCH3
H CO
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H CO
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OTBDMS
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H CO
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OCH3
H CO
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OCH3
OH
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OTBDMS
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OCH3
OCH3
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CA-4
H CO
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g
h
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OCH3
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OCH3
OCH3
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H N
Cl
Cl
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H CO
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OCH3
OCH3
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OCH3
O
H2
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N
O
O
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Cl
Cl
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Pt
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N
N
Cl
H
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HPLC Analyses On the Pt(IV) Complexes Released Ability. To investigate whether
the activities of the synthetic Pt(IV) complexes were released Pt(II) equivalents as
planned under reduction, complex 13 in a solution of acetonitrile and water (1.5:1) to
release compound 9 and cisplatin under reduction with ascorbic acid was studied by
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HPLC. As shown in Figure S1, complex 13 was gradually reduced to release compound
9
and cisplatin as the time passed, accompanied by the falling down peak of complex 13
and the rising peak of compound 9. It was noted in the HPLC chromatograms that
cisplatin was not observed due to its weak chromophore under the ultraviolet detecting
condition. Since oxaliplatin could be observed under the ultraviolet detecting condition,
we further investigated the behavior of Pt(IV) complex 15 under the same condition. As
illustrated in Figure S1, similar trend was observed. These results suggested that the
Pt(IV) complexes were easily reduced to their Pt(II) equivalents in the presence of
ascorbic acid at the room temperature, implying its potential biological activity.
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In Vitro Cytotoxicity Assay. The cytotoxicity of target complexes were first screened
in vitro against five different human cancer cell lines representative of MCFꢀ7 (breast),
HCTꢀ116 (colon), HepGꢀ2 (hepatoma), Belꢀ7404 (hepatoma), NCIꢀH460 (lung) and LO2
(human normal liver cell line) with cisplatin, oxaliplatin, DACHPt and CAꢀ4 as reference
controls using MTT assay. The corresponding IC values obtained after 72 h exposure,
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are summarized in Table 1. As shown in Table 1, the compound CAꢀ4 analogue 9 showed
a low cytotoxicity against the tested tumor cell lines than positive drug CAꢀ4. However,
compounds 13ꢀ15, the Pt(IV) derivatives of cisplatin, oxaliplatin, or DACHPt with one
CAꢀ4 analogue ligand in the axial position, exhibited significant antitumor activity
against all tested cell lines. The Pt(IV) derivative of cisplatin, complex 13, was
significantly more potent than the commercial anticancer drug cisplatin with IC values
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in the range of 0.35–3.01 ꢁM, and simultaneously showed low cytotoxicity against LO2
cells with IC50 value of 36.06±3.21 ꢁM compared with the positive drug cisplatin
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(
3.54±0.26 ꢁM) and CAꢀ4 (0.58±0.11 ꢁM), respectively, making it as a good antitumor
drug candidate. The Pt(IV) derivatives of DACHPt and oxaliplatin, complexes 14 and
5, displayed better cytotoxicity against the tested cell lines than their references
DACHPt and oxaliplatin, with IC values in the range of 0.87–5.95 and 4.69–9.53 ꢁM,
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respectively. Especially, both 14 and 15 also exhibited low cytotoxicity against LO2 cells
with IC values of 37.84±2.25 and 38.84±2.23 ꢁM compared with DACHPt (2.66±0.23,
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0
ꢁ
M) and oxaliplatin (4.71±0.41 ꢁM), respectively.
Table 1. Effect of Target Compounds against Cell Viability of Different Cell Lines
Compd.
