Combination of 7-hydroxycoumarin in a platinum(IV) complex derived from cisplatin enhanced cytotoxicity with multiple mechanisms of action

Article abstract of DOI:10.1016/j.jinorgbio.2018.05.015

A novel compound, Cou-platin, composed of 7-hydroxycoumarin and a platinum(IV) moiety derived from cisplatin was designed and synthesized. Significantly, Cou-platin exhibited more potent in vitro antitumor activity against all tested cancer cell lines than that of cisplatin, which was mainly attributed to the liberation of cisplatin and 7-hydroxycoumarin upon reduction with a biomolecular agent. Besides, cellular accumulation of Cou-platin was dramatically increased among several cancer cells in contrast to cisplatin. Flow cytometry study revealed that Cou-platin arrested cell cycle at G2 phase and induced cell apoptosis. Western blots results indicated that it not only activated cell apoptosis pathway, but also inhibited extracellular regulated protein kinases/mitogen-activated protein kinase pathway. In vivo tests showed that Cou-platin, at equimolar dose to cisplatin, could inhibit tumor growth in nude mouse HCT116 tumor xenograft models almost as cisplatin and oxaliplatin, but with less toxicity.

Full text of DOI:10.1016/j.jinorgbio.2018.05.015

Journal of Inorganic Biochemistry 186 (2018) 17–23
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Combination of 7-hydroxycoumarin in a platinum(IV) complex derived from
cisplatin enhanced cytotoxicity with multiple mechanisms of action
Wuyang Huaa,1, Jian Zhaoa,b,1, Weiwei Hua,b, Shaohua Goua,b,⁎
a
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Platinum(IV) complexes
A novel compound, Cou-platin, composed of 7-hydroxycoumarin and a platinum(IV) moiety derived from cis-
platin was designed and synthesized. Significantly, Cou-platin exhibited more potent in vitro antitumor activity
against all tested cancer cell lines than that of cisplatin, which was mainly attributed to the liberation of cisplatin
and 7-hydroxycoumarin upon reduction with a biomolecular agent. Besides, cellular accumulation of Cou-platin
was dramatically increased among several cancer cells in contrast to cisplatin. Flow cytometry study revealed
that Cou-platin arrested cell cycle at G2 phase and induced cell apoptosis. Western blots results indicated that it
not only activated cell apoptosis pathway, but also inhibited extracellular regulated protein kinases/mitogen-
activated protein kinase pathway. In vivo tests showed that Cou-platin, at equimolar dose to cisplatin, could
inhibit tumor growth in nude mouse HCT116 tumor xenograft models almost as cisplatin and oxaliplatin, but
with less toxicity.
7
-Hydroxycoumarin
Cou-platin
Anticancer
1. Introduction
cancer cells [13–17].
The extracellular regulated protein kinases (ERK)/mitogen-acti-
Cisplatin has become one of the most popular agents in cancer
vated protein kinase (MAPK) pathway plays a central role in regulating
cell growth, proliferation and survival. Hence, ERK/MAPK pathway has
emerged as a promising target for tumor therapy [18,19]. Coumarins, a
wide class of natural compounds, are well known for their various
biological functions, inclusive of antiviral [20], anti-inflammatory [21]
and anticancer activities [22]. It has been found that some 7-hydro-
xycoumarin derivatives can induce cancer cell apoptosis through ERK/
MAPK signaling pathway [23].
chemotherapy since it was approved by FDA in 1978. After that, other
cisplatin analogues, carboplatin and oxaliplatin have been successively
approved (Fig. 1). Principal indications for cisplatin are metastatic
testicular cancer, metastatic ovarian cancer, and transitional bladder
cancer [1]. By cross-linking DNA and inhibiting DNA transcription [2],
cisplatin can kill cancer cells in a relative high level and cure more
patients compared with the drugs used before [3,4]. However cisplatin
faces some embarrassments, such as drug resistance and side effects.
Meanwhile, it has negligible effect on some certain kinds of cancer in
vivo. Therefore, development of new platinum based anticancer agents
with higher anticancer activity but less toxicity is very meaningful.
