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Synthesis and anticancer properties of ruthenium (II) complexes as potent apoptosis inducers through mitochondrial disruption
Accepted Manuscript Synthesis and anticancer properties of ruthenium (II) complexes as potent apoptosis inducers through mitochondrial disruption Dan Wan, Bing Tang, Yang-Jie Wang, Bo-Hong Guo, Hui Yin, Qiao-Yan Yi, Yun-Jun Liu PII:
S0223-5234(17)30588-3
DOI:
10.1016/j.ejmech.2017.07.066
Reference:
EJMECH 9629
To appear in:
European Journal of Medicinal Chemistry
Received Date: 13 May 2017 Revised Date:
24 July 2017
Accepted Date: 27 July 2017
Please cite this article as: D. Wan, B. Tang, Y.-J. Wang, B.-H. Guo, H. Yin, Q.-Y. Yi, Y.-J. Liu, Synthesis and anticancer properties of ruthenium (II) complexes as potent apoptosis inducers through mitochondrial disruption, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.07.066. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract Three ruthenium (II) complexes were synthesized and characterized. The anticancer activity was evaluated by cytotoxicity in vitro, apoptosis, cell invasion, cell cycle
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arrest, ROS, mitochondrial membrane potential and western blot.
ACCEPTED MANUSCRIPT
Submitted to Eur. J. Med. Chem.
Synthesis and anticancer properties of ruthenium (II) complexes as
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potent apoptosis inducers through mitochondrial disruption
Dan Wana, Bing Tanga, Yang-Jie Wanga, Bo-Hong Guoa,*, Hui Yinc,*, Qiao-Yan
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006,
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a
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Yia, Yun-Jun Liua,b,*
PR China c
Guangdong Cosmetics Engineering & Technology Research Center, Guangzhou,
510006, PR China
Department of Microbiology and Immunology, Guangdong Pharmaceutical
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c
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University, Guangzhou 510006, PR China
*Corresponding
author.
E-mail
address:
[email protected]
[email protected] (H. Yin); [email protected] (Y.J. Liu). 1
(B.H.
Guo);
ACCEPTED MANUSCRIPT Abstract
A
new
ligand
MHPIP
(MHPIP
=
2-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its three ruthenium
(II)
complexes
[Ru(N-N)2(MHPIP)](ClO4)2
(N-N
=
phen:
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1,10-phenanthroline 1; dmp = 2,9-dimethyl-1,10-phenanthroline 2; ttbpy = 4,4'-ditertiarybutyl-2,2'-bipyridine 3) were synthesized and characterized. The cytotoxic activity in vitro was studied by MTT method. The complexes 1-3 show
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moderate cytotoxic effects on the cell growth in HepG2 cells with an IC50 value of
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25.5 ± 3.5, 35.6 ± 1.9 and 27.4 ± 2.3 µM, respectively. The apoptosis was investigated with AO/EB and Annex V/PI staining methods and comet assay. The reactive oxygen species, mitochondrial membrane potential were investigated under a fluorescent microscope. Autophagy assay shows that the complexes can cause
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autophagy and up-regulate the expression of Beclin-1 protein. Additionally, the complexes inhibit the cell growth in HepG2 cells at G0/G1 phase, and the complexes can regulate the expression of caspase 3 and Bcl-2 family proteins. The studies
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demonstrate that the complexes induce apoptosis in HepG2 cells through DNA
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damage and ROS-mediated mitochondrial dysfunction pathways.
Keywords: Ruthenium(II) complexes; DNA damage; apoptosis; autophagy; cell invasion; Bcl-2 family proteins.