IC (ꢁM)
50
MCFꢀ7
HepGꢀ2
HCTꢀ116
Belꢀ7404
NCIꢀH460
LO2
9
25.21±2.18 11.79±0.84 27.29±2.43 15.59±0.54 12.56±0.57 48.42±2.92
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3
2.14±0.15
4.43±0.45
9.53±0.87
0.13±0.13
5.90±0.35
0.35±0.11
0.87±0.71
6.89±0.52
0.23±0.13
3.96±0.28
3.01±0.27
3.89±0.42
4.89±0.51
0.25±0.17
2.17±1.56
5.95±1.03
8.94±0.15
1.16±0.35
0.56±1.19 36.06±3.21
0.91±1.85 37.84±2.25
4.69±0.23 38.84±2.23
14
15
CAꢀ4
0.33±0.15
0.58±0.11
a
CDDP
7.78±0.63 12.47±0.49 18.93±1.08 3.54±0.26
b
OXP
DACHPt
10.73±0.89 21.65±1.78 3.98±0.26 17.90±2.01 14.36±1.25 4.71±0.41
c
24.46±1.95 21.47±2.53 8.18±0.74 20.75±1.75 19.51±1.81 2.66±0.23
a
b
c
Cisplatin
,
Oxaliplatin
,
Dichloro(transꢀ1,2ꢀdiaminocyclohexane)platinum.
Notably, complex 13 showed up to 11.3ꢀfold increased cytotoxicity compared with
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cisplatin in HepGꢀ2 cells and exhibited almost similar antitumor activity to CAꢀ4. The
similar trend was also observed in NCIꢀH460 cells. This Pt(IV) compound showed high
potency against the tested cancer cell lines, which can be due to the “synergistic
accumulation” of both the Pt moiety and the CAꢀ4 analogue 9, respectively. Interestingly,
all resulting Pt(IV) complexes displayed low cytotoxicity than their corresponding
positive controls (cisplatin, oxaliplatin and DACHPt) against human normal LO2 cell line,
indicating that these complexes have a selective toxicity for the tumor cells over the
normal cell.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. In Vitro Growth Inhibitory Effect of Selected Compounds 13ꢀ15 on
CisplatinꢀResistant Cell Lines SGCꢀ7901 and A549
Compd.
IC (ꢁM)
resistant
IC (ꢁM)
resistant
5
0
50
d
d
SGCꢀ7901 SGCꢀ7901/CDDP
factor
A549
A549/CDDP
factor
9
58.93±5.26
0.89±0.07
3.98±0.28
7.37±0.62
84.54±6.69
1.11±0.09
15.21±1.03
12.36±1.07
c
61.15±5.26
1.62±0.09
7.91±1.24
9.53±0.87
77.68±5.23
12.86±0.73
21.65±1.78
21.47±2.53
1.04
1.82
1.99
1.29
ꢀ
21.36±0.58 29.68±0.71
1.44
1.36
2.89
1.87
14.89
4.03
3.32
3.63
1
1
1
3
4
5
2.04±0.92
2.93±0.47
5.56±1.53
0.18±0.49
6.29±1.59
9.81±0.82
8.37±0.36
2.77±1.66
8.47±1.52
10.38±1.35
2.68±0.31
25.33±0.73
32.54±0.97
30.41±1.34
d
CAꢀ4
a
CDDP
11.58
1.42
1.74
b
OXP
DACHPt
c
a
b
Cisplatin
ratio between IC determined in resistant and nonresistant cell lines
, Oxaliplatin, Dichloro(transꢀ1,2ꢀdiaminocyclohexane)platinum, The values express the
.
5
0
Effects of Complexes 13ꢀ15 on Drug Resistant Cell Lines. Drug resistance is a
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critical therapeutic problem that limited the efficacies of cisplatin for a variety of human
cancer cells. According to the above biological result, we further evaluated sensitivity to
the selected complexes (13ꢀ15) of two cisplatin resistant and nonꢀresistant cancer cells
10
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13
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(
SGCꢀ7901 human gastric cancer cell lines ) and human lung epithelial cells (A549). As
shown in Table 2, the IC values of cisplatin against SGCꢀ7901/CDDP and A549/CDDP
5
0
resistant cell lines were increased to 12.86 and 25.33 ꢁM, respectively. Interestingly, the
activity of compound 13 was not markedly changed for these two cisplatin resistant
cancer cell lines compared with the sensitive ones, its IC50 values against cisplatin
resistant SGCꢀ7901 and A549 cellꢀlines were 1.62 and 2.04 ꢁM, respectively. It was of
much significance to observe that compound 13 had a much lower resistance factor (1.82
for resistant SGCꢀ7901 cell line and 1.36 for resistant A549 cell line) than cisplatin
(11.58 for resistant SGCꢀ7901 cell line and 4.03 for resistant A549 cell line). Moreover,
1
5 and 14 have potent cytotoxicity against parental cells and cells resistant to cisplatin
comparable to that of oxaliplatin and DACHPt, and have smaller resistant factors,
suggesting that these compounds might be useful in the treatment of drug refractory
tumors resistant to other platinum drugs.