Platinum(IV) complexes with an octahedral geometry showed great
promising due to their improved cytotoxicity and relatively lower
toxicity [5–10]. Moreover, it has been reported that some platinum(IV)
complexes were sensitive to some cisplatin-resistant cancer cell lines
Based on the above, we herein report a novel dual-targeting pla-
tinum(IV) anticancer prodrug named Cou-platin, cis,cis,trans-{Pt
(NH
cing 7-hydroxycoumarin to the axial position of cis,cis,trans-[Pt
(NH Cl (OH) ] derived from cisplatin, whose anticancer activities
3 2 2
) Cl (OH)[2-((2-oxo-2H-chromen-7-yl)oxy)acetate]}, by introdu-
3
)
2
2
2
and underlying mechanisms were studied.
2. Results and discussion
[
11,12], indicating that platinum(IV) complexes can overcome cisplatin
resistance. Usually, platinum(IV) complexes were generally designed as
prodrugs by modification at the axial position with different functional
groups, which may improve the anticancer efficacy, kinetic perfor-
mance or selectivity of the platinum(IV) complexes against certain
2.1. Synthesis and characterization of Cou-platin
The preparation of Cou-platin is shown in Scheme 1. 7-Hydro-
xycoumarin was first treated with a linker, bromoacetic acid to produce
⁎
Corresponding author at: Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, China.
E-mail address: sgou@seu.edu.cn (S. Gou).
Wuyang Hua and Jian Zhao contributed equally to this work.
1
https://doi.org/10.1016/j.jinorgbio.2018.05.015
Received 29 November 2017; Received in revised form 3 May 2018; Accepted 23 May 2018
Available online 23 May 2018
0162-0134/ © 2018 Elsevier Inc. All rights reserved.

W. Hua et al.
Journal of Inorganic Biochemistry 186 (2018) 17–23
Fig. 1. FDA approved platinum(II) anticancer agents.
H2
N
O
O
O
O
O
O
H N
Cl
Cl
H N
O
O
3
3
Pt
Pt
Pt
H N
H N
3
3
N
H2
Carboplatin
Oxaliplatin
Cisplatin
future.
HO
O
O
Br
HOOC
O
O
O
The stability of Cou-platin was also studied by HPLC under both
phosphate buffered saline (PBS, pH = 7.4) and biological conditions. As
shown in Fig. S3, Cou-platin was stable in PBS solution for 12 h, and no
disassociation was observed. However, no useful information was ob-
tained in the solution of cell culture medium, because the peak of Cou-
platin was overlapped by the constituent components of the medium,
and we could not find a suitable condition to separate them for further
analysis (Fig. S4).
OH
K CO , KI, acetone
2
3
1
O
O
O
O
O
-
cis,cis,trans [Pt(NH3)2Cl2(OH)2]
H N
3
Cl
Cl
Pt
TBTU, TEA, DMF
H N
3
2.3. In vitro cytotoxicity
Scheme 1. Synthesis of the target compound, Cou-platin.
The in vitro cytotoxicity of Cou-platin was evaluated by 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
against six human cancer cell lines: HepG2 (hepatoma), HCT116
intermediate 1. By taking the carboxylic acid of the linker, Cou-platin
was then prepared by treatment of cis,cis,trans-[Pt(NH Cl (OH) ] with
3
)
2
2
2
(
(
colon), A549 (lung), MCF7 (breast), SGC7901 (stomach), SGC7901/cis
cisplatin resistant) and HUVEC (human umbilical vein endothelial
equivalent molar amount of intermediate 1 in the presence of O-(Ben-
zotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU)
and triethylamine (TEA) in dried DMF. The final product of Cou-platin
cells) with cisplatin and 7-hydroxycoumarin as positive controls. The
corresponding half maximal inhibitory concentration (IC50) values are
given in Table 1. As predicted, the cytotoxicity of 7-hydroxycoumarin
was negligible. It is noted that Cou-platin showed superior cytotoxicity
to cisplatin against all tested cancer cell lines. Particularly, Cou-platin
showed significantly enhanced inhibitory effect against HCT116 cells
with a 30-fold higher cytotoxicity than cisplatin. Besides, Cou-platin
also exhibited increased toxicity against HepG2, SGC7901 and
SGC7901/cis cancer cells with 29-fold, 23-fold and 19-fold lower IC50
values than cisplatin, respectively. Despite Cou-platin showed con-
siderable anti-proliferation activity against cisplatin resistant
SGC7901/cis cells but it did not overcome the drug tolerance. The re-
sistance index of Cou-platin was calculated as 5.37, which was higher
than that of cisplatin (resistance index = 4.38). However, the IC50 value
against HUVEC cells of Cou-platin was 4-fold higher than that of cis-
platin, suggesting that, in contrast to cisplatin, Cou-platin may be se-
lectively cytotoxic for human cancer cells. These in vitro results en-
couraged us to further explore the mechanism of inhibitory effect of
Cou-platin upon cancer cells.