1. Introduction The clinical success of cisplatin along with oxaliplatin and carboplatin as anticancer drugs has raised significant interest in the development of a wide range of transition 2
ACCEPTED MANUSCRIPT metal complexes with DNA/protein binding ability as well as potential anticancer activity [1]. However, these drugs exhibit side effects, severe toxicity and drug resistance [2,3]. Based on these reasons, many scientists are actively searching for
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other transition metal complexes as platinum alternative as antitumor candidates [4-10]. Among these metal complexes, ruthenium complexes, owing to their rich photochemical and photophysical properties and low cytotoxicity, have been paid
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great attention [11-24]. Recently, the studies of the ruthenium polypyridyl complexes
2,9-dimethyl-1,10-phenanthroline,
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on the bioactivity have made great progress. [Ru(dmp)2(dhbn)](ClO4)2 (dmp = dhbn
12,13-diphenyl-4,5,9,11,14,16-hexaazadibenzo[a,c]naphthacene)
= can
effectively
inhibit the cell growth in HepG2 cells with an IC50 value of 17.7 ± 1.1 µM. This
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complex induces apoptosis in HepG2 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway [25]. Chen et al. reported that RuPOP, a ruthenium polypyridyl complex with potent antitumor activity, was able to effectively
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inhibit growth and metastasis of MDA-MB-231 cells and synergistically enhance
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TRAIL-induced apoptosis [26]. Wang et al. reported that dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers could enter HeLa cells efficiently and localize within lysosomes [27]. [Ru(MeIm)(npip)]2+ (npip = 2-(4-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline) induces A549 cells apoptosis by acting on both mitochondrial homeostasis destruction and death receptor signal pathways. This complex may be a candidate as a chemotherapeutic agent against human tumor [28]. To obtain more information on the anticancer 3
ACCEPTED MANUSCRIPT activity of ruthenium complexes, in this article, a new ligand MHPIP (MHPIP = 2-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its three ruthenium(II)
complexes
[Ru(N-N)2(MHPIP)](ClO4)2
(N-N
=
phen:
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1,10-phenanthroline 1; dmp = 2,9-dimethyl-1,10-phenanthroline 2; ttbpy = 4,4'-ditertiarybutyl-2,2'-bipyridine 3) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1H NMR and
13
C NMR. The anticancer activity of
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the complexes was investigated by cytotoxicity in vitro, apoptosis, reactive oxygen
family proteins expression.
2. Results and discussion
The
ligand
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2.1. Synthesis and characterization
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species, mitochondrial membrane potential, cell cycle arrest, autophagy and Bcl-2
MHPIP
was
prepared
through
condensation
of
1,10-phenanthroline-5,6-dione with 1-methyl-1H-pyrazole-4-carboxaldehyde using a
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similar method to that described by Steck and Day [29]. The corresponding Ru(II)
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complexes were synthesized by direct reaction of MHPIP with the appropriate precursor complexes in ethylene glycol. The desired Ru(II) complexes were isolated as the perchlorates and purified by column chromatography. Each synthetic step involved here is straightforward and provides a relatively high yield of the desired product in pure form. In the ESI-MS spectra for the Ru(II) complexes, all of the expected signals [M-2ClO4-H]+ and [M-2ClO4]2+ were observed. The measured molecular weights were consistent with the expected values. 4
ACCEPTED MANUSCRIPT The absorption spectra of 10 µM of complexes 1-3 mainly consist of three resolved bands in range 200–600 nm (Fig. S1). The bands at 270-290 nm is attributed to intraligand (IL) π–π* transitions, the band at 340-360 nm is attributed to the π–π*
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transition and the lowest energy bands at 450-465 nm is assigned to the metal-to-ligand charge transfer (MLCT) transitions. Complexes 1 and 3 can emit in PBS solution at room temperature with a maximum appearing at 583 nm for 1 and
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608 nm for 3, respectively, whereas complex 2 can not emit at ambient temperature
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(Fig. S1b).
The stability of the complexes in PBS solution was studied by UV-Vis spectra at room temperature. As shown in Fig. S2, no obvious changes in absorbance of 10 µM complexes 1 (a), 2 (b) and 3 (c) at 0 h and 24 h were observed, which indicates that
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the complexes are stable in PBS solution.
2.2. The cytotoxic activity evaluation in vitro of the complexes
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The cytotoxic activity of the complexes 1-3 was evaluated against a set of cancer
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cell lines (HepG2, HeLa, A549, BEL-7402, SiHa, SGC-7901) and human normal hepatocyte LO2 cells. Cisplatin was used as a control, the IC50 values are listed in Table 1. As expectation, the ligand shows no cytotoxic activity against HepG2, A549, BEL-7402, SiHa, SGC-7901 and LO2 cells. The complex 3 displays cytotoxicity toward most selected cancer cell lines. Complexes 1 and 2 have no cytotoxic activity against A549 and SiHa cell lines. This may be caused by larger hydrophobicity of ancillary ligand ttbpy than those of phen and dmp. Thus, it is easy for complex 3 to 5
ACCEPTED MANUSCRIPT enter into the cells compared with complexes 1 and 2 under the same condition. The cytotoxic effect of complexes 1-3 is lower against the selected cancer cells than cisplatin under identical condition, but their cytotoxic activity is comparable to that of
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[Ru(dmp)2(AQTP)]2+ (IC50 = 32.9 ± 3.1 µM) toward HepG2 cells [13]. Interestingly, complexes 1 and 2 have no cytotoxic activity, complex 3 exhibits low cytotoxic activity against human normal hepatocyte LO2 cells. The results reveal that the ligand
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bonded to metal to form metal complexes, the cytotoxic activity can be enhanced.
