Synthesis of a novel platinum(II) complex with 6,7-dichloro-5,8-quinolinedione and the study of its antitumor mechanism in testicular seminoma

Article abstract of DOI:10.1016/j.jinorgbio.2019.110701

A new platinum(II) complex, [Pt(ClClQ)(DMSO)Cl] (1), utilizing 6,7-dichloro-5,8-quinolinedione (ClClQ) as a ligand, has been synthesized and fully characterized. Single-crystal X-ray diffraction and other spectroscopic and analytical methods revealed that the coordination geometry of Pt(II) in complex 1 can also be described as a four-coordinated square planar geometry. The aim of the study was to explore the in vitro anticancer properties of complex 1. Our studies showed that complex 1 can regulate the viability of testicular seminoma cells in vitro, including cell proliferation and apoptosis. We further observed negative regulation by complex 1 of the expression levels of the key elements in the phosphoinositide-3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK3β) pathway, including phosphorylated phosphoinositide-3 kinase (p-PI3K), phosphorylated protein kinase B(p-Akt) and phosphorylated glycogen synthase kinase-3β (p-GSK3β). Moreover, the negative effect of complex 1 was reversed by LiCl, a GSK3β-specific inhibitor of the PI3K signaling pathway. Meanwhile, the levels of Bcl2 associated death promoter (Bad), cytochrome c, active-caspase-3 and active-caspase-9 increased significantly. In conclusion, we observed that complex 1 can regulate the viability of testicular seminoma cells through the PI3K/Akt/GSK3β signaling pathway and the mitochondria-mediated apoptotic pathway in vitro, and thus, complex 1 may have potential for use as a drug in the treatment of testicular germ cell tumors.

Full text of DOI:10.1016/j.jinorgbio.2019.110701

Accepted Manuscript
Synthesis of
a
novel platinum(II) complex with
6
,7-dichloro-5,8-quinolinedione and the study of its antitumor
mechanism in testicular seminoma
Zitaiyu Li, Jun Zhou, Yu Gan, Yinghao Yin, Wuchao Zhang,
Jianfu Yang, Yuxin Tang, Yingbo Dai
PII:
S0162-0134(19)30057-1
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110701
Article Number:
Reference:
110701
JIB 110701
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
25 January 2019
25 April 2019
25 April 2019
Please cite this article as: Z. Li, J. Zhou, Y. Gan, et al., Synthesis of a novel platinum(II)
complex with 6,7-dichloro-5,8-quinolinedione and the study of its antitumor mechanism
in testicular seminoma, Journal of Inorganic Biochemistry, https://doi.org/10.1016/
j.jinorgbio.2019.110701
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ACCEPTED MANUSCRIPT
Synthesis of a novel platinum(II) complex with
6
,7-dichloro-5,8-quinolinedione and the study of its antitumor
mechanism in testicular seminoma
a
a
b
a
a
a
Zitaiyu Li , Jun Zhou , Yu Gan , Yinghao Yin , Wuchao Zhang , Jianfu Yang ,
⁎
c,
c
Yuxin Tang , and Yingbo Dai
a
Department of Urology, The Third Xiangya Hospital of Central South University, Central South
University, Changsha 410000, China
b
Department of Urology, Xiangya Hospital of Central South University, Central South University,
Changsha 410000, China;
c
Department of Urology, The Fifth Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen
University, Zhuhai 519000, China.
⁎
Corresponding author: Yuxin Tang. mmcct@126.com, Fax: +86-0756-2528308
Abstract
A new platinum(II) complex, [Pt(ClClQ)(DMSO)Cl] (1), utilizing
6
,7-dichloro-5,8-quinolinedione (ClClQ) as a ligand, has been
synthesized and fully characterized. Single-crystal X-ray diffraction and
other spectroscopic and analytical methods revealed that the coordination
geometry of Pt(II) in complex 1 can also be described as a
four-coordinated square planar geometry. The aim of the study was to
explore the in vitro anticancer properties of complex 1. Our studies
showed that complex 1 can regulate the viability of testicular seminoma
cells in vitro, including cell proliferation and apoptosis. We further
observed negative regulation by complex 1 of the expression levels of the
key elements in the phosphoinositide-3 kinase(PI3K)/protein kinase B
(
Akt)/glycogen synthase kinase-3β(GSK3β) pathway, including
phosphorylated phosphoinositide-3 kinase(p-PI3K), phosphorylated
protein kinase B(p-Akt) and phosphorylated glycogen synthase kinase-3β
(
p-GSK3β). Moreover, the negative effect of complex 1 was reversed by
LiCl, a GSK3β-specific inhibitor of the PI3K signaling pathway.
1

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Meanwhile, the levels of Bcl2 associated death promoter(Bad),
cytochrome c, active-caspase-3 and active-caspase-9 increased
significantly. In conclusion, we observed that complex 1 can regulate the
viability of testicular seminoma cells through the PI3K/Akt/GSK3β
signaling pathway and the mitochondria-mediated apoptotic pathway in
vitro, and thus, complex 1 may have potential for use as a drug in the
treatment of testicular germ cell tumors.
Keywords: 6,7-dichloro-5,8-quinolinedione, platinum(II) complex, cell
apoptosis, Akt, GSK3β
1
. Introduction
Platinum-based anticancer drugs, such as cisplatin and carboplatin,
have proven to be highly successful in the area of anticancer chemistry,
stimulating the development of new drugs that utilize this metal host[1].
Testicular seminoma is the most commonly occurring solid tumor type in
males[2, 3]. Furthermore, the standard first-line cisplatin-based
chemotherapy regimens are the most common therapy for testicular
seminoma in clinical practice[4-6]. However, platinum-based drugs lead
to severe side effects during the course of treatment, which has motivated
chemists to find new metal-based anticancer drugs[7]. Cisplatin has been
reported to possess anticancer activity by inducing apoptosis in cancer
cells[8, 9]. Therefore, the design and development of novel drugs with the
ability to trigger apoptosis is now considered the most promising strategy
for the discovery of new anticancer drugs for use in the treatment of
testicular seminoma[10-12].
Quinoline and its derivatives have attracted the attention of both
synthetic and biological chemists on account of the diversity of their
chemical and pharmacological properties[13, 14]. The suite of quinoline
derivatives possesses a wide range of biological activities, such as
antibacterial properties, anti-inflammatory functions, anticancer activities,
etc.[15-20] The 5,8-quinolinedione moiety was shown to exhibit
2