1
13
was characterized by microanalysis, H NMR and C NMR spectra
together with ESI-MS mass spectrometry. The purity of Cou-platin was
determined to be > 97% by reverse-phase HPLC (Fig. S1).
2.2. Reduction and stability study of Cou-platin
Ascorbic acid and glutathione are overexpressed in cancer cells
[
24], which gives platinum(IV) based anticancer drugs a target point so
that they could be reduced by ascorbic acid or glutathione and release
active free drugs. As Cou-platin was expected to be reduced by bio-
molecule such as ascorbic acid or glutathione, the reduction of Cou-
platin was investigated in the presence of ascorbic acid by HPLC. As
shown in Fig. 2, the reduction reaction of Cou-platin by ascorbic acid
proceeded gradually. It was noted that the compound released from
Cou-plaitn was 7-hydroxycoumarin instead of intermediate 1 as refer-
ring to the HPLC diagram of 7-hydroxycoumarin (Fig. S2), this might
arise from the instability of phenolic ether bond in the presence of
nucleophilic agents. Actually, the solution behavior of platinum(IV)
complexes is very complicated because not only the reduction occurs at
the axial position, but also the hydrolysis at the equatorial position also
happens in the aqueous solution [16]. Despite cisplatin was not found
due to its weak chromophore under the ultraviolet detecting condition
in Fig. 2, those results indicated that Cou-platin as a platinum(IV) based
anticancer prodrug could be reduced by ascorbic acid to release cyto-
toxic agents, which was consistent with our design. However, the spe-
cific reduction mechanism of Cou-platin needs further investigation in
2.4. Cellular accumulation
To figure out the underlying mechanism of the considerable cyto-
toxicity of Cou-platin, inductively coupled plasma mass spectrum (ICP-
MS) was used to evaluate the cellular accumulation of Cou-platin in
cancer cells. As shown in Fig. 3 and Table 2, platinum amount in
6
HCT116 cells treated with Cou-platin was 1232 ± 65 ng/(10 cells),
which was 37-fold higher than that of cisplatin. In other two cancer
Fig. 2. HPLC analysis of Cou-platin incubated with ascorbic acid.
18

W. Hua et al.
Journal of Inorganic Biochemistry 186 (2018) 17–23
Table 1
cells, Cou-platin also possessed dramatically increased platinum
IC50 values of Cou-platin and cisplatin against six human cancer cells.a
6
amount compared with cisplatin, 1023 ± 49 ng/(10 cells) in
6
SGC7901 and 1109 ± 57 ng/(10 cells) in SGC7901/cis. Just like other
Cell types
Compounds, IC50 (μM)
platinum(IV) based anticancer drugs reported before, Cou-platin also
has a lipophilic group so that it can enter cancer cells more efficiently
by passive diffusion and has such enhanced cellular accumulation
pattern [25–29]. These results demonstrated that Cou-platin can enter
cancer cells more efficiently than cisplatin after a treatment for 24 h
and exhibits high cellular persistence [30]. Compared with the in vitro
cytotoxicity results (Table 1), Cou-platin showed high anti-proliferation
activity against both wild and resistant SGC7901 cells. This is in tune
with the results of cellular accumulation. But it did not overcome the
drug resistance. This implies that chemoresistance does not come from
different drug accumulation.
