Synthesis, characterization and ROS-mediated antitumor effects of palladium(II) complexes of curcuminoids

Article abstract of DOI:10.1016/j.ejmech.2017.12.027

Based on the synthesis of curcumin and its derivatives from aromatic aldehydes, a novel series of palladium(II) complexes with curcumin (or its derivatives) and 2,2′-bipyridine have been synthesized through a directed self-assembly approach that involves spontaneous deprotonation of the curcuminoid ligands in H₂O/acetone solution. These complexes have been characterized by ¹H (¹³C) NMR, HRMS and elemental analysis. Crystal structure of 3h has been determined by X-ray diffraction analysis. Their cytotoxicity was tested by MTT. The preliminary results showed that complexes 3d, 3f, 3h have significant inhibition on proliferation of three carcinoma cells such as MCF-7, HeLa and A549 cells, which were more active than cisplatin. Further mechanistic studies indicated that the tested complex 3h arrested the cell cycle in the S phase and can disrupted mitochondrial membrane potential and induced tumor cell apoptosis through reactive oxygen species (ROS)-dependent pathway.

Full text of DOI:10.1016/j.ejmech.2017.12.027

Accepted Manuscript
Synthesis, characterization and ROS-mediated antitumor effects of palladium(II)
complexes of curcuminoids
Yanci Li, Zhenyu Gu, Can Zhang, Shenghui Li, Liang Zhang, Guoqiang Zhou,
Shuxiang Wang, Jinchao Zhang
PII:
S0223-5234(17)31033-4
10.1016/j.ejmech.2017.12.027
EJMECH 10005
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 July 2017
Revised Date: 7 December 2017
Accepted Date: 7 December 2017
Please cite this article as: Y. Li, Z. Gu, C. Zhang, S. Li, L. Zhang, G. Zhou, S. Wang, J. Zhang,
Synthesis, characterization and ROS-mediated antitumor effects of palladium(II) complexes of
curcuminoids, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.027.
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graphical abstract：
New palladium(II) complexes of curcuminoids have been synthesized and characterized
spectroscopically and by X-ray analysis. Some of the obtained complexes exhibit significant
antiproliferative activity by ROS-mediated apoptosis.

ACCEPTED MANUSCRIPT
Synthesis, characterization and ROS-mediated antitumor effects
of palladium(II) complexes of curcuminoids
Yanci Li, Zhenyu Gu, Can Zhang, Shenghui Li*, Liang Zhang, Guoqiang Zhou*, Shuxiang
Wang, Jinchao Zhang
Key Laboratory of Chemical Biology of Hebei Province; Key Laboratory of Medicinal Chemistry
and Molecular Diagnosis, Ministry of Education; College of Chemistry & Environmental
Science, Hebei University, Baoding 071002, China
Abstract
Based on the synthesis of curcumin and its derivatives from aromatic aldehydes, a novel series of
palladium(II) complexes with curcumin (or its derivatives) and 2,2’-bipyridine have been
synthesized through a directed self-assembly approach that involves spontaneous deprotonation of
the curcuminoid ligands in H2O/acetone solution. These complexes have been characterized by 1H
(13C) NMR, HRMS and elemental analysis. Crystal structure of 3h has been determined by X-ray
diffraction analysis. Their cytotoxicity was tested by MTT. The preliminary results showed that
complexes 3d, 3f, 3h have significant inhibition on proliferation of three carcinoma cells such as
MCF-7, HeLa and A549 cells, which were more active than cisplatin. Further mechanistic studies
indicated that the tested complex 3h arrested the cell cycle in the S phase and can disrupted
mitochondrial membrane potential and induced tumor cell apoptosis through reactive oxygen
species (ROS)-dependent pathway.
Keywords: Palladium(II) complexes; Curcuminoids, Bisdehydroxycurcumin, Reactive oxygen
species (ROS); Apoptosis; Antitumor.
*Corresponding authors. Tel./fax: +86 0312 5079005.
E-mail address: lish@hbu.cn (S. Li), zhougq1982@163.com (G. Zhou)
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1. Introduciton
Cancer remains one of the leading causes of death worldwide, and it is estimated that more
than 10 million new cancer cases are diagnosed every year which represents a real crisis for public
health in the world [1]. Many efforts in anticancer agent research have been devoted to developing
molecular-targeted drugs by exploiting the genetic differences between normal cells and cancer
cells. By the use of such molecular-targeted drugs, survival rates in some cases have increased [2].
However, due to the acquired drug resistance and the genomic instability of cancer cells, there still
remains the need for improved therapeutic activity and selectivity.
Compared with their normal counterparts, cancer cells often exhibit increased levels of ROS
and an altered redox status to maintain their malignant phenotypes. Although the increased ROS
stress can promote cell proliferation, angiogenesis, and metastasis, it may also makes the cancer
cells more susceptible for ROS-induced toxicity. Thus, exploiting increased ROS levels and
altered redox status in cancer cells has become a novel target strategy to improve therapeutic
activity and selectivity in the development of anticancer agents [3-8].
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), the major
component of three curcuminoids obtained from the rhizome of Curcuma longa L, is an important
prooxidant cancer chemopreventive agent [9], which has attracted the special attention of
scientists due to its wide spectrum of pharmacological activities such as antioxidant,
anti-inflammatory, antimicrobial and anticancer [10,11]. In addition, curcumin is nontoxic to
humans up to a dose of 10 g/day, with almost no adverse effects. It is considered to be a potential
chemopreventive agent and has been used in clinical trials. Unfortunately, the pharmacology
application of curcumin is severely limited by its extremely low bioavailability due to the low
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water solubility, poor absorption, rapid metabolism and systemic elimination. Although some
approaches, such as the use of adjuvants, liposomal curcumin, and the preparation of curcumin
derivatives and analogues have been applied to improve the efficacy of curcumin-based treatments,
there is still an urgent need for new strategy to broad the application of curcumin [12,13].
It is believed that the instability, partly due to the presence of theβ-diketone moiety in its
structure, attributes to the low bioavailability of curcumin. Coordination of curcumin in
deprotonated form with a metal ion often makes curcumin hydrolytically stable. By virtue of this
property, curcumin and its analogues in their deprotonated form have been used as supporting
ligands for palladium (II) [14-19], platinum(II) [20-22], copper(II) [23,24], zinc(II) [25,26],
oxovanadium (IV) [27-31], cobalt(III) [32], organometallic ruthenium(II) [33-36], and
organometallic rhodium(III)/iridium(III) [37] anticancer agents. Importantly, many of these metal
curcumin complexes exhibit very diverse and highly promising health effects. The last decade has
witnessed their exciting and promising medicinal applications such as selective
(photo)cytotoxicity and anticancer activity, anti-Alzheimer’s disease activity, and
antioxidative/neuroprotective effects [38]. Already it has become clear that the potential medicinal
applications of metal complexes of curcumin are as diverse as those of curcumin itself [39,40].