Cellular Uptake. Since complexes 13 and 14 exhibited better cytotoxicity, they were
selected to carry out the cellular uptake test in HepGꢀ2 cells by using the inductively
coupled plasma mass spectrometry (ICPꢀMS). As shown in Figure 4 and Table 3, treating
HepGꢀ2 cells with the complexes (10.0 and 20.0 ꢁM) for 12 h resulted in a substantial
increase in the content of cellular platinum in a concentration dependent manner,
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suggesting facile internalization of the complexes within 12 h. Particularly, the uptake of
complex 13 was significantly higher than those of cisplatin and complex 14. After
exposure to 20.0 ꢁM of complex 13 for 12 h, the concentration of cellular platinum rose
0
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2
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4
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8
9
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0
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to 606 ng/10 cells, which is nearly two times as much as that of cisplatin. Upon the
results from the cytotoxicity assay and cellular uptake tests, it seems that there might be a
positive correlation between these two tests, namely, the enhanced cellular uptake can
result in the increase of the cytotoxicity. However, the platinum accumulation of complex
14 on the tested cells was not always high in comparison with cisplatin, which did not
correlate with the cytotoxicity against HepGꢀ2 cells. The unꢀcorrelation between the
intracellular platinum levels and the cytotoxicity of complexes may be due to the fact that
the intracellular platinum level is, although important, not the only factor deciding the
cytotoxicity of the complexes.
Table 3. Cellular Uptake of 13 and 14 in HepGꢀ2 Cells after 12 h of Incubation
6
Complex
Pt content (ng/10 cells)
HepGꢀ2
1
1
1
1
3 (10 ꢁM/L)
3 (20 ꢁM/L)
4 (10 ꢁM/L)
4 (20 ꢁM/L)
235±21
606±61
140±12
220±19
157±21
351±18
CDDP (10 ꢁM/L)
CDDP (20 ꢁM/L)
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10 µM/L
20 µM/L
Figure 4. Intracellular accumulation of cisplatin, 13 and 14 (10, 20 ꢁM) in HepGꢀ2 cells
6
after 12 h. Each value shown in the table is in nanograms of platinum per 10 cells.
Results are expressed as the mean ± SD for three independent experiments.
Effects of complex 13 on Tubulin Polymerization in vitro. Microtubule perturbing
agents can either enhance or inhibit the assembly of microtubules. It is wellꢀknown that
the mechanism of tubulinꢀbinding agents can be divided into two types: stimulating
3
7
agents (e.g. paclitaxel) and inhibiting agents (e.g. CAꢀ4). As platinum conjugates with
CAꢀ4, compound 13 was expected to inhibit the formation of microtubules by interacting
with the colchicines binding site of tubulin. To prove this, we investigated complex 13
that acts as an inhibitor of tubulin polymerization at doseꢀdependent manner in a tubulin
polymerization assay. As shown in Figure 5, incubation with 13, paclitaxel or CAꢀ4
resulted in various degrees of inhibition of tubulin polymerization, depending on the
property of each compound and the dose in the time dependency of this process. Among
them, the positive drug paclitaxel (10 ꢁM) was found to stimulate tubulin polymerization
and CAꢀ4 (10 ꢁM) nearly completely inhibited tubulin polymerization as expected. For
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compound 13, an obvious inhibition of polymerization was observed at two
concentrations, and the rate of assembly as well as the final amount of microtubules was
lower than the control. However, cisplatin at 10 ꢁM was barely effective in inhibition or
stimulation of tubulin polymerization under the similar conditions compared with the
control.