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Because all complexes 1-3 show moderate cytotoxic effect on the cell growth in HepG2, this cell was selected to undergo the following experiments.
2.3. DNA damage studies
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DNA fragmentation is a hallmark of apoptosis, mitotic catastrophe or both [30]. The DNA damage was investigated by single cell gel electrophoresis (comet assay) in an agarose gel matrix. As shown in Fig. 1, in the control (a), HepG2 cells do not show
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any comet like appearance. After HepG2 cells were treated with 25 µM of the
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complexes 1 (b), 2 (c) and 3 (d) for 24 h, the cell nuclei were stained with EB, the HepG2 cell demonstrates statistically significant and well-formed comets, and the length of the comet tail represents the extent of DNA damage. These results indicate that the three complexes indeed induce DNA fragmentation, which is further evidence of apoptosis.
2.4. Apoptosis assay with AO/EB and Annex V/PI double staining methods 6
ACCEPTED MANUSCRIPT The comet assay suggests that the complexes can induce apoptosis in HepG2 cells. Apoptosis and necrosis, two major types of cell death, are delineated in response to a death stimulus. Disruption or inappropriate regulation of apoptotic and necrosis
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processes can result in several diseases including cancer [31,32]. The morphological changes of the cells was assayed with acridine orange (AO)/ethidium bromide (EB) stained cells. As shown in Fig. 2A, in the control (a), the living cells show bright
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green with intact cell nuclei. After the treatment of HepG2 cells with 25 µM of
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complexes 1 (b), 2 (c) and 3 (d) for 24 h, the apoptotic cells with apoptotic features such as cell blebbing, nuclear shrinkage and chromatin condensation were observed. To quantitatively compare the apoptotic effect of the complexes, Annex V/PI double staining was used to evaluate the percentage of apoptotic cells. As shown in Fig. 2B,
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HepG2 cells were treated with 25 µM of complexes 1 (c), 2 (e) and 3 (g) for 24 h, the percentage of apoptotic (Q3) cells was increased to 5.44% for 1, 3.18% for 2 and 16.5% for 3 in comparison with the control (1.58%). The apoptotic effects follow the order
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cell.
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of 3 > 1 > 2. The results indicate that the complexes can induce apoptosis in HepG2
2.5. Autophagy induced by the complexes Autophagy is a lysosomal degradation pathway and it is essential for
homeostasis under normal conditions. Autophagy is not only a survival response to growth factor or nutrient deprivation but also a way for tumor cell suppression [33,34]. To investigate the autophagic effect of the complexes on HepG2 cells, the cells were 7
ACCEPTED MANUSCRIPT treated with complexes 1-3 for 24 h, then the cells were stained with monodansylcadaverine (MDC) [35]. As shown in Fig. 3A, in the control (a), no obvious green fluorescent points were observed. After the treatment of HepG2 cell
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with 25 µM of complexes 1, 2 and 3 for 24 h, a number of bright green fluorescent points were observed, which indicates that the autophagic vacuoles were formed. The MDC fluorescent intensity is proportional to the autophagic effects. As shown in Fig.
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3B, exposure of HepG2 cell to 25.0 µM of complexes 1-3 led to an increase of MDC
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fluorescent intensity of 170.81 times for 1, 2.72 times for 2 and 3.83 times for 3 than that of control, respectively. The increase of MDC fluorescent intensity demonstrates the increasing number of autophagic cells. Moreover, the autophagic effect shows a concentration-dependent manner. In addition, the conversion of LC3-I to LC3-II is a
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hallmark of autophagy, and Beclin-1 is necessary to form autophagosomes in autophagy. As seen in Fig. 3C, the conversion of LC3-I to LC3-II occurred and there were significant increases in the expression of Beclin-1 protein in HepG2 cells treated
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with 25 µM of the complexes 1-3. Taken the above results together, the results
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indicate that the complexes can induce autophagy in HepG2 cells.