In addition to photocytotoxic curcumin complexes [21-23,27-31,41], designing curcumin
metal complexes with anticancer activities by a prooxidant strategy is relatively scarce to our
knowledge, and little information is available concerning how to design them based on this
strategy. Recently, a heteroleptic palladium (II) complex, with the metal center coordinated to pure
curcumin and the 4,4′-dinonyl-2,2′-bipyridine was reported by valentini and co-workers, which
could induce both cell growth inhibition and apoptosis of human prostate cancer cells. The
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apoptosis was associated with mitochondrial membrane depolarisation and modulation of Bcl-2
protein levels. The generated ROS was attributed to its ability to induce JNK phosphorylation,
GSTp1 (glutathione S-transferase P1) down-regulation and GSH depletion [15]. However, this
heteroleptic palladium (II) complex displayed, in prostate cancer cells, lower oxidant properties
than pure curcumin.
It has been suggested that the presence of the phenolic, β-diketone, as well as the methoxy
groups contribute to the free-radical-scavenging activity of curcumin [10]. To obtain enhanced
prooxidative ability, a logical approach would be the blocking (or removing) of the free phenolic
–OH groups at the 4-positions of the phenyl rings and binding β-diketone group to a metal ion at
the same time. This deduction, along with the reported antitumoral mechanism and oxidant
property of curcumin palladium complex structure [15], made us conduct further studies on the
cytotoxicity of palladium complexes of natural and synthetic curcuminoids.
To gain more insights into the structure–activity relationships (SAR) and cytotoxic
mechanisms, a series of heteroleptic palladium (II) complex of curcumin or synthetic
curcuminoids has been synthesized through a directed self-assembly approach that involves
spontaneous deprotonation of the curcuminoid ligands in H2O/acetone solution. Moreover,
antiproliferative activity, ROS production, induction of apoptosis, morphological observation of
nuclear change, cell cycle distribution, and mitochondrial membrane potentials assay were
investigated.
2. Results and discussion
2.1. Synthesis and Characterization
A series of curcumin and synthetic curcuminoids with changes in aryl substitution pattern
were prepared by a method reported earlier [42-44]. The precursor complex (bipy)Pd(ONO2)2
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(where bipy = 2, 2-bipyridine) was prepared according to literature procedures [45]. As shown in
Scheme 1, a facile combination of (bipy)Pd(ONO2)2 with curcuminoid ligands in a 1:1 molar ratio
in H2O and acetone affords the formation of heteroleptic palladium(II) complex in quantitative
yield. Complexes 3a-3h were obtained by adding 6-fold excess KPF6 to the above obtained
aqueous solution.
1
These complexes have been fully characterized by H NMR, 13C NMR, ESI-MS and
elemental analysis, confirming the hypothetic stoichiometry (chemical structure). The presence of
hexafluorophosphate as the counterion for 3a-3h was verified by an ESI-MS m/z peak
corresponding to the characteristic of the molecular cation [M-PF6]+. In the mass spectra of 3a-3h,
only the molecular ion arising from the cationic palladium complex was observed. Elemental
analyses of 3a-3h are in agreement with the anticipated molecular formulas. The 1H NMR spectra
of 3a-3h clearly show that a 1:1 (bipy)Pd to a curcuminoid anion complex formed and
1
deprotonation from curcuminoid ligands has occurred spontaneously. The patterns of H and 13C
NMR spectra are consistent with the presence of the unique mononuclear palladium (II)
curcuminoid complex structure in solution.
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2a, 3a: R1 = H, R2 = OCH3, R3 = OH; 2b, 3b: R1 = H, R2 = R3 = OCH3; 2c, 3c: R1= R2 = H, R3 = OH;
2d, 3d: R1 = R2 = H, R3 = OCH3; 2e, 3e: R1 = R3 = H, R2 = OH; 2f, 3f: R1 = R3 = H, R2 = OCH3;
2g, 3g: R1 = OH, R2 = R3 = H; 2h, 3h: R1 = OCH3, R2 = R3 = H
Scheme 1. Synthesis routes for complexes 3a-3h.
2.2. Crystal structure analysis by X-ray diffraction
Complex 3h was structurally characterized by single crystal X-ray diffraction technique.
Single crystal sample suitable for X-ray analysis was grown in 1,4-dioxane/acetonitrile. The
structure and atom numbering scheme of complex are shown in Fig. 1. The selected
crystallographic information of the complex 3h are summarized in Table 1 and Table 2. Complex
3h crystallizes in the orthorhombic space group Pbca and shows a discrete mononuclear species
with the Pd(II) center in a four-coordinate PdO2N2 coordination geometry. Both the bipyridyl and
the bisdehydroxycurcumin analog-2 (BDHC-A2) ligands show a bidentate mode of binding. The
Pd–O and Pd–N distances are in the ranges of 1.975(4) to 1.978(4) A˚ and 1.989(5) to 1.994(5) A˚,
respectively (Table 2). The angle between planar N(1)-Pd(1)-N(2) and planar O(1)-Pd(1)-O(2) is
0.626(167)°, which indicates that the complex adopts approximately square planar geometry.
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Fig. 1. Molecular structure and atom-labeling scheme for complex 3h.
Table 1
Crystallographic data for complex 3h
Formula
C35H35F6N2O6PPd
Fw
T(K)
831.02
298(2)
Cryst syst
Space group
a(Å)
Orthorhombic
Pbca
13.8754(13)
18.5046(17)
28.473(3)
1312.3(9)
8
b(Å)
c(Å)
V(nm3)
Z
Dc(Mg m-3)
1.510
F(000)
3376
Cryst dimens(mm)
θ Range(deg)
0.45 x 0.37 x 0.23
2.31 -25.02
-16<h<16
-22<k<21
-25<l<33
6454 / 462
1.072
hkl ranges
Data/parameters
Goodness-of-fit on F2
R1 = 0.0536
wR2= 0.1230
Final R indices[I>2σ(I)]
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Table 2
Selected bond lengths (Å) and angles (°) for complex 3h.