Cisplatin
7-Hydroxycoumarin
Cou-platin
HepG2
HCT116
A549
MCF7
SGC7901
SGC7901/cis
HUVEC
6.94 ± 0.48
9.34 ± 0.85
4.51 ± 0.35
6.40 ± 0.48
1.86 ± 0.15
8.15 ± 0.63
7.86 ± 0.34
4.38
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.24 ± 0.05
0.31 ± 0.02
4.02 ± 0.26
2.46 ± 0.18
0.08 ± 0.01
0.43 ± 0.02
29.79 ± 1.58
5.38
b
Resistance index
–
a
Cancer cells were treated with a series dose of drugs for 72 h and measured
with IC50 values. The data were presented as the mean ± SD for three in-
dependent experiments.
2.5. Cell image
b
Resistance index = (IC50 value of SGC7901/cis)/(IC50 value of SGC7901).
Due to the existence of a coumarin moiety, Cou-platin can be used
as a track agent to show the drug uptake in cells under the laser
scanning confocal microscopy (LSCM). HCT116 cells were incubated
with Cou-platin (30 μM) for 3 h. Then the free drug was washed off and
the treated cells were analyzed by LSCM. As shown in Fig. 4, with ex-
citation by using 405 nm light source, green fluorescence was observed
in HCT116 cells, indicating that Cou-platin could be uptaken by
HCT116 cells. Besides, the morphology of HCT116 cells became round
from spindle-shaped (Fig. 4A), demonstrating that the cells were un-
dergoing apoptosis or death. The result was consistent with the corre-
sponding cytotoxicity by MTT assay.
2.6. Cell cycle
Flow cytometry was used to investigate cell cycle block pattern of
Fig. 3. Cellular accumulation of Cou-platin and cisplatin in three cancer cells.
All tested cancer cells were incubated with cisplatin and Cou-platin (15 μM
respectively) for 24 h at 37 °C. The Data were expressed as the mean ± SD for
three independent experiments.
Cou-platin with cisplatin at equitoxic concentration (at concentration of
IC50 value). Cou-platin arrested HCT116 cells in G2 phase as cisplatin
did, which is a common phenomenon caused by anticancer drugs
31,32]. Unlike cisplatin reducing the portion of G1 phase of HCT116
cells dramatically, Cou-platin showed almost no effect on this phase
Fig. 5). Besides, after treatment of cisplatin, the percentage of S phase
of cells was reduced to 54.44% compared with the negative control
(58.92%). Interestingly, Cou-platin reduced the portion of S phase of
HCT116 cells by 14.80%. This result was in line with an anticancer drug
that contained a 7-hydroxycoumarin moiety reported previously [33].
Hence, both cisplatin and 7-hydroxycoumarin may exert their effect in
the cell cycle of HCT116 cells.
[
Table 2
(
a
Cellular platinum amount of Cou-platin and cisplatin in three cancer cell lines.
Compounds
Pt (ng/106 cells) in cells
HCT116 SGC7901
SGC7901/cis
Cisplatin
33 ±
3
230 ± 17
222 ± 10
Cou-plaitn
1232 ± 65
1023 ± 49
1109 ± 57
a
All tested cancer cells were incubated with cisplatin and Cou-platin (15 μM
respectively) for 24 h at 37 °C. Results are expressed as the mean ± SD for
three independent experiments.
2
.7. Apoptosis studies
To further study the underlying anticancer mechanism of Cou-
platin, the analysis of apoptosis of our compound and cisplatin toward
Fig. 4. Cell image of HCT116 cells treated with Cou-platin. (A) Bright field; (B) Fluorescence image; (C) Overlay of the merged images of (A) and (B). HCT116 cells
were treated with 30 μM Cou-platin for 3 h. The excitation wavelength was 405 nm and emission wavelength was 488 nm.
19

W. Hua et al.
Journal of Inorganic Biochemistry 186 (2018) 17–23
Fig. 5. Cell cycle distribution of HCT116 cells treated with (A) DMF; (B) cisplatin and (C) Cou-platin. HCT116 cells were incubated with 9.3 μM cisplatin and 0.3 μM
Cou-platin for 24 h at 37 °C.
Fig. 6. Cell apoptosis results of HCT116 cells treated with Cou-platin and cisplatin. (A) DMF; (B) Cisplatin and (C) Cou-platin are graphical representations; (D)
Digital representation of apoptosis rate. HCT116 cell line was incubated with 9.3 μM cisplatin and 0.3 μM Cou-platin for 24 h at 37 °C.