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cytotoxic activity against cancer cell lines such as melanoma and breast
cancer cells. Thus, the anticancer activities of 5,8-quinolinedione have
attracted significant attention in recent years[21]. A series of
quinoline-derived transition metal complexes, such as quilamine-iron
chelators, clioquinol copper(II) and zinc(II) complexes, copper(II) and
iron(II) complexes and ruthenium(II) complexes, were shown to possess
good anticancer ability[22-26]. The anticancer mechanism of the
quinoline derivatives is possibly due to their action as a
phosphoinositide-3 kinase(PI3K) / protein kinase B (Akt) inhibitor,
mammalian target of rapamycin(mTOR) inhibitor, vascular endothelial
growth factor receptor 2(VEGFR-2) inhibitor, cellular-mesenchymal to
epithelial transition factor(c-Met) inhibitor, etc.[27-29]
The PI3K/Akt pathway is one of the crucial signaling cascades in
humans. This pathway participates in many physiologic processes, such
as cell survival, proliferation and apoptosis[30, 31]. Several studies have
reported the abnormal activation of this signaling pathway relative to the
formation of seminoma[32, 33]. However, few studies have explored the
application of platinum-based agents with quinoline derivatives in the
treatment of testicular seminomas.
Therefore, we synthesized a new platinum(II) complex,
[Pt(ClClQ)(DMSO)Cl] (1), utilizing 6,7-dichloro-5,8-quinolinedione
(ClClQ) as a ligand. The biological activity and action mechanism of
complex 1, such as its cytotoxicity, cell cycle and cell apoptosis, were
studied.
2
. Results and discussion
2
.1. Synthesis
5
-Nitroso-8-hydroxyquinoline
hydrochloride
and
5
-amino-8-hydroxyquinoline sulfate were synthesized according to
reported procedures[34]. In addition, the synthesis procedures of
[Pt(ClClQ)(DMSO)Cl] (1) are shown in Scheme 1. Complex 1 was
3

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obtained by the reaction of the 6,7-dichloro-5,8-quinolinedione (ClClQ)
ligand and cis-Pt(DMSO) Cl in a 1:1 ratio in CH OH and acetone
2
2
3
(
v:v=20 mL:1 mL) and in CH OH and DMSO (v:v=20 mL:1 mL) under
3
refluxing (Scheme 1). The 6,7-dichloro-5,8-quinolinedione ligand and
1
13
Pt(II) complex 1 were characterized by IR, H and C NMR spectrometry,
electrospray ionization mass spectrometry(ESI-MS), single-crystal X-ray
diffraction analyses and elemental analyses (Figs. S1−S6).
Scheme 1. Synthetic routes for the preparation of 6,7-dichloro-5,8-quinolinedione and complex 1.
Reagents and solvents are the following: (a) NaNO (30 g), concentrated HCl (37.0 mL), H O
2
2
(
150.0 mL), 4 °C, 1 h; (b) 5 M NaOH solution (269.0 mL), H O (160.0 mL), Na S O (95.0 g),
2 2 2 4
8
0 °C, 2 h; 6.0 M H SO (250 mL), 2 h; (c) KCIO (5.5 g), concentrated HCl (69.0 mL), 60 °C, 30
2 4 3
min; (d) CH OH and acetone (v:v= 20 mL:1 mL), refluxing, 24 h.
3
2
.2. Crystal Structures
The molecular structure of complex 1 is illustrated in Fig. 1. The
detailed crystallographic data and structural refinement parameters are
summarized in Table 1. Selected bond lengths and bond angles are listed
in Tables 2−3. From Fig. 1, it can be seen that in each case, the Pt(II)
∧
center is four-coordinated by the chelating ClClQ ligand (N O-ClClQ)
via the O atom and heterocyclic nitrogen (N) atom, one DMSO ligand via
the S atom, and one Cl atom, to form a square-planar geometry. The bite
angles of the chelate rings (N(1)–Pt(1)–Cl(1), N(1)–Pt(1)–S(1) and
N(1)–Pt(1)–O(1)) were 170.20(18), 100.66(18) and 80.7(2)°, respectively.
The bond lengths of Pt–O (2.057(5)Å) and Pt–N (2.041(6)Å) were
significantly shorter than those of Pt–Cl (2.286(2)Å) and Pt–S
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(2.2120(18)Å), which fell within the normal range.
Figure 1. ORTEP view of the molecular structures and atomic labeling of complex 1
Table 1. Crystal data and structure refinement details for complex 1.
Empirical formula
C H Cl NO PtS
11 9 3 3
Formula weight
536.69
296.15
Temperature/K
Crystal system
monoclinic
Space group
P2 /c
1
a/Å
b/Å
c/Å
α/°
β/°
11.9240(2)
13.4714(2)
8.8326(2)
90
103.974(2)
90
γ/°
3
Volume/Å
1376.82(5)
4
Z
3
ρ g/cm
calc
2.589
-
1
μ/mm
10.928
1004.0
F(000)
3
Crystal size/mm
Radiation
0.22 × 0.2 × 0.18
MoKα (λ = 0.71073)
2
Θ range for data collection/°7 to 52.728
5