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.45
.40
.35
Control
Paclitaxel (10 µM)
CAꢀ4 (10 µM)
Cisplatin (10 µM)
13 (10 µM)
0.30
0
.25
.20
13 (20 µM)
0
0.15
0
.10
.05
0
0.00
0
10
20
30
40
50
60
70
Time (min)
Figure 5. Effects of compound 13 on microtubule dynamics. Polymerization of tubulin at
7 °C in the presence of paclitaxel (10 ꢁM), CAꢀ4 (10 ꢁM), cisplatin (10 ꢁM) and 13 (10
M and 20 ꢁM) were monitored continuously by recording the absorbance at 340 nm
3
ꢁ
over 60 min. The reaction was initiated by the addition of tubulin to a final concentration
of 3.0 mg/mL.
Molecular Modeling. To elucidate the binding mode of platinum(IV) complexes, we
performed a molecular docking postulated that they have the same binding site as
colchicine and CAꢀ4. The binding modes of these complexes in the colchicine binding
site of tubulin are depicted in Figure 6 and the Surflex docking scores obtained are
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summarized in Table 4. The Surflex docking scores are 7.83 for CAꢀ4, 11.29 for 13,
10.37 for 15 and 10.68 for 14, where higher scores indicate greater binding affinity. The
order of the docking scores correlates with the IC50 values of the complexes for the
growth inhibition of HepGꢀ2 human liver cancer cells. Here we examine the docking
details of complex 13 (whose IC values is 0.35 ꢁM in HepGꢀ2 cells) as compared with
10
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12
13
14
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59
60
5
0
those of 3E22ꢀcolchicine, CAꢀ4 and compound 9 (CAꢀ4 derivative).
Table 4. Docking Scores (kcal/mol) for All Studied Compounds
Compd.
Total Crash polar D_Score PMF_Score GꢀScore Chem_Score Cscore
Score
1
3
11.29 ꢀ2.25 3.11 ꢀ235.69
10.37 ꢀ2.28 4.91 ꢀ225.40
10.68 ꢀ5.26 6.06 ꢀ262.27
6.71 ꢀ0.98 1.79 ꢀ162.88
7.83 ꢀ2.44 2.48 ꢀ137.09
ꢀ5.03
ꢀ9.48
ꢀ392.55
ꢀ419.01
ꢀ440.78
ꢀ221.82
ꢀ236.47
ꢀ281.20
ꢀ35.51
ꢀ42.66
ꢀ38.01
ꢀ25.39
ꢀ23.67
ꢀ26.83
4
5
5
4
4
4
14
1
5
ꢀ19.15
24.82
10.98
18.38
9
CAꢀ4
3
E22ꢀligand 6.70 ꢀ4.60 1.85 ꢀ158.29
Figure 6 A shows that the interacting mode of the cocrystallized 3E22ꢀcolchicine in
the binding site, with 3,4,5ꢀtrimethoxyꢀphenyl rings placed in proximity to residue
Cys241. In particular, 3E22ꢀcolchicine formed three hydrogen bonds with the polar
amino acids Asn249, Ala 250 and Asn258, suggesting a probable stronger electrostatic
interaction with the protein. In addition, the hydrophobic moiety of the 3E22ꢀcolchicine
is well embedded in a pocket interacting with several hydrophobic residues making
3
E22ꢀcolchicine bind tightly to tubulin. Not surprisingly, the accommodation of complex
1
3 in the binding site is similar to colchicine (Figure 7). Also in this case, docking
simulations showed that the 3,4,5ꢀtrimethoxyꢀphenyl rings of complex 13 like colchicine
can also be accommodated in the same hydrophobic groove, adopting an energetically
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stable conformation. Moreover, the methoxy group in 13 as an acceptor establishes one
hydrogen bond with Asn258, which is consistent with the observation that colchicine
stabilizes the tubulin heterodimer and further confirms that this moiety is also crucial for
binding. It is interesting to note that the crucial electrostatic interactions between the
ammine of the Pt(IV) unit and residues Thr179, Ser178, Glu183 and Asn101 of the
neighboring αꢀsubunit were observed in the binding pocket, demonstrating a plausible
competitive mechanism of action at the colchicine site.