HCT116 cells was studied at equitoxic concentration (at concentration
of IC50 value) by Annexin V-FITC/Propidium Iodide (PI) assay. Cells
treated with DMF were used as negative control. As shown in Fig. 6,
Cou-platin caused 47.70% cells apoptosis, which was in accordance
with MTT results, indicating that Cou-platin can inhibit HCT116 cells
via apoptosis pathway more effectively than cisplatin.
2.8. Western blot assay
Based on the cell apoptosis and cycle results, Western blot technique
was applied to investigate the influence of Cou-platin exerted on the
related protein expression. HCT116 cells were incubated with cisplatin
and Cou-platin for 24 h, respectively. The expression of apoptosis re-
lated protein in HCT116 cells was shown in Fig. 7A and B. It was no-
ticed that Cou-platin upregulated the expression of Bax, cleaved
20

W. Hua et al.
Journal of Inorganic Biochemistry 186 (2018) 17–23
Fig. 7. Cell apoptosis pathway Western blot
analysis of HCT116 cells incubated with cis-
platin and Cou-platin. (A) Blots; (B) Relative
gray intensity analysis. HCT116 cells were in-
cubated with 9.3 μM cisplatin and 0.3 and
1
.0 μM Cou-platin for 24 h at 37 °C. Relative
gray intensity = (gray intensity of indicated
protein)/(gray intensity of β-actin-1). The data
are representative of three independent experi-
ments and shown as the mean ± S.D.
Fig. 8. ERK/MAPK pathway Western blot ana-
lysis of HCT116 cells incubated with 7-hydro-
xycoumarin and Cou-platin. (A) Blots; (B)
Relative gray intensity analysis. HCT116 cells
were incubated with 7-hydroxycoumarin and
Cou-platin (0.5, 1.0, 5.0 μM) for 24 h at 37 °C.
Relative gray intensity = (gray intensity of in-
dicated protein)/(gray intensity of β-actin-1).
The data are representative of three in-
dependent experiments and shown as the
mean ± S.D.
caspase3, cleaved PARP and cyto-c (Fig. S5) in a concentration de-
pendent manner, which indicated that Cou-platin could activate the
apoptosis pathway and finally inhibited the proliferation of HCT116
cells. Taken the cellular accumulation results, this may come from the
enhanced drug accumulation of Cou-platin [34,35]. Moreover, we
further investigated the inhibitory effect of Cou-platin on phosphor-
ylation of mitogen-activated extracellular signal-regulated kinase 1
of body weight of mice treated with Cou-platin in contrast to that with
the positive drugs (Fig. 9C).
3. Conclusions
In summary, a novel compound, named Cou-platin, comprising 7-
hydroxycoumarin and a platinum(IV) moiety derived from cisplatin
was designed and obtained via a linker. HPLC assay showed that the
compound could release cisplain and 7-hydroxycoumarin upon reduc-
tion by ascorbic acid as expected. But the specific reduction mechanism
of Cou-platin needs a further studied. In vitro test indicated that Cou-
platin had stronger anticancer activity against all the tested cancer cell
lines than cisplatin. Related experiment exhibited that introduction of
7-hydroxycoumarin led to the increased cellular accumulation of Cou-
platin in several cancer cells in contrast to cisplatin. Both cell cycle and
cell apoptosis analysis indicated that Cou-platin arrested HCT116 cell
cycle in a mix manner of cisplatin and 7-hydroxycoumarin and could
induce cell apoptosis more efficiently than cisplatin. Western blot
analysis revealed that Cou-platin inhibited HCT116 cells via cell
apoptosis pathway and the ERK/MAPK pathway. In vivo tests showed
that Cou-platin, at equimolar dose to cisplatin, could effectively inhibit
tumor growth in nude mouse HCT116 tumor xenograft models almost
the same as cisplatin and oxaliplatin, but with less toxicity. However,
we are aware that the limitation of using the equimolar dose in the in
vivo test. Consequently, our work demonstrated that 7-hydro-
xycoumarin and cisplatin have a jointed effect on the antitumor activity
of the resulting platinum(IV) complex, which can be useful for devel-
oping new anticancer agents.