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Index ranges
-14 ≤ h ≤ 14, -16 ≤ k ≤ 15, -11 ≤ l ≤ 10
Reflections collected
9140
Independent reflections
Data/restraints/parameters
2807 [R = 0.0268, R
= 0.0298]
sigma
int
2807/0/183
1.053
2
Goodness-of-fit on F
Final R indexes [I>=2σ (I)] R = 0.0324, wR = 0.0835
1
2
Final R indexes [all data]
R = 0.0395, wR = 0.0881
1 2
-3
Largest diff. peak/hole / e Å 1.59/-1.92
a
|; b wR
2
2)2/Σw(F
2 2 ½
R
1
= Σ ||F
o
| – |F
c
||/Σ|F
o
2
= [Σw(F
o
– F
c
o
) ] .
Table 2. Selected bond lengths (Å) for complex 1.
AtomAtom Length/Å AtomAtom Length/Å
Pt01 S1
Pt01 Cl1
Pt01 O2
Pt01 N1
S1 O1
S1 C2
S1 C1
Cl2 C9
O2 C10
N1 C11
N1 C3
2.2120(18) Cl3 C8
2.286(2) C11 C10
2.057(5) C11 C6
2.041(6) C10 C9
1.465(6) O3 C7
1.775(8) C3 C4
1.775(8) C9 C8
1.728(8) C6 C7
1.299(9) C6 C5
1.358(9) C8 C7
1.345(10) C4 C5
1.234(9)
1.478(10)
1.382(10)
1.374(11)
1.196(10)
1.376(12)
1.441(11)
1.487(11)
1.371(12)
1.500(12)
1.390(12)
Table 3. Selected bond angles (°) for complex 1.
Angle/˚ AtomAtomAtom
AtomAtomAtom
S1 Pt01 Cl1
O2 Pt01 S1
O2 Pt01 Cl1
Angle/˚
117.3(6)
89.13(7) O2 C10 C11
178.35(15) O2 C10 C9
89.54(15) C9 C10 C11
123.8(7)
118.9(7)
6

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N1 Pt01 S1
100.66(18) N1 C3 C4
170.20(18) C10 C9 Cl2
80.7(2) C10 C9 C8
117.0(3) C8 C9 Cl2
107.9(4) C11 C6 C7
107.5(4) C5 C6 C11
110.1(3) C5 C6 C7
103.3(4) Cl3 C8 C9
110.3(3) Cl3 C8 C7
113.7(5) C9 C8 C7
113.1(5) O3 C7 C6
128.9(5) O3 C7 C8
118.0(6) C6 C7 C8
115.2(6) C3 C4 C5
122.2(7) C6 C5 C4
122.7(7)
122.9(8)
119.8(6)
122.1(7)
118.1(6)
119.3(7)
118.8(7)
121.9(7)
123.1(8)
117.6(7)
119.3(6)
121.3(8)
121.6(7)
117.1(6)
118.2(8)
119.9(7)
N1 Pt01 Cl1
N1 Pt01 O2
O1 S1 Pt01
O1 S1 C2
O1 S1 C1
C2 S1 Pt01
C2 S1 C1
C1 S1 Pt01
C10 O2 Pt01
C11 N1 Pt01
C3 N1 Pt01
C3 N1 C11
N1 C11 C10
N1 C11 C6
C6 C11 C10
2
.3. Complex 1 modulated the viability and invasive ability of testicular
seminoma cells
The cytotoxicity of complex 1 against the testicular seminoma TCam-2
cells
and
SEM-1
cells
was
tested
by
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide(MTT)
assays. The concentration of DMSO is approximately 0.1% after
calculation, thus 0.1% concentration of DMSO was used as a vehicle
control. As demonstrated in Fig. 2, after incubation with different
concentrations of complex 1 for 0, 24, 48, and 72 h, both cell lines
presented a time-dependent and a dose-dependent inhibition of cell
viability. The in vitro antitumor activity of complex 1 was further
quantified by determining its corresponding half maximal inhibitory
concentration(IC50) levels. The effect of 0.1% concentration of DMSO
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on cells was negligible. As shown in Table 4, the IC50 values for
complex 1 against TCam-2 cells and SEM-1 cells were 9.53 ± 0.43 μM
and 8.89 ± 0.47 μM, respectively, which exhibited higher cytotoxicity
against cisplatin. The impact of complex 1 on tumor cell invasion was
tested by cell invasion assays at 48 h. Compared with untreated cells, the
viability and invasive ability of the tumor cells were significantly
weakened after incubation with complex 1 in both testicular seminoma
cell lines (Fig. 2 and Fig. 3).
Table 4. IC50 (μM) values of complex 1 on testicular seminoma cells for 48 h
IC50 (μM)
Compounds
TCam-2 cells
9.53 ± 0.43
SEM-1 cells
8.89 ± 0.47
Complex 1
cisplatin
14.73± 0.37
12.38± 0.41
8

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Figure 2. The cytotoxicity of complex 1 against testicular seminoma TCam-2 cells and SEM-1 cells. (A and C)
Viability of TCam-2 cells after incubation with different concentrations of complex 1 and cisplatin for 0, 24, 48,
and 72 h. (B and D) Viability of SEM-1 cells after incubation with different concentrations of complex 1 and
cisplatin for 0, 24, 48, and 72 h. (E and F) Viability of TCam-2 cells and SEM-1 cells after incubation with 0.1%
concentration of DMSO for 0, 24, 48, and 72 h.
9