Length/Å or Angle/°
Pd(1)-O(1)
Pd(1)-O(2)
Pd(1)-N(1)
Pd(1)-N(2)
N(1)-C(22)
N(1)-C(26)
N(2)-C(31)
O(1)-Pd(1)-O(2)
O(1)-Pd(1)-N(1)
O(2)-Pd(1)-N(1)
O(1)-Pd(1)-N(2)
O(2)-Pd(1)-N(2)
N(1)-Pd(1)-N(2)
C(31)-N(2)-Pd(1)
94.56(15)
173.6(2)
1.975(4)
1.978(4)
1.989(5)
1.994(5)
1.332(7)
1.351(7)
1.326(7)
91.85(19)
91.8(2)
173.64(19)
81.8(2)
126.0(4)
114.1(4)
N(2)-C(27)
O(1)-C(3)
O(2)-C(5)
C(27)-N(2)-Pd(1)
C(3)-O(1)-Pd(1)
C(5)-O(2)-Pd(1)
1.364(7)
1.265(6)
1.285(6)
123.6(4)
122.7(3)
2.3. Cytotoxicity in vitro assay by MTT method
The in vitro cytotoxicity of the ligands 2a-2h, as well as the as-prepared ionic complexes
3a-3h, against a panel of human carcinoma cell lines including MCF-7 (human mammary cancer
cells), HeLa (human cervical carcinoma cells), and A549 (human lung cancer cells) were
evaluated by MTT assay (MTT = 3-(4,5-dimetyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide). The IC50 values were listed in Table 3 and Table 4 and cisplatin was used as a positive
control. As reported in the Table 3 and Table 4, we observed that the cytotoxicity of curcumin
palladium complex (3a) was comparable with that of curcumin itself (2a) against these human
carcinoma cells. It is worth noting that the activity of the palladium complex of curcumin and
curcumin derivatives depended strongly on the substituents. Generally, the hydroxyl groups on the
benzene rings have no special impact on cytotoxicity, while the methoxy substituent gave
significant rise to activity. For example, the three palladium (II) complexes (3d, 3f and 3h) with
bisdehydroxycurcumin serial ligand exhibited better activity than complexes (3c, 3e and 3g) with
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bisdemethoxycurcumin serial ligand against all of these three human carcinoma cells. It seems that
there may be a potential correlation between the activity of these complexes and the activity of
curcumin. It’s widely accepted that hydroxyl groups in curcumin are responsible for its
antioxidant activity, while the methoxy groups are essential for its antiinflammatory and
antiproliferative activity [46-48]. Interestingly, the palladium complex (3h) with the
bisdehydroxycurcumin analog-2 (BDHC-A2) ligand was the most potent anticarcinogen among
these bisdehydroxycurcumin palladium complexes. We speculated that this complex might
overcome the GSH detoxification because the а, β-unsaturated carbonyl group is hidden by not
only the metal fragment but also the methoxyl groups on the phenyl ring [15].
Table 3
IC50 Values of curcumin and curcuminoids (2a-2h) toward MCF-7, HeLa and A549 cell lines
IC50(µM)
Compounds
MCF-7
HeLa
A549
2a
2b
21.14 ± 2.68
18.52 ± 2.34
20.33 ± 2.47
24.01 ± 1.96
12.95 ± 1.88
28.92 ± 2.16
146.31 ± 10.95
19.74 ± 2.76
19.65 ± 1.11
27.30 ± 3.25
22.85 ± 2.76
25.81 ± 1.22
25.67 ± 2.57
30.94 ± 4.09
32.99 ± 5.24
84.97 ± 7.28
30.21 ± 3.62
16.72 ± 1.56
31.71 ± 3.81
29.90 ± 4.17
28.52 ± 3.08
27.45 ± 1.73
26.04 ± 3.42
30.31 ± 3.79
121.48 ± 13.06
25.41 ± 2.48
20.10 ± 2.42
2c
2d
2e
2f
2g
2h
Cisplatin
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Table 4
IC50 Values of complexes 3a-3h toward MCF-7, HeLa and A549 cell lines
IC50(µM)
Complexes
MCF-7
HeLa
A549
3a
3b
22.00 ± 3.12
26.50 ± 2.84
20.79 ± 1.88
3.86 ± 0.36
20.61 ± 2.55
3.69 ± 0.22
37.33 ± 5.16
3.32 ± 0.41
19.65 ± 1.11
25.43 ± 2.56
14.60 ± 1.62
22.75 ± 3.27
6.06 ± 0.91
23.50 ± 2.06
2.35 ± 0.37
36.79 ± 4.25
1.73 ± 0.29
16.72 ± 1.56
34.52 ± 3.73
25.88 ± 2.95
20.19 ± 2.03
8.27 ± 0.64
19.31 ± 1.38
6.15 ± 0.82
> 100
3c
3d
3e
3f
3g
3h
4.29 ± 0.53
20.10 ± 2.42
Cisplatin
2.4. Effect of complexes on ROS induction
It’s well known that ROS play an important role in cancer cell death and apoptosis, and a
curcumin palladium (II) complex comparable to those investigated in this study was already
shown to produce ROS [15]. Consequently, we determined to investigate whether ROS production
is underlying the cytotoxic activities of the investigated complexes. The effects on intracellular
ROS levels of these complexes were studied by 2′,7′-dichlorodihydrofluorescein diacetate
(DCFH-DA) assay [49]. The induction of ROS in HeLa cells exposed for 48 h to selected
complexes 3c, 3d, 3f, 3g and 3h at 5 µM and 10µM values using flow cytometry was determined
and compared this to the ROS level observed in H2O2 under similar conditions. As shown in Fig. 2
and Fig. 3, upon treatment of HeLa cells for 48h with complex 3d, 3f and 3h, a significant
concentration-dependent increase of ROS generation was observed. However, complex 3c and 3g
at the 10 µm value showed no evident influence on the generation of ROS compared with the
control. These observations indicate that complexes 3c and 3g with phenolic hydroxyl display, in
HeLa cells, lower oxidant properties than complexes 3d, 3f and 3h with methoxy group. These
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maybe due to the fact that the hydroxyl group of curcumin is partly responsible for its well-known
antioxidant property [10]. In addition, in order to further identify the correlation between ROS
production and the cytotoxicity of these complexes, cell viability of HeLa cells exposed to
complex 3h for 24, 36, and 48 hrs following pretreatment with the widely-used ROS scavenger
N-acetyl-L-cysteine (NAC) was evaluated [50-52]. As shown in Fig. 4, we found that the viability
of cells pretreated with NAc significantly improved in HeLa (85.3 ± 3.7% vs. 104.7 ± 6.6%, p <
0.05; 34.9 ± 0.67% vs. 80.5 ± 4.3%, p < 0.001; 28.8 ± 0.87% vs. 79.0 ± 5.6%, p < 0.001). The
cytotoxicity of, and ROS generation by, curcumin complexes were reduced by the addition of
NAC, suggesting a possible link between cytotoxicity and ROS, just as curcumin itself does [53].