(
MEK1, key node of ERK/MAPK pathway) and its unique downstream
target ERK1 [18]. As shown in Fig. 8A and B, Cou-platin indeed
downregulated the phosphorylation of MEK1 and ERK1 in a con-
centration dependent manner and shared the same trend and degree as
the positive control. It should be noted that cisplatin also possess the
ERK/MAPK pathway modulation ability [36]. As a derivative of 7-hy-
droxycoumarin, Cou-platin could inhibited ERK/MAPK pathway effi-
ciently.
2.9. In vivo antitumor activity
Encouraged by the in vitro results, we further investigated the in
vivo tumor inhibitory effect of Cou-platin in nude mouse HCT116
tumor xenograft models. Mice with tumors were divided into five
groups including (1) control, (2) cisplatin (5 mg/kg), (3) oxaliplatin
(
(
5 mg/kg), (4) Cou-plaitn (5 mg/kg, equal mass dose to cisplatin), and
5) Cou-plaitn (9 mg/kg, equimolar dose to cisplatin), which were
treated by tail vein injection once a week for 21 days. Tumor volumes
and weights are illustrated in Fig. 9A and B. In the equal mass dose,
unfortunately, Cou-plaitn exhibited a little weaker inhibitory activity
than cisplatin and oxaliplatin. While in the equimolar dose, the in vivo
antitumor activity of Cou-plaitn was promoted, which was nearly
comparable to that of cisplatin. However, it was of interest to note that
Cou-platin was less toxic than cisplatin and oxaliplatin upon the change
21

W. Hua et al.
Journal of Inorganic Biochemistry 186 (2018) 17–23
2.5
2.0
1.5
1.0
0.5
0.0
Control
Cisliatin 5mg/kg
Oxaliplatin 5mg/kg
Cou-platin 5mg/kg
Cou-platin 9mg/kg
3.0
2.5
2.0
1
.5
.0
1
0.5
0.0
Control
Cisliatin
5mg/kg
Oxaliplatin
5mg/kg
Cou-platin
5mg/kg
Cou-platin
9mg/kg
1
3
5
7
9
11 13 15 17 19 21
Days
2
2
2
2
2
1
1
1
4.5
3.5
2.5
1.5
0.5
9.5
8.5
7.5
Control
Cisliatin 5mg/kg
Oxaliplatin 5mg/kg
Cou-platin 5mg/kg
Cou-platin 9mg/kg
1
3
5
7
9
11 13
Days
15 17
19 21
Fig. 9. In vivo antitumor activity of Cou-platin in nude mouse HCT116 tumor xenograft models. (A) Tumor volume of mice during the administration period; (B)
Tumor weight of mice; (C) Curve of mice body weight recorded during administration period. Mice were sacrificed and tumors were weighed after 21-day treated
with drugs. The data were presented as the mean ± SEM. *P < 0.05.
4. Materials and methods
platin separately at a series of concentrations and incubated for 72 h at
7 °C. Then the cell viability was evaluated by the MTT method ac-
3
4.1. HPLC experiments
cording to literature method [37].
For reduction experiment, 0.5 ml of Cou-platin (0.3 mM) in PBS
4.4. Cellular accumulation
(
pH = 7.4, contains 10% DMF) and 0.5 ml of ascorbic acid (3.0 mM) in
PBS (pH = 7.4) were mixed and incubated at 37 °C in dark for 0 h, 1 h,
h, 3 h, 4 h, 5 h, 6 h, 7 h and 8 h respectively and then studied by HPLC
with an eluent comprised of MeOH and H O (1:10, v:v, containing 1%
Drug uptake of HCT116, SGC7901 and SGC7901/cis cells against
cisplatin and Cou-platin was evaluated by ICP-MS. After an overnight
culturing in 6-well plates, cancer cells were incubated with cisplatin
and Cou-platin (15 μM respectively) for 24 hat 37 °C. Then cancer cells
were washed by PBS buffer (pH 7.4) for three times, and harvested after
trypsinization. The cells were concentrated and digested by addition of
HNO3 to obtain the samples. Finally, the samples were examined by
ICP-MS.
2
2
trifluoroacetic acid) at r.t. Flow rate was 1.0 ml/min. Reversed-phase
HPLC was carried out on a ODS column (250 × 4.5 mm). The wave-
length of UV detector was 280 nm. For stability experiment, 0.5 ml of
Cou-platin (3.0 mM) in PBS (pH = 7.4, contains 10% DMF) was in-
cubated at 37 °C in dark and studied by HPLC every 3 h with the same
condition as reduction experiment.