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Figure 3. The invasive ability of the testicular seminoma TCam-2 cells and SEM-1 cells after incubation with
complex 1 (1 μM) for 24 h. Control group means no drug treatment, complex (+) group means treatment with
complex 1. *p < 0.05 compared to negative control values.
2
.4. Complex 1 regulates the cell cycle transition of seminoma cells
Complex 1 was found to reduce the cell proliferation results based on
the slower progression of cells through the cell cycle. To evaluate these
mechanisms, we first investigated the cell cycle distribution changes
under different treatments. Glycogen synthase kinase-3β(GSK3β) is
involved in the regulation of cellular differentiation, proliferation and
apoptosis of cancer cells by certain signaling pathways[35, 36]. Herein,
through a series of experiments, we utilized the GSK3β inhibitor, LiCl, as
a reference drug to clarify whether GSK3β was involved in the antitumor
mechanism of complex 1[37]. As shown in Fig. 4, the cytometric profiles
of the TCam-2 cells and SEM-1 cells treated with Complex 1 for 48 h
indicated that the respective S phase populations were 16.55 % and
1
7.22%. Compared with the untreated cells, their respective S phase
populations were 13.56% and 11.91% lower than that of the untreated
cells, respectively. The data showed that complex 1 induces cell cycle
arrest in G0/G1 phase in both cell lines with a lower proliferation index,
while the GSK3β specific inhibitor accelerates cell cycle progression by
promoting the transition from G0/G1 phase to S phase. These results
suggested that complex 1 significantly arrested the cell cycle in G0/G1
phase in the seminomas. In addition, GSK3β, which is a downstream
target of the PI3K/Akt signaling pathway, may be involved in the
antitumor mechanism of complex 1.
1
0

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Figure 4. Complex 1 regulates the cell cycle transition of seminoma cells. (A) Cell cycle distribution in seminoma
TCam-2 cells and SEM-1 cells following complex 1 (10 μM) treatment for 48 h was analyzed by flow cytometry.
(B) Quantitative summary of the cell cycle distribution in different groups.
2
.5. Complex 1 promotes apoptosis of seminoma cells
Cell cycle arrest and cell apoptosis induction were considered to be
closely correlated in tumor cells, and thus, we next chose to examine cell
apoptosis in the testicular seminoma cells as induced by complex 1 by
flow cytometry. As shown in Fig. 5, compared with that in the control
group, complex 1 significantly induced apoptosis in both testicular
seminoma cell lines. The significant increase in the percentages of
apoptotic cells and early apoptotic cells strongly suggested that complex
1
could effectively induce apoptosis in seminoma cells. Interestingly,
LiCl was able to decrease the ability of complex 1 to induce cell
apoptosis.
Figure 5. The effect of complex 1 on early cell apoptosis.ꢀ(A) Cells were treated with complex 1 (1 μM) followed
by staining with Annexin V-fluorescein isothiocyanate and propidium iodide and then were analyzed by flow
cytometry. (B) Quantitative summary of early apoptosis rate in different groups. Complex 1 was able to
significantly induce apoptosis in both testicular seminoma cell lines. LiCl decreased the apoptosis rate after cells
were treated with complex 1. Compared as indicated, **p < 0.01.
2
.6. Complex 1 exhibits antitumor activity through the PI3K/Akt/GSK3β
signaling pathway in seminoma cells
Our previous study indicates that the development and migration of
seminoma cells via regulation of the PI3K/Akt signaling pathway[38, 39].
1
1

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Akt is a crucial component in PI3K signaling and has the ability to
regulate downstream targets, such as mTOR and GSK3β, indicating that
Akt is involved in many important physiologic processes[40]. Therefore,
based on the previous results, we employed Western blotting(WB) to
explore whether PI3K/Akt/GSK3β signaling was involved in the complex
1
antitumor mechanism with regard to testicular seminoma. GSK-3β
played a crucial role in negative expression of cyclin D1 via
phosphorylation of cyclin D1, and phosphorylation of GSK-3β was the
inactivated form of GSK-3β. However, LiCl could promote the
phosphorylation of GSK-3β, which has positive modulation on
expression of cyclin D1. As shown in Fig. 6, we found that the expression
levels of phosphorylated PI3K, Akt and GSK-3β in TCam-2 cells and
SEM-1 cells were decreased after complex 1 treatment for 48 h, while
there were not significant changes in PI3K or AKT. As expected, the
expression levels of cyclin D1 were inhibited in two seminoma cells. It
has previously been reported that cyclin D1 is an up-regulator to cell
cycle progression and drives the G1/S phase transition. Compared with
merely complex 1 treatment groups, the expression levels of
phosphorylated GSK-3β and cyclin D1 were significantly increased in
complex 1 plus LiCl treatment for 48 h, while the expression levels of
phosphorylated PI3K and Akt just had a mild increase. These results
suggested that the PI3K/Akt/GSK3β signaling pathway might be
involved in the antitumor mechanism of complex 1 in seminoma cells.
2
.7. Mitochondria-mediated apoptotic pathway is involved in complex 1
antitumor mechanism
The mitochondria-associated apoptotic pathway is one of the major
apoptosis pathways and participates in the cellular response of tumors to
many
anticancer
drugs[41].
To
determine
whether
the
mitochondria-associated apoptotic pathway is involved in the antitumor
activity of complex 1 in seminoma cells, we performed an analysis of
pro-apoptotic proteins (Bcl2 associated death promoter(Bad), cytochrome
c)[42]. Moreover, it is also believed that the activation of caspase-9 and
1
2