Fig. 2 Oxidative activity of the selected complexes. (A) HeLa cells were incubated with 5 or 10 µm/L of
complexes 3g and 3h for 48 h and intracellular ROS levels were determined with DCFH-DA staining. (B) ROS
levels (relative to untreated cells) after exposing HeLa cells to the indicated concentrations of complexes 3g and
3h for 48 h. Differences versus untreated control cells were considered significant at ***p ≤ 0.001. Data
represents the mean ±SD from three independent experiments.
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Fig. 3 Oxidative activity of the selected complexes. (A) HeLa cells were incubated with 10 µm/L of complexes 3c,
3d and 3f for 48 h and intracellular ROS levels were determined with DCFH-DA staining. (B) ROS levels (relative
to untreated cells) after exposing HeLa cells to the indicated concentrations of complexes 3c, 3d and 3f for 48 h.
Differences versus untreated control cells were considered significant at ***p ≤ 0.001. Data represents the mean
±SD from three independent experiments.
Fig. 4 Complex 3h-induced cytotoxicity is rescued by the inhibition of ROS. HeLa cells were pre-incubated for 2
hr with 10 mM NAc prior to 24, 36, and 48 hr treatment with 10 µM complex 3h, and cell viability was assessed
by trypan blue exclusion. Values and error bars shown in the graphs represent mean and SD respectively (***p ≤
0.001 -*p ≤ 0.05, compared with controls).
2.5 Morphological observation of nuclear change and flow cytometric analysis of apoptosis
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To examine whether complex 3h-mediated ROS generation induced apoptosis,
morphological observation and flow cytometric analysis were conducted [54,55]. Morphological
observation of nuclear change was observed by Hoechst 33342 staining fluorescence microscopy.
HeLa cells were treated with or without complex 3h, as shown in Fig. S1A, the nuclei of untreated
control HeLa cells were stained in less bright blue and homogeneous color. By contrast, after
treatment with 10 µM complex 3h for 24 h, most cells displayed some typical apoptotic
morphological features, such as decrease in cells number, loss of cell volume (cell shrinkage),
chromatin condensation, nuclear fragmentation, and appearance of apoptotic bodies. The annexin
V/propidium iodide (PI) dual staining method was also used to further determine whether the
death of cancer cells incubated with complex 3h was induced by apoptosis. As shown in Fig. S1B,
a small percentage of untreated HeLa (8.16%) cells bound annexinV-FITC. In contrast, the
percentage of annexinV-FITC binding HeLa cells significantly inhanced in
a
concentration-dependent manner after treatment with 5 µM complex 3h (10.01%) and 10 µM
complex 3h (27.55%).
2.6. Effect of complex on the cell cycle
Cell cycle arrest is one of the predominant regulatory mechanisms of apoptosis [56], and it’s
well known that curcumin may assert its anti-tumor activity by altering the deregulated cell cycle
of cancer cells [11]. To explore the possible correlation between the cytotoxicity of 3h and its cell
cycle arrest, the effect on cell cycle progression was examined using propidiumiodide (PI) staining
by flow cytometry analysis in HeLa cells. As shown in Fig. 5, after treatment of Hela cells with
complex 3h at 5 µm and 10µm, the percentages of cells in G0/G1 (Control: 42.04%; 10 µm of
complex 3h: 15.36%) and G2/M (Control: 25.25%; 10 µm of complex 3h: 12.55%) decreased,
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while there was an obvious accumulation of cells in the S phase (Control: 32.71%; 10 µm of
complex 3h: 72.09%). These results indicated that complex 3h mainly arrested S phase cell cycle
in HeLa cells.
Fig. 5. Effects of complex 3h on cell cycle distribution in Hela cells. Cells were untreated or treated with complex
3h at 10 µm/L for 24 h, stained with propidium iodide (PI). Then cell cycle was analyzed by flow cytometry.
Experiments were performed in duplicates. Data show percentage of live cells in each phase of the cell cycle
(G0/G1, S, and G2/M).
2.7. Effect of complex on mitochondrial membrane potential
In the apoptotic process of cells, mitochondrion plays a fundamental role, and the level of
mitochondrial membrane potential (∆ψmit) is considered to be an indicator of apoptosis [57,58].
The well-known fluorescent dye rhodamine 123 (RH-123), which could selectively accumulate in
mitochondria dependent on the membrane potential, was used to evaluate the changes of the
mitochondrial membrane potential with flow cytometry and fluorescent imaging. It’s well
accepted that the potential-dependent uptake of RH-123 often results in extensive,
concentration-dependent, quenching of the dye fluorescence, due to the aggregation or stacking of
the dye in the mitochondria [59,60]. The changes of ∆ψmit in HeLa cells inducted by complex 3h
at different concentration (1 µM, 5 µM and 10 µM) were evaluated by measuring RH-123
fluorescence quenching. As shown in Fig. 6, expectedly, after exposure to complex 3h for 48 h,
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there was a significant and dose-dependent decrease in ∆Ψmit, indicated the dysfunction of
mitochondria membrane potential and the impaired mitochondrial integrity.