2.6. Intracellular reactive oxygen species levels determination It has been reported that apoptosis can be triggered by increasing intracellular
ROS levels [36], and Ru(II) polypyridyl complexes can markedly increase the intracellular ROS levels [4, 37-39]. To study whether the cell death induced by the complexes is dependent on the ROS levels, HepG2 cells were exposed to the 8
ACCEPTED MANUSCRIPT complexes for 24 h, and then the ROS levels were determined using a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as fluorescent probe [40,41]. This dye is cleaved by intracellular esterases into its non-fluorescent form (DCFH).
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Then the non-fluorescent substrate is oxidized by intracellular free radicals to produce a fluorescent product, dichlorofluorescein (DCF). The DCF fluorescent intensity was determined by flow cytometry. As shown in Fig. 4, after the treatment of HepG2 cells
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(a) with 25 µM of complexes 1 (b), 2 (d) and 3 (f) for 24 h, the DCF fluorescent
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intensity increases 6.22 times for 1, 1.39 times for 2 and 1.33 times for 3 than that of control. Exposure HepG2 cells to 50 µM of complexes 1 (c), 2 (e) and 3 (g) for 24 h, the DCF fluorescent intensity increases 13.62, 1.45 and 1.54 times than the original, respectively. The intracellular ROS levels depend on the balance of generation and
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consumption of ROS induced by the complexes. Complex 1 induces the highest enhancement in the ROS levels among the three complexes. This may be caused by higher generation and lower consumption of intracellular ROS levels of complex 1
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than those of complexes 2 and 3. These data reveal that the complexes can increase
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the intracellular ROS levels.
2.7. Location assay of the complexes and evaluation of the mitochondrial membrane potential changes
Mitochondrial changes, including loss of mitochondrial membrane potential (∆Ψm), are key events that take place during drug-induced apoptosis [42]. The maintenance of mitochondrial membrane potential (∆Ψm) is important for 9
ACCEPTED MANUSCRIPT mitochondrial integrity and bioenergetic function [43]. To study the location of the complexes in the mitochondria, Mito Tracker ® Deep Green FM (ThermoFisher, 100 nM) was used as red fluorescent probe. As shown in Fig. 5A, in the control, the
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mitochondria were stained in green. After the treatment of HepG2 cells with 12.5 µM of complexes 1-3 for 4 h, the complexes emit red fluorescence. The merge of the red and green indicates that the complexes entered into the mitochondria. The
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mitochondrial membrane potential induced by the complexes was investigated using
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JC-1 as a fluorescent probe. As shown in Fig. 5B, in the control (a), JC-1 emits red fluorescence corresponding to high mitochondrial membrane potential. However, HepG2 cells were incubated with cccp (b, positive control) and 25.0 µM of complexes 1 (c), 2 (d) and 3 (e) for 24 h, JC-1 emits bright green fluorescence, which
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corresponds to low mitochondrial membrane potential. To compare the effect of the complexes on the changes of mitochondrial membrane potential, the ratio of the green/red fluorescent intensity was determined by flow cytometry. As shown in Fig.
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5C, in the control, the ratio of green/red fluorescence is 2.26. Treatment of the cells
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with 50.0 µM of complexes 1-3, the ratios of green/red fluorescence are 3.69, 3.64 and 5.51, respectively. The increases of the ratio demonstrate that the green fluorescent intensities increase and the red fluorescent intensities reduce. The efficiency of the complexes on mitochondrial membrane potential exhibits a concentration-dependent manner and follows the order of 3 > 2 > 1. The results show that the complexes can induce a decrease in the mitochondrial membrane potential.
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ACCEPTED MANUSCRIPT 2.8. Cell invasion studies Metastasis, the main cause of death of cancer patients, is the spread of tumour cells from a primary tumour source to other organs, and remains as one of the ultimate
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challenges in cancer treatment [44]. Thus, it is urgent to find an effective drug to inhibit cell invasion. The effects of the complexes on the cell invasion were studied by Matrigel invasion assay. As shown in Fig. 6A and 6B, HepG2 cells were treated
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with 50 µM of complexes 1, 2 and 3 for 24 h, the percentage of inhibiting the cell
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invasion reaches 86.5%, 81.9% and 60.9%, respectively. Complex 1 displays the highest inhibiting efficiency among the three complexes. Furthermore, the complexes reveal a concentration-dependent manner to inhibit the cell invasion.