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caspase-3 stimulates mitochondrial cell death signals; thus, we also
investigated the changes in the expression levels of these two proteins. In
Fig. 6, compared to the control cells, the WB results showed a significant
increase in Bad, cytochrome c, caspase-9 ratio (activated/total) and
caspase-3 ratio (activated/total) levels after complex 1 treatment in
TCam-2 cells and SEM-1 cells for 48 h. It has been reported that Akt
negatively regulated the expression of caspase-9 and caspase-3. However,
as shown in WB results, there were significant decrease in caspase-9 and
caspase-3, and no significant changes in Akt, which indicates that lower
expression of caspase-9 and caspase-3 was not relevant to the Akt. These
observations suggested that apoptosis, when exposed to complex 1, could
be attributed to the mitochondria-mediated apoptotic pathway in
seminoma cells.
Figure 6. Protein expression of PI3K, p-PI3K, Akt, p-Akt, GSK-3β, p-GSK-3β and cyclin D1 was measured by
protein gel blotting. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) served as the internal control. The
1
3

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expression levels of phosphorylated PI3K, Akt and GSK-3β and cyclin D1 in TCam-2 cells and SEM-1 cells
decreased after complex 1 treatment for 48 h. Moreover, the expression levels of Bad, cytochrome c,
active-caspase-9 and active-caspase-3 were significantly increased. *p < 0.05, **p < 0.01, ***p < 0.001.
3
. Conclusions
A new platinum complex, known as complex 1 and utilizing the
,7-dichloro-5,8-quinolinedione ligand, was synthesized and fully
6
characterized. This complex exhibited significant antitumor activity and
cytotoxicity against human seminoma cells. In detail, complex 1 could
weaken the viability and invasive ability of tumor cells. Moreover,
complex 1 induces testicular seminoma cell cycle arrest in the G0/G1
phase. The molecular mechanisms of the antitumor activity of complex 1
include its ability to induce tumor cell apoptosis through the PI3K/Akt
signaling pathway and the mitochondria-mediated apoptotic pathway.
This complex may serve as a potential drug in the treatment of testicular
germ cell tumors.
4
. Experimental Methods
4
.1. Synthesis
4
.1.1. Synthesis of 6,7-dichloro-5,8-quinolinedione
5
-Nitroso-8-hydroxyquinoline hydrochloride and
5
-amino-8-hydroxyquinoline sulfate were synthesized according to the
reported procedures. Consequently, KClO (5.5 g) was added to a mixture
3
of 5-amino-8-hydroxyquinoline sulfate (9.0 g, 0.35 mol) in concentrated
HCl (69.0 mL) at 60°C for 30 min. The mixture was poured into ice
water (500 mL). The yellow 6,7-dichloro-5,8-quinolinedione product was
prepared in methanol/CHCl3 (v：v= 100:1) under solvothermal conditions
and suitable for structural characterization. Yield (7.5 g, 90.0%). ESI-MS
+
1
m/z: 360.2 [M+DMSO+CH CN+H] . H NMR (400 MHz, DMSO-d ) δ
3
6
9
.05 (dd, J = 4.7, 1.7 Hz, 1H), 8.46 (dd, J = 7.9, 1.7 Hz, 1H), 7.89 (dd, J =
1
4

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1
3
7
1
1
.9, 4.7 Hz, 1H). C NMR (100 MHz, DMSO-d ) δ 176.8, 174.7, 154.9,
6
47.5, 143.6, 142.1, 135.4, 129.0, 128.8. IR (KBr): 3367, 1693, 1680,
−1
573, 1559, 1288, 1198, 1140, 896, 826, 725, 695, 637, 478 cm .
Elemental analysis calculated (%) for C H Cl NO : C 47.40, H 1.33, N
9
3
2
2
6
.14; found: C 47.43, H 1.38, N 6.10.
4
.1.2 Synthesis of complex 1
Complex 1 was prepared by treating cis-Pt(DMSO) Cl (0.0422 g,
2
2
0
.1 mmol) with 6,7-dichloro-5,8-quinolinedione (0.0227 g, 0.1 mmol) in
CH OH and acetone (v:v=20 mL:1 mL) under refluxing. And the
3
resulting brown product suitable for structural characterization were
isolated, washed with ethanol and ether, and air-dried, respectively. Yield
(0.0459 g, 85.69%). IR (KBr): 3405, 3082, 3023, 2931, 1711, 1650, 1611,
1
539, 1465, 1367, 1301, 1170, 1136, 1024, 854, 691, 654, 632, 537, 452
−1
cm . 13C NMR (126 MHz, DMSO) δ 175.03, 173.05, 170.71, 158.44,
51.69, 138.30, 137.52, 128.11, 40.92. 1H NMR (500 MHz, DMSO-d6)
Ä 9.42 (dd, J = 6.1, 1.3 Hz, 1H), 8.59 ¨C 8.54 (m, 1H), 7.95 (m, J = 7.8,
.4 Hz, 1H), 2.54(s,6H). Elemental analysis calculated (%) for
C H Cl NO PtS: C 24.57, H 1.87, N 2.60; found: C 24.62, H 1.90, N
1
¦
5
11
10
3
3
2
.57.
4
.2 Materials and methods for X-ray Crystallography
Single-crystal X-ray crystallography of complex 1 was performed
using a SuperNova CCD diffractometer equipped with graphite
monochromated Mo Kα radiation (λ = 0.710 73 Å) at room temperature.
The structures were solved with direct methods and refined using
SHELX-97 programs. The nonhydrogen atoms were located using
successive difference Fourier synthesis. The final refinement was
performed using the full-matrix least-squares methods with anisotropic
thermal parameters for no-hydrogen atoms on F . The hydrogen atoms
2
were added theoretically to the atoms of concern. The parameters used
intensity collection, and the associated refinements are summarized in
1
5