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(A)
(B)
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(
C)
Figure 6. Molecular modeling of CAꢀ4, 3E22ꢀcolchicine and 13 in complex with tubulin.
Illustrated is the proposed binding mode and interaction between tubulin and selected
compounds, (A) 13, (B) CAꢀ4 and (C) 3E22ꢀcolchicine. The compounds and important
amino acids in the binding pockets are shown in stick model, whereas tubulin is depicted
in the ribbon model.
Figure 7. Comparison of the crystallographic structure of colchicines (in green), CAꢀ4 (in
orange) in complex with tubulin (Protein Data Bank code 3E22) and the energetically
most favorable pose of 13 (in gray) obtained by molecular docking simulation. Hydrogen
atoms are omitted.
Competitive Binding to Colchicine Binding Site of Tubulin. To understand whether
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the Pt(IV) conjugates can competitively bind to colchicine site of tubulin, we studied our
3
representative compound to evaluate its effect on the binding of [ H] colchicine to tubulin.
The result is given in Table 5. For comparison, CAꢀ4 was examined in contemporaneous
experiments. In the colchicine binding studies, both compounds 9 and 13 are capable of
inhibiting tubulin polymerization with calculated IC values of 7.56 ꢁM and 7.91 ꢁM,
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9
0
5
0
3
respectively. In addition, compound 13 showed the ability to compete with [ H]
colchicine in binding to tubulin. The binding potency of 13 (53.5%) to the tubulin
colchicine binding site, comparable to compound 9 (55.8%), was much stronger than that
of cisplatin but less than that of CAꢀ4 (98%).
Table 5. Inhibition of Tubulin Polymerization and Colchicine Binding by Tested
Compounds
Compounds
(10 ꢁM)
Tubilin assembly
IC (ꢁM
Colchicines binding
(% inhibition)
)
5
0
9
7.56±1.2
7.91±1.1
>100
55.8±1.1
53.5±1.4
1.2±0.3
1
3
a
CDDP
CAꢀ4
1.2±0.2
98.0±0.5
a
Cisplatin
Effect on Cell Cycle Arrest. To investigate the effect of the synthetic Pt(IV)
complexes on cell cycle arrest, we used flow cytometry to analyze the cell cycle
distribution of HepGꢀ2 cells following a 24 h treatment with complex 13 at different
concentrations. Untreated cells were used as a negative control, and cells treated with
cisplatin were used as a positive control. As shown in Figure 8, the most potent complex
1
3 was found to be as effective in arresting the cell cycle at G2/M phase as cisplatin.
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With the untreated cells, the percentage of cells in the G0/G1 phase was at 73.34% with
only 2.44% in the G2/M phase. After treatment with complex 13, the percentage of cells
in the G2/M phase increased to 18.09% (1 ꢁM) and 63.30% (2 ꢁM), respectively. These
results compare favorably to 23.28% in the G2/M phase for cells treated with cisplatin
(10 ꢁM).
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Control
Cisplatin (10 ꢁM)
13 (1 ꢁM)
13 (2 ꢁM)
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)
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(
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G0/G1
G2/M
S
Figure 8. Effects of 13 on cell cycle phase arrest in HepGꢀ2 cells. Cells were treated with
and 2 ꢁM of 13 for 24 h. Then the cells were fixed and stained with PI to analyze DNA
1
content by flow cytometry.
Figure 9. Western blot analysis of cyclin B1, CDK1, p21 and p53 after treatment of
HepGꢀ2 cells with 13 at the indicated concentrations and for the indicated times. βꢀactin
antibody was used as reference control.
Furthermore, the molecular events involved in cellular response to the effective
compound were investigated. The levels of regulatory proteins implicated in G2 arrest,
including p21, CDK1, cyclin B1 and p53, were examined (Figure 9). Protein p21 plays a
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cdc2
significant role in G2 arrest through accumulation of inactive cyclin B1/p34
38ꢀ40
complex.