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2.9. The cell cycle distribution analysis
Cell cycle and control of apoptosis are the significant regulatory mechanisms of cell growth, development, and differentiation. Cell cycle checkpoints guarantee the
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maintenance of genomic integrity by preventing DNA damage and incomplete DNA
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cells from further cell division [45]. The effect of the complexes on cell cycle progression of HepG2 cells was examined by flow cytometry. As shown in Fig. 7, in the control, the percentage in the cells at G0/G1 is 54.9%. The treatment of HepG2 cells led to an increase of 4.1% for 1, 10.7% for 2 and 5.3% for 3 in the cells at G0/G1 phase, which was accompanied by corresponding reduction of percentage in the cells at G2/M phase. The data demonstrate that complexes 1, 2 and 3 inhibit cell proliferation of HepG2 cells at G0/G1 phase. From these results we can conclude that 11
ACCEPTED MANUSCRIPT growth inhibition of HepG2 cells by the complexes involves a profound cell cycle arrest in G0/G1 phase, indicating its potential of interaction with DNA.
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2.10. The effect of the complexes on the expression of Bcl-2 family proteins Caspase 3 is one of the key executioners of apoptosis. The activation of caspase 3 is generally regarded as one of the most obvious characteristics in the apoptosis
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stage for many cells [46]. The effect of the complexes on the expression of caspase is
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shown in Fig. 8, treatment of HepG2 cells with 25 µM of complexes 1-3 for 24 h resulted in an increase in the expression levels of caspase 3. As expectation, the complexes treated the HepG2 cells for 24 h and down-regulated the expression of anti-apoptotic proteins Bcl-2 and Bcl-x. The activation of pro-apoptotic proteins is
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helpful for inducing apoptosis. HepG2 cells were incubated with 25 µM of complexes 1-3, the expression levels of Bak and Bax were up-regulated. Taken above together, we conclude that the complexes induce apoptosis in HepG2 cell through the following
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two pathways (Fig. 9): (I) the complexes enhance ROS levels, and induce a decrease
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in the mitochondrial membrane potential. The changes of mitochondrial membrane potential activate the expression of caspase 3. Then the activation of caspase 3 stimulates the cell apoptosis. (II) The complexes can cause DNA damage, and then the complexes inhibit the cell growth at G0/G1 phase. Finally, the complexes induce cell apoptosis. This work will be helpful for design and synthesis of new ruthenium(II) complexes as potent anticancer drugs.
12
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3. Conclusions Three complexes [Ru(phen)2(MHPIP)](ClO4)2 1, [Ru(dmp)2(MHPIP)](ClO4)2 2 and [Ru(ttbpy)2(MHPIP)](ClO4)2 3 were synthesized and characterized. The
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cytotoxicity in vitro shows that the complexes display moderate cytotoxic activity toward HepG2 cells. Complexes 1-3 can induce apoptosis, enhance the intracellular ROS levels and induce a decrease in the mitochondrial membrane potential. MDC
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staining demonstrates that the complexes can cause autophagy and up-regulate the
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expression of Beclin-1 protein. The cell cycle distribution indicates that the complexes inhibit the cell growth in HepG2 cells at G0/G1 phase. The cell invasion shows that the complexes can effectively inhibit the cell invasion. In addition, the complexes can activate caspase 3, down-regulate the expression of Bcl-2 and Bcl-x
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proteins, and up-regulate the expression levels of proapoptotic proteins Bak and Bax. In summary, the complexes induce apoptosis through DNA damage and
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ROS-mediated mitochondrial dysfunction pathways.
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4. Experimental
4.1. Materials and methods All reagents and solvents were purchased commercially and used without further
purification unless otherwise indicated. Ultrapure MilliQ water was used in all experiments. DMSO, 4,4'-ditertiary-butyl-2,2′-bipyridine and RPMI 1640 were purchased from Sigma. 1,10-phenanthroline was obtained from the Guangzhou Chemical Reagent Factory. Cell lines of HepG2 (human hepatocellular carcinoma), 13
ACCEPTED MANUSCRIPT HeLa (human (hepatocellular),
cervical cancer), SiHa
A549
(human
cervical
(human lungcarcinoma), carcinone),
SGC-7901
BEL-7402 (gastric
adenocarcinoma) and human normal hepatocyte LO2 cells were purchased from the
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American Type Culture Collection. RuCl3·3H2O was purchased from the Kunming Institution of Precious Metals. Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental analyzer. Electrospray ionization mass spectra
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(ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA) using acetonitrile
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as mobile phase. The spray voltage, tube lens offset, capillary voltage and capillary temperature were set at 4.50 kV, 30.00 V, 23.00 V and 200 °C, respectively, and the quoted m/z values are for the major peaks in the isotope distribution. 1H NMR and 13C NMR spectra were recorded on a Varian-500 spectrometer with DMSO-d6 as solvent
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and tetramethylsilane (TMS) as an internal standard at 500 MHz at room temperature.