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Tables 1−3 together with the crystal data. The crystallographic data of
complex 1 is available free of charge from The Cambridge
Crystallographic Data Centre: CCDC 1839014.
4
.3 Chemical compounds and antibodies
LiCl (Sigma-Aldrich, St. Louis, MO, USA) stock solution was
prepared in water and then was sterilized and added to culture medium
until a final concentration of 20 mM. Antibodies against PI3K (the p85
subunit, 1:8000), p-PI3K (Tyr607 of the p85 subunit, 1:8000), p-Akt
(Ser473, 1:4000), cytochrome c (1:200) and caspase-3 (1:200) were
obtained from Abcam Biotechnology (Cambridge, UK); antibodies
against caspase-9 (1:150) and GAPDH (1:8000) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA). Akt (1:600) and
antibodies were purchased from Signalway Antibody Biotechnology
(Baltimore, MD, USA), Cell Signaling Technology (Boston, MA, USA),
Acris Biotechnology (San Diego, CA, USA) and Gen-eTex (Irvine, CA,
USA), respectively.
4
.4 Cell culture and treatment
The tumor cell lines TCam-2 cells and SEM-1 were kindly gifted
from Dr. Riko Kitazawa (Department of Diagnostic Pathology, Ehime
University Hospital, Matsuyama, Japan). All cell lines were maintained in
complete medium (Roswell Park Memorial Institute Medium-1640 with
1
0% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100
μg/mL streptomycin) in a humidified 5% CO , 37°C incubator. A stock
2
solution of complex 1 dissolved in DMSO at a concentration of 2.0 mM
was prepared. The stock was diluted by phosphate buffer saline(PBS) to
the required concentration immediately before use.
4
.5 Cell viability assay
The cytotoxicity of complex 1 against the testicular seminoma cell
lines TCam-2 and SEM-1 was measured by MTT assay. Cells were
1
6

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seeded at a density of 104 cells/well in 96-well plates for 24 h. For
time-dependent and dose-dependent assays, the testicular seminoma
TCam-2 and SEM-1 cell lines were treated with different concentrations
(
0 μM, 0.1 μM, 0.5 μM, 1.0 μM, 5 μM, 10 μM, 20 μM, and 50 μM) of
complex 1 for 0 h, 24 h, 48 h, and 72 h. Then, the cells were incubated
with 20 ml of MTT solution (5 mg/ml, Sigma-Aldrich) for 4 h at 37°C. At
different time points (0, 24, 48, 72 h), MTT was added as described
above. Then, DMSO was added to each well to dissolve the purple-blue
formazan crystals by gentle agitation. For each well, the absorbance at
5
70 nm (A570) was estimated using a Microplate Reader, with DMSO
used as the blank.
4
8
.6 Cell invasion assay
A cell invasion assay was performed using Transwell chambers with
-μm pore inserts. Cell suspensions were seeded in the upper chamber,
and 500 μL of Dulbecco’s Modified Eagle’s Medium containing fetal
bovine serum was added to the lower chamber. The noninvading cells
were removed with a cotton-tipped swab after 24 h of incubation, and the
invading cells on the bottom surface of the membrane were stained with
0
.1% crystal violet. The invading cells were quantified by counting ten
random fields at ×200 magnification.
4
.7 Cell apoptosis analysis
Apoptosis was assessed with an Annexin V- fluorescein
isothiocyanate(FITC)/ propidium iodide (PI) Apoptosis Detection Kit
Beyotime Institute of Biotechnology, Shanghai, People’s Republic of China)
(
according to the manufacturer’s instructions. Briefly, after complex 1
treatment and incubation for 48 h, cells were collected, washed with PBS
and labeled with Annexin V and propidium iodide in the dark using an
Annexin V-FITC apoptosis detection kit (Beyotime Biotechnology,
Shanghai, China). Cell apoptosis was subsequently analyzed by a
FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA).
1
7

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4
.8 Cell cycle analysis by flow cytometry
Cell cycle analysis was performed using the PI single staining method
as described previously[43]. Briefly, two cell lines were incubated in 10%
fatal bovine serun(FBS)-supplemented culture medium with complex
1
(10 μM) for 48 h at 37°C and 5% CO . Then, cells were collected,
2
washed and centrifuged at 1000 rpm and finally were fixed with 75%
cold ethanol at 4°C overnight. After staining in
phosphatidylinositol(PtdIns) and ribonuclease(RNase) A at room
temperature for 30 min in the dark, the percentage of cells in each cell
cycle phase was determined with Cell Quest software and ModiFit
(Verity Software House, Topsham, USA) by using a flow cytometer (BD
Biosciences).
4
.9 Western blotting
Western blotting (WB) was implemented as described previously[43].
Briefly, the cells were lysed in lysis buffer containing protease inhibitors.
The protein concentration of lysates was determined by the bicinchoninic
acid assay (Beyotime Biotechnology). Equal amounts of protein (20 mg)
were separated by 10% sodium dodecylsulphate-polyacrylamide gel
electrophoresis(SDS-PAGE) electrophoresis and subsequently transferred
onto poly vinylidene fluoride(PVDF) membranes. Semi dry transfer was
done in the WB protocol. The membranes were blocked with 5% non-fat
dry milk in 0.2% Tween-20 in Tris-buffered saline (TBS-T) for 1 h at
room temperature and then were probed with primary antibodies.
Immunoreactivity was detected after incubation with a horseradish
peroxidase-conjugated secondary antibody by the enhanced
chemiluminescence method according to the instructions (Thermo
Scientific, Waltham, MA, USA). GAPDH was stained as a loading
control. The positive bands were analyzed by Gel-pro analyzer software,
and the value of integrated optical density (IOD) was measured.
4
.10 Data processing
1
8