(A)
(B)
Fig. 6. The MMP of HeLa cells after incubation with complex 3h at 1 µM, 5 µM and 10 µM for 48 h. (A) the
images were observed with fluorescence microscope (Bar = 200 µm); (B) the MMP was measured by flow
cytometry. **p ≤ 0.05 compared with control group, ***p ≤ 0.001 compared with control group
3. Conclusions
In conclusion, eight new palladium(II) complexes containing curcuminoid ligands have been
synthesized and characterized, which demonstrated antitumor activity against MCF-7, HeLa, and
A549 tumor cells. Interestingly, the three palladium (II) complexes (3d, 3f and 3h) with
bisdehydroxycurcumin serial ligand possess higher antitumor activity than complexes (3a, 3c, 3e
and 3g) with curcumin or bisdemethoxycurcumin serial ligand, which are also more active than
cisplatin. The DCFDA assay revealed a potent ROS induction for complexes (3d, 3f and 3h) but
not the other complexes. Presence of NAC was able to reduce ROS generation and improve
significantly the survival rate of HeLa cells treated by complex 3h, indicating that the antitumor
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effect by complexes 3h is mediated mainly through oxidative stress. Mechanism studies show that
complex 3h may disrupt mitochondrial membrane potential, alter the deregulated cell cycle and
induce apoptosis of tumor cells. In conclusion, our findings on palladium(II) complexes of
bisdehydroxycurcumin as an inducer of ROS may be useful in designing novel therapeutic agent
for cancer treatment through prooxidant strategy.
4. Experimental section
4.1 Material
All reagents and chemicals purchased were of reagent grade and used without further
purification. Dulbecco's Modified minimal essential medium (DMEM) and fetal bovine serum
(FBS) were purchased from Gibco (Grand Island, NY, USA). MTT, trypsin, RH-123, DCFH-DA,
and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Elemental
analysis (C, H and N) was performed on an Elementar Vario EL III elemental analyzer. The 1H and
13C NMR spectra were obtained on a Bruker AVIII 600 spectrometer. The proton and carbon
chemical shifts (δ) are given with respect to tetramethylsilane (TMS). Melting points were
determined on a XT-4 microscopic melting-point spectrometer and are uncorrected.
High-resolution mass spectra (HRMS) were recorded on a Bruker Apex Ultra 7.0T FTMS mass
spectrometer.
4.2. Preparation of Curcuminoids (2a–2h)
Curcumin and its analogues (2a–2h) were synthesized by referring to the available methods
[42-44]. Generally, 2,4-pentanedione (1.0 g, 0.01 mol) and boron oxide (0.5 g, 0.007 mol) were
added to a solution of tributyl borate solution(3 mL) in a well dried three neck flask and stirred for
0.5 h at 90 ℃. White turbid solution will be found followed by cooling to 60 ℃ and different
substituted benzaldehyde (0.02 mol) and tributyl borate (2 mL) were added. Then n-butylamine
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(0.7 mL) in tributyl borate solution (1 mL) was added dropwise during 30 min. After further
stirring for 1.5 h at 100 ℃, the mixture was treated by HCl (0.4 M) and pH is adjusted to 3-4.
Then the aqueous layer extracted three times with EtOAc. The organic layers were washed with
saturated salt water and dried over Na2SO4. Organic solvent was evaporated under reduced
pressure and the product was purified by silica gel column chromatography using petroleum and
ethyl acetate as eluent.
4.2.1. 1,7-Bis-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (curcumin)(2a)
Orange solid, yield: 58%, mp: 180-181℃.1H NMR (600 MHz, CDCl3 ) δ (ppm): 7.59 (d, J =
15.6 Hz, 2H, -CH=CH), 7.12 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 2H, Ar-H), 7.05 (d, J = 1.8 Hz, 2H,
Ar-H), 6.93 (d, J = 8.4 Hz, 2H, Ar-H), 6.48 (d, J = 15.6 Hz, 2H, CH=CH-), 5.80 (s, 1H, = CH-C),
3.94 (s, 6H, -OCH3).
4.2.2. 1,7-Bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (dimethoxycurcumin) (2b)
Yellow solid, Yield: 53%, mp: 119-120℃.1H NMR (600 MHz, CDCl3) δ (ppm): 7.62 (d, J =
15.6 Hz, 2H, -CH=CH), 7.16 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 2H, Ar-H), 7.10 (d, J = 1.8 Hz, Ar-H),
6.90 (d, J = 8.4 Hz, 2H, Ar-H), 6.52 (d, J = 15.6 Hz, 2H, CH=CH-), 5.84 (s, 1H, =CH-C), 3.95 (s,
6H, -OCH3), 3.94 (s, 6H, -OCH3)
4.2.3.1,7-Bis(4-hydroxyphenyl)hepta-1,6-diene-3,5-dione (bisdemethoxycurcumin, BDMC) (2c)
Yellow solid, yield: 38%, mp: 147-148℃.1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.07 (s,
2H, Ar-OH), 7.58-7.54 (m, 6H, -CH=CH, Ar-H), 6.82 (d, J = 8.4 Hz, 2H, Ar-H), 6.70 (d, J = 15.6
Hz, 2H, CH=CH-), 6.04 (s, 1H, =CH-CO).
4.2.4. 1,7-Bis(4-methoxyphenyl)hepta-1,6-diene-3,5-dione (bisdehydroxycurcumin analog-1,
BDHC-A1) (2d)
Yellow solid, yield: 55%, mp: 147-148℃.1H NMR (600 MHz, CDCl3) δ (ppm): 7.64 (d, J =
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15.7 Hz, 2H, -CH=CH), 7.53 (d, J = 8.6 Hz, 4H, Ar-H), 6.93 (d, J = 8.6 Hz, 4H, Ar-H), 6.52 (d, J
= 15.7 Hz, 2H, CH=CH-), 5.80 (s, 1H, =CH-C), 3.87 (s, 6H, -OCH3).
4.2.5. 1,7-Bis(3-hydroxyphenyl)hepta-1,6-diene-3,5-dione (bisdemethoxycurcumin analog-1,
BDMC-A1) (2e)
Yellow solid, yield: 39%, mp: 186-187℃.1H NMR (600 MHz, DMSO-d6) δ (ppm): 9.66 (s,
2H, Ar-OH), 7.57 (d, J = 15.6 Hz, 2H, -CH=CH), 7.25 (t, J = 7.8 Hz, 2H, Ar-H), 7.15 (d, J = 7.2
Hz, 2H, Ar-H), 7.07 (s, 2H, Ar-H), 6.85 (d, J = 15.6 Hz, 2H, CH=CH-), 6.84 (dd, J1 = 8.4 Hz, J2 =
2.4 Hz, 2H, Ar-H), 6.23 (s, 1H, =CH-CO).
4.2.6. 1,7-Bis(3-methoxyphenyl)hepta-1,6-diene-3,5-dione (bisdehydroxycurcumin, BDHC) (2f)
Yellow solid, yield: 54%, mp: 64-65℃. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.63 (d, J =
15.6 Hz, 2H, -CH=CH), 7.31 (t, J = 7.8 Hz, 2H, Ar-H), 7.16 (d, J =7.2 Hz, 2H, Ar-H), 7.07 (s, 2H,
Ar-H), 6.93 (dd, J1 = 7.8 Hz, J2 = 1.8 Hz, 2H, Ar-H), 6.62 (d, J = 15.6 Hz, 2H, CH=CH-),5.86 (s,
1H, =CH-C), 3.85 (s, 6H, -OCH3).