4.2. Stability of the complexes in buffer
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For stability studies, all the complexes were first dissolved in a minimum amount
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of DMSO (0.5% of the final volume) and then diluted with PBS to a required concentration. The stability was analyzed by monitoring the electronic spectra of the complexes over 24 h.
4.3. Synthesis of ligand and complexes 4.3.1. Preparation of ligand MHPIP A mixture of 1,10-phenthroline-5,6-dione (0.210 g, 1.00 mmol) [47], 14
ACCEPTED MANUSCRIPT 1-methyl-1H-pyrazole-4-carboxaldehyde (0.165 g, 1.5 mmol), ammonium acetate (2.31 g, 30 mmol) and glacial acetic acid (20 mL) was refluxed with stirring for 2 h. The cooled solution was diluted with water and neutralized with concentrated aqueous
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ammonia. The brown precipitate was collected and purified by column chromatography on silica gel (60–100 mesh) with ethanol as eluent to give the compound as a brown yellow powder. Yield: 0.360 g, 80%. Anal. Calc for C17H12N6:
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C, 67.99; H, 4.03; N, 27.98%. Found: C, 67.81; H, 4.20; N, 27.87%. IR (KBr, cm−1):
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3117, 2811, 1614, 1581, 1567, 1505, 1432, 1399, 1357, 1182, 1118, 1073, 1018, 861, 803, 738. FAB-MS: m/z = 301 [M + 1].
4.3.2. Synthesis of complex [Ru(phen)2(MHPIP)](ClO4)2 (1)
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A mixture of [Ru(phen)2Cl2]·2H2O [48] (0.280 g , 0.5 mmol) and MHPIP (0.150 g, 0.5 mmol) in ethylene glycol (20 mL) was heated at 150 oC under argon for 8 h to give a clear red solution. After cooling to room temperature, a red precipitate
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was obtained by the addition of an excess of saturated aqueous NaClO4 solution. The
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crude product was purified by column chromatography on neutral alumina with a mixture of CH3CN-toluene (3:1, v/v) as eluent. The red band was collected. The solvent was removed under reduced pressure and a red powder was obtained. Yield: 0.336 g, 70%. Anal. Anal. Calc for C41H28N10Cl2O8Ru: C, 51.26; H, 2.94; N, 14.58%. Found: C, 51.15; H, 3.08; N, 14.41%. IR (KBr, cm−1): 3390, 3049, 1615, 1496, 1476, 1426, 1364, 1307, 1197, 1143, 1121, 1086, 1013, 846, 721, 625. 1H NMR: 8.95 (d, 2H, J = 8.0 Hz), 8.77 (dd, 4H, J = 8.5, J = 8.5 Hz), 8.49 (s, 1H), 8.39 (s, 4H), 8.18 (s, 1H), 15
ACCEPTED MANUSCRIPT 8.12 (dd, 2H, J = 5.5, J = 5.5 Hz), 8.08 (dd, 2H, J = 5.5, J = 5.0 Hz), 7.98 (dd, 2H, J = 5.5, J = 5.5 Hz), 7.78-7.75 (m, 6H), 3.98 (s, 3H).
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C NMR (DMSO-d6, 125 Hz):
152.77, 152.62, 150.03, 147.83, 147.24, 147.16, 145.06, 137.43, 136.78, 130.44,
4.3.3. Synthesis of [Ru(dmp)2(MHPIP)](ClO4)2 (2)
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([M-2ClO4]2+).