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The data are expressed as the mean ± standard deviation. A statistical
analysis of the data was performed using SPSS 17.0 (SPSS Inc., Chicago,
IL, USA). Statistical significance was determined using an analysis of
variance followed by Student’s t-tests. A value of P<0.05 was considered
to indicate a statistically significant difference.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Funding
This research was funded by the National Nature Science Foundation
of China (81372181, J. Yang; 81571432, Y. Tang).
Abbreviations
PBS
phosphate buffer salineꢀ
MTT
IC50
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideꢀ
half maximal inhibitory concentrationꢀ
TCam-2 cells
SEM-1 cells
human testicular seminoma cell line
human testicular seminoma cell line
FITC
PI
fluorescein isothiocyanateꢀ
propidium iodideꢀ
Acknowledgments
The authors thank Mr. Xiaohua Li for technical support.
References
[
1] V. Milacic, D. Chen, L. Ronconi, K.R. Landis-Piwowar, D. Fregona, Q.P. Dou,
1
9

ACCEPTED MANUSCRIPT
Cancer Research, vol. 66, 2006, pp. 10478-10486.
2] N. Ismaili, S. Naciri, S. Afqir, N. Mellas, I. Bekkouch, S. Elmajjaoui, O. Masbah,
N. Othmani, A. Flechon, J. Droz, H. Errihani, Cases Journal, vol. 1, 2008, pp. 357.
3] V.M. Chia, Y. Li, S.M. Quraishi, B.I. Graubard, J.D. Figueroa, J.P. Weber, S.J.
[
[
Chanock, M.V. Rubertone, R.L. Erickson, K.A. McGlynn, International Journal of
Andrology, 2009.
[
4] M. Juliachs, C. Munoz, C.A. Moutinho, A. Vidal, E. Condom, M. Esteller, M.
Graupera, O. Casanovas, J.R. Germa, A. Villanueva, F. Vinals, Clin Cancer Res, vol.
0, 2014, pp. 658-667.
5] T. Kobayashi, T. Fujii, Y. Jo, K. Kinugawa, M. Fujisawa, The Journal of Urology,
vol. 171, 2004, pp. 1929-1933.
6] C.M. Squillante, D.J. Vaughn, Urologic Oncology: Seminars and Original
Investigations, vol. 33, 2015, pp. 363-369.
7] P. Giannatempo, T. Greco, L. Mariani, N. Nicolai, S. Tana, E. Fare, D. Raggi, L.
2
[
[
[
Piva, M. Catanzaro, D. Biasoni, T. Torelli, S. Stagni, B. Avuzzi, M. Maffezzini, G.
Landoni, F. De Braud, A.M. Gianni, G. Sonpavde, R. Salvioni, A. Necchi, Ann Oncol,
vol. 26, 2015, pp. 657-668.
[
8] Chatterjee.B. D., Mitra. A., De.G. S., Platinum Metals Review, vol. 50, 2006, pp.
-12.
2
[
[
9] Wong. E., Giandomenico.C. M., Chemical reviews, vol. 99, 1999, pp. 2451-2466.
10] P.U. Maheswari., M.v.d. Ster., S. Smulders., S. Barends., G.P.v. Wezel., C.
Massera., S. Roy., H.d. Dulk., P. Gamez., J. Reedijk, Inorganic Chemistry vol. 47,
008, pp. 3719-3727.
11] Y. Jin., J.A. Cowan., Journal of the American Chemical Society, vol. 127, 2005,
pp. 8408-8415.
12] F. Mancin, P. Scrimin, P. Tecilla, U. Tonellato, Chem Commun (Camb), 2005, pp.
540-2548.
13] Swenson.R. E., Sowin.T. J., Zhang.H. Q, The Journal of organic chemistry, vol.
7, 2002, pp. 9182-9185.
14] Madapa. S., Tusi. Z., Batra. S., Current Organic Chemistry, vol. 12, 2008, pp.
116-1183.
15] S. Kumar, S. Bawa, H. Gupta, Mini-Reviews in Medicinal Chemistry, vol. 9,
2
[
[
2
[
6
[
1
[
2
0