4.5. Cell image
4.2. Cell culture
HCT116 cells were cultured in a six well plate containing a cover-
Human cancer cell lines HepG2, HCT-116, A549, MCF7, SGC-7901,
SGC-7901/cis and HUVEC were purchased from Jiangsu KeyGEN
BioTECH company (China). HepG2, A549, MCF7 and HUVEC cell lines
slip. Then the cells were incubated with 30 μM Cou-platin for 3 h. After
that, the cells were washed with PBS for three times to wipe out the free
drug. Then the coverslip was taken out and put on a glass slid. The glass
slid was detected by LSCM operating at a 405 nm excitation wavelength
and at 488 nm detecting emission wavelength.
were cultured in 5% CO
tomycin (Beyotime, Nantong, China) and 10% fetal bovine serum (FBS)
Hyclone, Lifescience, USA) at 37 °C. HCT116 and SGC-7901 cell lines
were cultured in 5% CO with RPMI-1640 added by 1% penicillin/
2
with DMEM added by 1% penicillin/strep-
(
2
4.6. Cell cycle
streptomycin and 10% FBS. SGC-7901/cis cell line was cultured and
screened in RPMI-1640 added by 1% penicillin/streptomycin, 10% FBS
and 800 ng/ml cisplatin before use. Cancer cell lines were passed every
Cell cycle was carried out by flow cytometry (FCM) according to a
method report previously [38]. Data were collected and analyzed by
FlowJo software (TreeStar, Inc.).
2
days and recovered from frozen stocks upon reaching pass number 20.
4.3. MTT assay
4.7. Cell apoptosis
In vitro anticancer activity of cisplatin and Cou-platin against
According to manufacturer's protocol (Shanghai Yisheng
Biotechnology Co. Ltd., 40302ES50) of Annexin V-FITC assay, HCT116
cells were treated with 9.3 μM cisplatin and 0.3 μM Cou-platin at 37 °C
for 24 h, respectively. Then the cells were collected and resuspended in
HepG2, HCT116, A549, MCF7, SGC7901 and SGC7901/cis cancer cell
lines were carried out by the MTT assay. Cancer cells were plated in 96-
5
well plates at density of 10 /ml per well. After an overnight culturing,
cells were exposed to culture medium containing cisplatin and Cou-
2
binding buffer (pH 7.5, 10 mM HEPES, 2.5 mM CaCl and 140 mM
22

W. Hua et al.
Journal of Inorganic Biochemistry 186 (2018) 17–23
NaCl). After that, cells were incubated with Annexin V-FITC and PI in
Appendix A. Supplementary data
dark for 10 min at r.t. Finally, cells were analyzed with flow cytometry
(
FACSCalibur, Becton Dickinson, USA) and computer station running
The synthesis procedure of Cou-platin in detail, HPLC analysis of
Cou-platin, standard sample of 7-hydroxycoumarin, stability study of
Cou-platin and cyto-C Western blot analysis. This material is available
free of charge via the Internet. Supplementary data to this article can be
found online at https://doi.org/10.1016/j.jinorgbio.2018.05.015.
Cell-Quest software (BD Biosciences, Franklin Lakes, NJ, USA).
4
.8. Western blot
HCT116 cells were cultured until the cell density reached 80%.
Then positive control (cisplatin or 7-hydroxycoumarin) and Cou-platin
were added, and the cells were cultured for 24 h at 37 °C. Proteins were
extracted by lysis buffer. The concentration of protein was measured
using the BCA (bicinchoninic acid) assay with a Varioskan multimode
microplate spectrophotometer (Thermo, Waltham, MA). Then protein
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Project of the National Science and Technology Major Foundation of
China (Grant No. 2015ZX09101032) for financial aids to this work. The
research was also supported by Jiangsu Province Natural Science
Foundation (Grant No. BK20160664) and the Fundamental Research
Funds for the Central Universities (Project 2242016k30020) for sup-
plying basic facilities to our key laboratory. We also want to express our
gratitude to the Priority Academic Program Development of Jiangsu
Higher Education Institutions for the construction of fundamental fa-
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23