4.2.7. 1,7-Bis(2-hydroxyphenyl)hepta-1,6-diene-3,5-dione (bisdemethoxycurcumin analog-2,
BDMC-A2) (2g)
Yellow solid, yield: 25%, mp: 144-146 ℃.1H NMR (600 MHz, CDCl3) δ (ppm): 16.29(s, 1H,
-OH), 10.24(s, 2H, Ar-OH), 7.88(d, J = 16.2 Hz, 2H, -CH=CH), 7.64(d, J = 7.8Hz, 2H, Ar-H),
7.25(t, J = 7.8Hz, 2H), 6.93(d, J = 16.2Hz, 2H, CH=CH-), 6.92(d, J = 8.4Hz, 2H, Ar-H), 6.86(t, J
= 7.8 Hz, 2H, Ar-H), 6.11(s, 1H, =CH-CO).
4.2.8. 1,7-Bis(2-methoxyphenyl)hepta-1,6-diene-3,5-dione (bisdehydroxycurcumin analog-2,
BDHC-A2) (2h)
Yellow solid, yield: 51%, mp: 120-121℃. 1H NMR (600 MHz, CDCl3) δ (ppm): 16.06 (s,
18

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1H, -OH), 8.00 (d, J = 16.2 Hz, 2H, -CH=CH), 7.58 (d, J = 7.2 Hz, 2H, Ar-H), 7.34 (t, J = 7.8 Hz,
2H, Ar-H), 6.97 (t, J = 7.8 Hz, 2H, Ar-H), 6.92 (d, J = 8.4 Hz, 2H, Ar-H), 6.75 (d, J = 16.2 Hz, 2H,
CH=CH-), 5.90 (s, 1H, =CH-C), 3.93 (s, 6H, -OCH3).
4.3. Synthesis of palladium(II) complexes of curcumin and curcuminoids (3a-3h)
(Bipy)Pd(ONO2)2 (58.0 mg, 0.15 mmol) was added to a suspension of the corresponding
compound (2a-2g) (0.15 mmol) in H2O-acetone (1:1, 10 mL), and the mixture was stirred at 50℃
for 20 h. The PF6 - salt of 3 (3a-3g) was obtained by adding a 6-fold excess of KPF6 to the above
aqueous solution at 60 °C, which resulted in the immediate deposition of 3 as yellow
microcrystals in moderate to quantitative yield. The crystals were filtered, washed with a
minimum amount of ethyl acetate and cold water, and then dried under vacuum.
4.3.1. [Pd(bipy)(curcumin)](PF6) (3a)
Orange solid, yield: 74%. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 8.74 (d, J = 5.4 Hz, 2H),
8.62 (d, J = 8.4 Hz, 2H), 8.44 (t, J = 7.8 Hz, 2H), 7.96 (t, J = 6.0 Hz, 2H), 7.66 (d, J = 15.6 Hz,
2H), 7.35 (d, J = 1.2 Hz, 2H), 7.24 (dd, J1 = 7.8 Hz, J2 = 1.2 Hz, 2H), 6.85 (d, J = 7.8 Hz, 2H),
6.84 (d, J = 15.6 Hz, 2H), 6.09 (s, 1H), 3.87 (s, 6H); 13C NMR (150 MHz, DMSO-d6) δ (ppm):
178.05, 155.53, 153.03, 147.01, 141.89, 141.00, 139.69, 130.15, 127.91, 123.88, 105.82, 60.14,
55.93; HRMS(ESI) m/z calcd for C31H27N2O6Pd [M-PF6]+: 629.0910; Found: 629.0912; Anal.
Calc. for C31H27F6N2O6PPd.H2O (%): C, 46.95; H, 3.69; N, 3.53. Found: C, 47.54; H, 3.52; N,
3.75.
4.3.2. [Pd(bipy)(dimethoxycurcumin)](PF6) (3b)
Orange solid, Yield: 62%. 1H NMR (600 MHz, DMSO-d6): δ(ppm) 8.70 (d, J = 5.4 Hz, 2H),
8.54 (d, J = 7.8 Hz, 2H), 8.40 (t, J = 7.8 Hz, 2H), 7.95 (t, J = 6.6 Hz, 2H), 7.65 (d, J = 15.6 Hz,
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2H), 7.30 (s, 2H), 7.29 (d, J = 7.2 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 15.6 Hz, 2H),
6.12 (s, 1H), 3.83 (s, 6H), 3.82 (s, 6H). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 178.14, 155.71,
151.20, 149.08, 147.24, 142.05, 141.28, 127.86, 127.66, 123.93, 123.48, 122.23, 111.60, 110.29,
105.64, 55.65, 55.61; HRMS (ESI) m/z calcd for C33H31F6N2O6PPd: 657.12207, [M-PF6]+; Found:
657.12238; Anal. Calc. for C33H31F6N2O6PPd·1.0H2O (%): C, 48.27; H, 4.05; N, 3.41. Found: C,
48.58; H, 3.82; N, 3.44.
4.3.3. [Pd(bipy)( BDMC)](PF6) (3c)
Orange solid, yield: 85%. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.14 (s, 2H), 8.62 (d, J
= 5.4 Hz, 2H), 8.54 (d, J = 7.8 Hz, 2H), 8.37 (t, J = 7.8 Hz, 2H), 7.87 (t, J = 6.6 Hz, 2H), 7.58 (d,
J = 8.4 Hz, 4H), 7.56 (d, J = 15.6 Hz, 2H), 6.85 (d, J = 8.4 Hz, 4H), 6.67 (d, J = 15.6 Hz, 2H),
5.92 (s, 1H); 13C NMR (150 MHz, DMSO-d6): δ(ppm) 177.63, 159.98, 155.42, 146.78, 141.76,
141.09, 130.51, 127.72, 125.87, 123.96, 120.84, 115.91, 105.23. HRMS (ESI) m/z calcd for
C29H23N2O4Pd [M-PF6]+: 569.0698; Found: 569.0694; Anal. Calc. for C29H23F6N2O4Pd.H2O (%):
C, 47.52; H, 3.44; N, 3.82. Found: C, 47.04; H, 2.90; N, 3.97.