ACCEPTED MANUSCRIPT
2
009, pp. 1648-1654.
[
16] X. Mao, X. Li, R. Sprangers, X. Wang, A. Venugopal, T. Wood, Y. Zhang, D.A.
Kuntz, E. Coe, S. Trudel, D. Rose, R.A. Batey, L.E. Kay, A.D. Schimmer, Leukemia,
vol. 23, 2009, pp. 585-590.
[
17] Chen.Z. F., Gu.Y. Q., Song.X. Y., European journal of medicinal chemistry, 2013,
pp. 194-202.
18] Z.F. Chen, Y. Peng, Y.Q. Gu, Y.C. Liu, M. Liu, K.B. Huang, K. Hu, H. Liang, Eur
J Med Chem, vol. 62, 2013, pp. 51-58.
19] Q.P. Qin, Z.F. Chen, J.L. Qin, X.J. He, Y.L. Li, Y.C. Liu, K.B. Huang, H. Liang,
Eur J Med Chem, vol. 92, 2015, pp. 302-313.
20] X. Jia, F.F. Yang, J. Li, J.Y. Liu, J.P. Xue, J Med Chem, vol. 56, 2013, pp.
797-5805.
21] M. Kadela, M. Jastrzębska, E. Bębenek, E. Chrobak, M. Latocha, J. Kusz, M.
Książek, S. Boryczka, Molecules, vol. 21, 2016, pp. 156.
22] V. Corce, S. Renaud, I. Cannie, K. Julienne, S.G. Gouin, O. Loreal, F. Gaboriau,
D. Deniaud, Bioconjug Chem, vol. 25, 2014, pp. 320-334.
23] Santos.C. M., Cabrera. S., Ríos-Luci. C., Dalton Transactions, vol. 42, 2013, pp.
3343-13348
24] L. Yang, J. Zhang, C. Wang, X. Qin, Q. Yu, Y. Zhou, J. Liu, Metallomics, vol. 6,
014, pp. 518-531.
25] M. Gobec, J. Kljun, I. Sosic, I. Mlinaric-Rascan, M. Ursic, S. Gobec, I. Turel,
Dalton Trans, vol. 43, 2014, pp. 9045-9051.
26] Y.C. Liu, J.H. Wei, Z.F. Chen, M. Liu, Y.Q. Gu, K.B. Huang, Z.Q. Li, H. Liang,
Eur J Med Chem, vol. 69, 2013, pp. 554-563.
[
[
[
5
[
[
[
1
[
2
[
[
[
[
27] R. Roskoski, Jr., Crit Rev Oncol Hematol, vol. 62, 2007, pp. 179-213.
28] Y. Wang, J. Ai, Y. Wang, Y. Chen, L. Wang, G. Liu, M. Geng, A. Zhang, Journal
of Medicinal Chemistry, vol. 54, 2011, pp. 2127-2142.
29] S.D. Knight, N.D. Adams, J.L. Burgess, A.M. Chaudhari, M.G. Darcy, C.A.
[
Donatelli, J.I. Luengo, K.A. Newlander, C.A. Parrish, L.H. Ridgers, M.A. Sarpong,
S.J. Schmidt, G.S. Van Aller, J.D. Carson, M.A. Diamond, P.A. Elkins, C.M. Gardiner,
E. Garver, S.A. Gilbert, R.R. Gontarek, J.R. Jackson, K.L. Kershner, L. Luo, K. Raha,
C.S. Sherk, C.M. Sung, D. Sutton, P.J. Tummino, R.J. Wegrzyn, K.R. Auger, D.
2
1

ACCEPTED MANUSCRIPT
Dhanak, ACS Med Chem Lett, vol. 1, 2010, pp. 39-43.
[
[
[
30] L.C. Cantley, Science, vol. 296, 2002, pp. 1655-1657.
31] Boudewijn.M. T., Coffer.P. J., Nature, vol. 376, 1995, pp. 599.
32] Y. Nakai, N. Nonomura, D. Oka, M. Shiba, Y. Arai, M. Nakayama, H. Inoue, K.
Nishimura, K. Aozasa, Y. Mizutani, T. Miki, A. Okuyama, Biochem Biophys Res
Commun, vol. 337, 2005, pp. 289-296.
[
[
[
33] D.A. Altomare, J.R. Testa, Oncogene, vol. 24, 2005, pp. 7455-7464.
34] Ryu.C. K., Kim.H. J., Archives of Pharmacal Research, vol. 17, 1994, pp. 139.
35] M. Sharma, W.W. Chuang, Z. Sun, J Biol Chem, vol. 277, 2002, pp.
3
0935-30941.
36] S. Attarha, A. Roy, B. Westermark, E. Tchougounova, Cellular Signalling, vol. 37,
017, pp. 81-92.
37] Z.-Q. Fu, Y. Yang, J. Song, Q. Jiang, Z.-C. Liu, Q. Wang, L.-Q. Zhu, J.-Z. Wang,
Q. Tian, Journal of Alzheimer's Disease, vol. 21, 2010, pp. 1107-1117.
38] X. Jiang, D. Li, J. Yang, J. Wen, H. Chen, X. Xiao, Y. Dai, J. Yang, Y. Tang, The
Tohoku Journal of Experimental Medicine, vol. 225, 2011, pp. 311-318.
39] H. Chen, J. Sun, Y. He, Q. Zou, Q. Wu, Y. Tang, Tohoku J Exp Med, vol. 235,
015, pp. 103-109.
40] D. Sun, A. Sawada, M. Nakashima, T. Kobayashi, O. Ogawa, Y. Matsui, Urol
Oncol, vol. 33, 2015, pp. 111 e117-126.
41] L. Yang, T. Shi, F. Liu, C. Ren, Z. Wang, Y. Li, X. Tu, G. Yang, X. Cheng, PLoS
One, vol. 10, 2015, pp. e0120334.
[
2
[
[
[
2
[
[
[
[
42] D.W. Nicholson., N. A., Science, vol. 299, 2003, pp. 214-215.
43] Y. Wang, Y. Gan, Z. Tan, J. Zhou, R. Kitazawa, X. Jiang, Y. Tang, J. Yang, Onco
Targets Ther, vol. 9, 2016, pp. 409-420.
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Graphical Abstract
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Highlights:
Platinum(II) complex was easily prepared and utilized;
The complex exhibited considerable antitumor activity;
The complex could regulate the viability of seminoma cells;
The complex may induce seminoma cells apoptosis.
The complex may serve as a potential drug of testicular cell tumors.
2
4