In fact, under the same conditions used for analysis of cell cycle
perturbation (24 h exposure to 1, 2 ꢁM for 13), there was a noteworthy increase of p21
and p53 expression accompanied with upꢀregulation of cyclin B1, indicating an activation
of the mitotic checkpoint following drug exposure. This effect was confirmed by the
appearance of slower migrating forms of phosphatase CDK1 at 1 ꢁM, followed by a
strong reduction at 2 ꢁM. Altogether these events are consistent with a pivotal role of p21
in G2 arrest as a consequence of inactivation of cyclin B1, and thus suggesting that the
cells are effectively arrested at G2 phase of the cell cycle.
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Complex 13 Induced Apoptotic Cell Death. In order to confirm whether the complex
13 induced reduction in cell viability was responsible for the induction of apoptosis,
HepGꢀ2 cells were coꢀstained with AnnexinꢀV FITC and PI, and the number of apoptotic
cells was estimated by flow cytometry. The tested complex was incubated with HepGꢀ2
cells for 24 h at the increasing concentrations, and cisplatin served as a positive control.
Q1−Q4 represent four different cell states: necrotic cells, late apoptotic or necrotic cells,
living cells, and apoptotic cells, respectively (Figure 10). A doseꢀdependent increase in
the percentage of apoptotic cells was noted after the cells were treated with complex 13
for 24 h at the concentrations of 2 ꢁM and 4 ꢁM. As shown in Figure 10, few (0.81%)
apoptotic cells were present in the control panel, in contrast, the population rose to
23.58% at the concentration of 2 ꢁM after treatment with 13 for 24 h. Further increase to
3
3.88% occurred after treatment with 13 at the concentration of 4 ꢁM, while cisplatin
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showed a lower population of apoptotic cells than complex 13. Overall, the results clearly
confirmed that complex 13 effectively induced apoptosis in HepGꢀ2 cells.
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Figure 10. Representative flow cytometric histograms of apoptotic HepGꢀ2 cells after 24
h treatment with 13. The cells were harvested and labeled with annexinꢀVꢀFITC and PI,
and analyzed by flow cytometry. Data are expressed as the mean ± SEM for five
independent experiments.
Morphological Examination. The ability of complex 13 to induce apoptosis was
further confirmed by analyzing the nuclear morphology of the exposed HepGꢀ2 cells.
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HepGꢀ2 cells were treated with complex 13 for 24 h and stained with
membraneꢀpermeable blue Hoechst 33258 to detect apoptosis morphologically. As shown
in Figure 11A, the Hoechst 33258 fluorescent photomicrographs of cultured HepGꢀ2
cells treated with 2.0 and 4.0 ꢁM complex 13 for 24 h, respectively, indicated that in the
control cultures, the nuclei of HepGꢀ2 cells appeared with regular contours and were
round, whereas smaller nuclei and condensed chromatin were rarely seen. Treatment with
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0.0 ꢁM cisplatin or 2.0 ꢁM complex 13 slightly changed the nuclear morphology
(Figure 11B and 11C). It should be noted that the numbers of apoptotic nuclei containing
condensed chromatin increased significantly as the result of treatment with 4.0 ꢁM
complex 13 (Figure 11D).
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Figure 11. Morphological changes in the nuclei (typical of apoptosis) of cultured HepGꢀ2
cancer cells. HepGꢀ2 cancer cells treated with 2.0 and 4.0 ꢁM 13 ( C and D) and treated
with 10.0 ꢁM cisplatin for 24 h, respectively, and stained with Hoechst 33258. Selected
fields illustrating occurrence of apoptosis were shown. Cells with condensed chromatin
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brightly stained) were defined as apoptotic HepGꢀ2 cancer cells. Images were acquired
using a Nikon Te2000 deconvolution microscope (magnification 200×).
Complex 13 Triggered ROS Generation. Reactive oxygen species (ROS) are highly
harmful elements to cells as they initiate oxidative stress and ultimately cause cellular
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damage. Excessive ROS generation renders cells vulnerable to apoptosis.