4.3.4. [Pd(bipy)( BDHC-A1)](PF6) (3d)
Orange solid, yield: 77%. 1H NMR (600 MHz, DMSO-d6): δ(ppm): 8.50 (d, J = 5.4 Hz, 2H),
8.43 (d, J = 7.8 Hz, 2H), 8.30 (t, J = 7.8 Hz, 2H), 7.82 (t, J = 6.6 Hz, 2H), 7.57 (d, J = 8.4 Hz, 4H),
7.47 (d, J = 15.6 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.63 (d, J = 15.6 Hz, 2H), 5.89 (s, 1H), 3.84 (s,
6H); 13C NMR (150 MHz, DMSO-d6): δ(ppm) 117.76, 161.12, 155.39, 146.83, 141.79, 140.68,
130.17, 127.71, 127.27, 123.90, 121.80, 114.34, 105.36, 55.34; HRMS (ESI) m/z calcd for
C31H27N2O4Pd [M-PF6]+: 597.1011; Founded: 597.1011; Anal. Calc. for C31H27N2O4Pd.H2O (%):
C, 48.93; H, 3.84; N, 3.68. Found: C, 48.58; H, 3.41; N, 3.61.
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4.3.5. [Pd(bipy)( BDMC-A1)](PF6) (3e)
Orange solid, yield: 91%.1H NMR (600 MHz, DMSO-d6) δ (ppm): 9.67 (s, 2H), 8.62 (d, J =
5.4 Hz, 2H), 8.56 (d, J = 8.4 Hz, 2H), 8.37 (t, J = 8.4 Hz, 2H), 7.87 (t, J = 6.6 Hz, 2H), 7.59 (d, J
= 15.6 Hz, 2H), 7.25 (t, J = 7.8 Hz, 2H), 7.15 (d, J = 7.8 Hz, 2H), 7.12 (s, 2H), 6.89 (dd, J1 = 7.8
Hz, J2 = 1.8 Hz, 2H), 6.82 (d, J = 15.6 Hz, 2H), 6.13 (s, 1H); 13C NMR (150 MHz, DMSO-d6) δ
(ppm): 178.13, 157.89, 155.56, 146.97, 142.08, 141.44, 136.14, 130.21, 127.99, 124.34, 124.21,
120.04, 117.96, 114.89, 106.07; HRMS (ESI) m/z calcd for C29H23N2O4Pd [M-PF6]+: 569.0698;
Found: 569.0693; Anal. Calc. for C29H23F6N2O4Pd.H2O (%): C, 47.52; H, 3.44; N, 3.82. Found: C,
47.35; H, 2.93; N, 3.80.
4.3.6. [Pd(bipy)( BDHC)](PF6) (3f)
Orange solid, yield: 91%. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 8.51 (d, J = 5.4 Hz, 2H),
8.45 (d, J = 7.8 Hz, 2H), 8.31 (t, J = 7.8 Hz, 2H), 7.82 (t, J = 6.6 Hz, 2H), 7.51 (d, J = 15.6 Hz,
2H), 7.30 (t, J = 7.8 Hz, 2H), 7.18 (d, J = 7.8 Hz, 2H), 7.16 (s, 2H), 7.00 (d, J = 8.4 Hz, 2H), 6.82
(d, J = 15.6 Hz, 2H), 6.04 (s, 1H), 3.79 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ (ppm): 177.90,
159.49, 155.26, 146.69, 141.76, 140.77, 135.83, 129.81, 127.71, 124.48, 123.90, 120.99, 115.93,
113.18, 105.92, 55.10; HRMS (ESI) m/z calcd for C31H27N2O4Pd [M-PF6]+: 597.1011; Founded:
597.1011; Anal. Calc. for C31H27N2O4Pd (%): C, 50.12; H, 3.66; N, 3.77. Found: C, 49.92; H, 3.57;
N, 3.65.
4.3.7. [Pd(bipy)( BDMC-A2)](PF6) (3g)
Orange solid, yield: 90%. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.34(s, 2H), 8.64(d, J =
4.8 Hz, 2H), 8.61(d, J = 7.8 Hz, 2H), 8.41(t, J = 7.2 Hz, 2H), 7.89(d, J = 4.8 Hz, 2H), 7.87(d, J =
15.6 Hz, 2H), 7.61(d, J = 7.8 Hz, 2H), 7.27(t, J = 7.2 Hz, 2H), 7.06(d, J = 15.6 Hz, 2H), 6.95(d, J
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= 7.8 Hz, 2H), 6.87(t, J = 7.2 Hz, 2H), 6.11(s, 1H). 13C NMR (150 MHz, DMSO-d6): δ (ppm):
178.52, 157.01, 155.70, 146.43, 141.99, 137.22, 131.62, 129.61, 127.50, 124.27, 124.12, 121.57,
119.45, 116.32, 99.80; HRMS (ESI) m/z calcd for C29H23N2O4Pd [M-PF6]+: 569.0698; Found:
569.0694; Anal. Calc. for C29H23F6N2O4Pd.H2O (%): C, 47.52; H, 3.44; N, 3.82. Found: C, 47.41;
H, 3.13; N, 3.90.
4.3.8. [Pd(bipy)( BDHC-A2)](PF6) (3h)
Yellow solid, yield: 88%. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 8.30 (d, J = 7.8 Hz, 2H),
8.24 (d, J = 5.4 Hz, 2H), 8.20 (t, J = 7.8 Hz, 2H), 7.70 (t, J = 6.6 Hz, 2H), 7.61 (d, J = 15.6 Hz,
2H), 7.46 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.8 Hz, 2H), 6.99 (d, J = 7.8 Hz, 2H), 6.91 (t, J = 7.2 Hz,
2H), 6.64 (d, J = 15.6 Hz, 2H), 5.65 (s, 1H), 3.75 (s, 6H); 13C NMR (150 MHz, DMSO-d6) δ
(ppm): 177.78, 157.83, 155.22, 145.73, 141.74, 135.75, 131.91, 128.42, 127.29, 124.00, 123.81,
122.60, 120.54, 111.59, 105.80, 55.50; HRMS (ESI) m/z calcd for C31H27N2O4Pd [M-PF6]+:
597.1011; Founded: 597.1010; Anal. Calc. for C31H27N2O4Pd.H2O (%): C, 48.93.12; H, 3.84; N,
3.68. Found: C, 49.54; H, 3.52; N, 3.75.
4.4. X-ray crystallography
The data collection of the complex 3h was performed on a Bruker SMART APEX II CCD
diffractometer equipped with a graphite monochromatized Mo Ka radiation (λ = 0.71073 Å) at
296(2) K. Multi-scan absorption corrections were applied using the SADABS program. The
structure was solved by the direct method using the SHELXS-97 program. Refinements on F2
were performed using SHELXL-97 by the full-matrix least-squares method with anisotropic
thermal parameters for all non-hydrogen atoms. X-ray crystallographic data for complex 3h have
been deposited in the Cambridge Crystallographic Data Centre under the accession number CCDC
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1564509.
4.5. Cell culture and in vitro cytotoxicity assays
Three human cancer cell lines used in the study, including MCF-7, Hela and A549, were
purchased from the American Type Culture Collection. Cells were cultured, according to the
instructions of the supplier, in DMEM supplemented with 10% of fetal bovine serum, 100 Units
per mL each of penicillin and streptomycin at 37°C and 5% CO2 in a humidified atmosphere.
Appropriate amount of the tested complexes were dissolved in DMSO to give 5 mM stock
solution.
In vitro cytotoxicity of the complexes was evaluated by MTT assay [61]. Briefly, the
harvested cells were seeded to 96-well culture plates at a density of 1.5 × 104 cells/well,
pre-incubated in complex-free media at 37°C for 24 h and then treated typically with the 0.1, 1.0
and 10 µM of complex solutions, prepared from the stock solutions by dilution with medium. To
avoid DMSO toxicity, the concentration of DMSO was less than 0.1% (v/v) in all experiments.
Wells containing culture medium and cisplatin were used as positive control for all the cell lines
used, while wells containing culture medium without cells served as control blanks. After 48 h
incubation, 10 µL of stock MTT dye solution (5 mg/mL) was added to each well and incubated for
additional 4 h, DMSO (100 µL) was added to solubilize the formazan. Finally, the optical density
(OD) was measured on a microplate reader (BioRad Model 3550, USA) at a wavelength of 570
nm. The IC50 value was determined from plot of % viability against dose of complex added. All
experiments were performed in quintuplicate.
4.6. Measurement of ROS generation
The ROS assay was carried out to study the influence of the selected complexes on the
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production of ROS level in HeLa cells. Cellular ROS content was determined using DCFH-DA
fluorescent probe. Briefly, cells at a density of 2.0 × 104 cells/well were seeded in 6-well plate and
cultured 12 h prior to treatments. Cells were treated with the tested complexes at different
concentration or vehicle alone as a control, for 24 h. Upon completion of the treatment, the
medium was discarded and cells were washed with PBS. The collected cells were incubated in
PBS with DCFH-DA (20 µM) at 37 °C for 30 mins in the dark. Then, the cells were washed with
medium and the fluorescence intensity of dichlorofluoroscein (DCF) was measured with
excitation at 488 nm and emission at 525 nm by a flow cytometry (BD FACS, USA).
4.7. Morphological observation of nuclear change
Morphological observation of nuclear change was assayed with Hoechst 33342. HeLa cells
(2×104 cells/mL) were seeded in 6-well plates. After reaching 80% confluence, the different
concentration complexes 3h were added and incubated for 24 h. Cells were washed with pure PBS
twice, fixed in 4% paraformaldehyde for 30 min and stained with 5 mg/mL Hoechst 33342 for 5
min at room temperature. The cells were observed by using inverted fluorescence microscope and
photographed.
4.8. Annexin V-FITC/PI apoptosis assay
Apoptosis induced by complex 3h was detected by flow cytometry using an Annexin
V-FITC/PI double labeling method. The Hela cells were seeded under usual conditions in a 6-well
culture plate, at 1.5 × 105 cells per well, in a 2 mL culture medium, and 12 h later the medium
containing the complexes 3h (5.0 µM and 10.0 µM) was added . After 24 h incubation, cells were
harvested and centrifuged at 2000 rpm for 5 min. Subsequently the cells were washed twice with
pure PBS, trypsinized, suspended and stained by using Annexin V-FITC/PI apoptosis assay Kit
(KeyGen, Nanjing, China) according to the manufacture’s instructions. The stained cells were
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performed on a flow cytometry (BD FACS, USA) within one hour. The cells without the treatment
were used as control.
4.9. Cell cycle arrest assay
HeLa cells were seeded into six-well plates at a density of 1.5 × 105 cells/mL and cultured in
DMEM medium supplemented with 10% of FBS for 24 h. The medium was removed and replaced
with medium (final DMSO concentration 0.05% v/v) containing different concentrations complex
3h. After 24 h of incubation, cells were washed twice with ice-cold PBS, collected, and
permeabilized with ice-cold 70% ethanol at −20°C overnight. The cells were treated with 1
mg/mL RNase A at 37 °C for 30 min, finally stained with 50 µg/mL propidium iodide (PI) in the
dark at 4 °C for 15 min in dark. The samples were then analyzed by a flow cytometry (BD FACS,
USA). The number of cells analyzed for each sample was 10,000.
4.10. Mitochondrial membrane potentials (MMP) assay
Mitochondrial membrane potential was assessed by flow cytometry using the cationic probe
RH-123. Briefly, HeLa cells (1.5×105 cells/mL) were cultured in 6-well plate for 12 h prior
exposure to complexes 3h at different concentration for 24 h, cells without complex 3h treatment
were used as a control. After removal of the medium, the collected cells were resuspended in 0.5
mL of PBS buffer containing 2 µg/mL of RH-123. After 30 min’ incubation at 37
℃ in the dark,
the cells were washed twice with PBS, and were analyzed immediately by flow cytometry
(FACScan, Bection Dickinson, San Jose, CA). 10,000 events were acquired for each sample. The
cell images were also obtained with fluorescence microscope (EVOS® FL Auto) to monitor
change in MMP.
4.11. Statistical analysis
The data were obtained from at least three separate experiments and expressed as mean ± SD.
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A one–way ANOVA followed by Tukey post-hoc analysis was used for multiple group
comparisons. A *P value of <0.05 was considered as significant.
Acknowledgements
This work was supported by the Nature Science Fund of Hebei Province (B2015201213) and
the Key Basic Research Special Foundation of Science Technology Ministry of Hebei Province
(15962602D).
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