To
determine whether 13 triggers ROS generation in HepGꢀ2 cells to induce apoptosis, the
ROS level was measured with and without (control) treatment of 13 (2.0, 4.0 and 6.0 ꢁM)
for 24 h, using the fluorescent probe 2,7ꢀdichlorofluorescein diacetate (DCFꢀDA) by flow
cytometry, and treatment of cisplatin (4.0 ꢁM) as a positive control, respectively. As
shown in Figure 12, the results indicated that complex 13 induced the production of
significant amounts of ROS in HepGꢀ2 cells. After exposure to 2.0 ꢁM of complex 13 for
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4 h, the ROS level was 33.6%, which is more than two times that of control and
cisplatin. In all, these results proved that 13 causes oxidative imbalance in HepGꢀ2 cells.
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Figure 12. Assessment of the ROS production in HepGꢀ2 cells. After 24 h incubations
with 13, cells were stained with DCFꢀDA and analyzed by flow cytometry. Data are
expressed as the mean ± SEM of three independent experiments.
Effect of Complex 13 on Mitochondrial Depolarization. The loss of mitochondrial
transꢀmembrane potential (ꢂψ ) is regarded as a limiting factor in the apoptotic
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pathway.
Therefore, the ability of mitochondria to maintain membrane potential after
incubation with complex 13 was measured in HepGꢀ2 cells using the fluorescent dyes
JCꢀ1. HepGꢀ2 cells treated with complex 13 (2.0 and 4.0 ꢁM) and cisplatin (4.0 ꢁM),
respectively, for 24 h, subsequently processed, which were stained with JCꢀ1 dye and
analyzed by flow cytometry. Complex 13 treated cells showed an increase in green/red
fluorescence intensity indicating increased mitochondrial membrane depolarization
compared with untreated cells (Figure 13). As shown in Figure 13 B, the cisplatin
induced dissipation of ꢂψ was significantly reduced in HepGꢀ2 cells as compared with
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3. The results revealed that the induction of apoptosis by complex 13 in HepGꢀ2 cells is
closely associated with mitochondrial function disruption. In fact, the dissipation of ꢂψm
is characteristic of apoptosis and has been observed with platinum conjugates in different
cell types.
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Figure 13. Assessment of mitochondrial membrane potential (ꢂψ ) after treatment of
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HepGꢀ2 cells with cisplatin (B) or compound 13 (C, D). Cells were treated with the
indicated concentration of the compound for 24 h and then stained with the fluorescent
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probe JCꢀ1 and analyzed by flow cytometry. Data are presented as the mean ± SEM for
three independent experiments.
Effect of Complex 13 Induced Apoptosis via an Intrinsic Apoptosis Pathway in
HepGꢀ2 Cells. The mitochondrial pathway is one of the major apoptosis pathways,
which is often related to the loss of ꢂΨm. The mitochondriaꢀdependent apoptotic
pathway is regulated by the Bclꢀ2 family of proꢀ and antiꢀapoptotic proteins, which
induce the permeabilization of the mitochondrial outer membrane and cytochrome c
released into the cytosol, resulting in the activation of the caspase cascade and the
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induction of apoptotic cell death.
To further understand the mechanism of action of
the newly synthesized complex, the mitochondrial related apoptotic proteins of Bax,
Bclꢀ2, cytochrome c, caspaseꢀ9, caspaseꢀ3 and PARP were tested in HepGꢀ2 cells treated
with complex 13 by the Western blot analysis. As shown in Figure 14, in comparison
with the control cells, complex 13 induced a significant increase in the expression of Bax
and a reduction in the levels of Bclꢀ2, in a time dependent fashion. The result indicated
that treatment with 13 could shift the ratio of Bax/ Bclꢀ2 proteins and thus lead to
collapse of the mitochondrial membrane potential. Moreover, after HepGꢀ2 cells were
treated with 5.0, and 10.0 ꢁM 13 for 24 h, the release of cytochrosome c increased in a
concentrationꢀcorresponding manner. Subsequently, the activation of downstream
caspases, including caspaseꢀ3 and caspaseꢀ9, was observed, all of which could trigger
apoptosis of tumor cells. Exposure of HepGꢀ2 cells to this complex caused a dramatic
increase in the levels of caspaseꢀ9, caspaseꢀ3 and cleavedꢀPARP, as compared with
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