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130.38, 130.17, 128.05, 126.32, 126.02, 112.91, 40.33. ESI-MS (CH3CN): m/z 381.1
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This complex was synthesized in a manner identical to that described for 1, with [Ru(dmp)2Cl2]·2H2O [49] in place of [Ru(phen)2Cl2]·2H2O. Yield: 0.366 g, 72%. Anal. Calc for C45H36N10Cl2O8Ru: C, 53.16; H, 3.57; N, 13.78%. Found: C, 53.26; H, 3.46; N, 13.91%. IR (KBr, cm−1): 3379, 3085, 1615, 1596, 1509, 1439, 1412, 1370,
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1349, 1196, 1013, 843, 726, 557. 1H NMR: 13.92 (s, 1H), 8.91 (d, 2H, J = 8.5 Hz), 8.79 (d, 2H, J = 8.0 Hz), 8.45 (s, 1H), 8.41 (d, 4H, J = 8.5 Hz), 8.24 (d, 2H, J = 8.5 Hz), 8.10 (s, 1H), 7.97 (d, 2H, J = 8.0 Hz), 7.52-7.46 (m, 2H), 7.36 (t, 2H, J = 5.5 Hz),
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7.22 (d, 2H, J = 6.0 Hz), 3.97 (s, 3H), 1.93 (s, 6H), 1.71 (s, 6H). 13C NMR (DMSO-d6,
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125 Hz): 167.94, 166.37, 158.26, 148.88, 147.79, 138.10, 137.36, 136.67, 130.34, 129.51, 128.91, 128.21, 127.52, 127.39, 127.11, 126.65, 126.53, 125.54, 125.32, 123.57, 112.66, 40.28, 25.55, 24.85, 24.51. ESI-MS (CH3CN): m/z 408.8 ([M-2ClO4]2+).
4.3.4. Synthesis of [Ru(ttbpy)2(MHPIP)](ClO4)2 (3) This complex was synthesized in a manner identical to that described for 1, with 16
ACCEPTED MANUSCRIPT [Ru(ttbpy)2Cl2]·2H2O [48] in place of [Ru(phen)2Cl2]·2H2O. Yield: 0.409 g, 71%. Anal. Calc for C53H60N10Cl2O8Ru: C, 55.98; H, 5.32; N, 12.32%. Found: C, 55.76; H, 5.43; N, 12.24%. IR (KBr, cm−1): 3368, 3087, 2961, 1615, 1598, 1539, 1482, 1414,
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1368, 1252, 1197, 1014, 839, 726, 558. 1H NMR: 8.96 (d, 2H, J = 7.5 Hz), 8.86 (dd, 4H, J = 8.0, J = 8.0 Hz), 8.48 (s, 1H), 8.17 (s, 1H), 7.98 (d, 2H, J = 5.5 Hz), 7.92 (t, 2H, J = 5.0 Hz), 7.66 (d, 2H, J = 6.5 Hz), 7.71 (dd, 2H, J = 6.5, J = 6.0 Hz), 7.45 (d,
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2H, J = 6.0 Hz), 7.32 (dd, 2H, J = 6.0, J = 6.0 Hz), 4.00 (s, 3H), 1.43 (s, 18H), 1.34 (s,
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18H). 13C NMR (DMSO-d6, 125 Hz): 161.91, 161.73, 156.57, 156.39, 150.83, 150.71, 147.84, 137.44, 130.43, 124.84, 124.48, 121.79, 112.82, 40.21, 35.56, 35.43, 30.11, 30.00. ESI-MS (CH3CN): 937.0 ([M-2ClO4-H]+), 469.0 ([M-2ClO4]2+).
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4.4. Cytotoxicity in vitro assay
The cytotoxicity in vitro was determined by MTT assay [50]. Cancer cells (8 × 103 cells per well) were seeded in 96-well for 24 h. Cells was incubated with the
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tested compounds to achieve final concentrations ranging from 10-6 to 10-4 M. Control
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wells were prepared by addition of culture medium (100 µL) and cisplatin was used as a positive control. After 48 h incubation, 10 µL of MTT dye solution (5 mg/mL−1) was added to each well. After incubation at 37 oC for 4 h, buffer (100 µL) containing dimethylformamide (50%) and sodium dodecyl sulfate (20%) was added to transform MTT to a purple formazan dye. The optical density of each well was then measured for three times to obtain the mean values. The IC50 values were analyzed by software of SPSS. 17
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4.5. Apoptosis studies with AO/EB staining method HepG2 cells (2 × 105) were exposed to 25 µM of the complexes and cultured in
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RPMI (Roswell Park Memorial Institute) 1640 with 10% of fetal bovine serum (FBS) and incubated at 37 °C in 5% CO2 for 24 h. The cells were washed with ice-cold phosphate buffer saline (PBS), and fixed with formalin (4%, w/v). Cell nuclei were
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counterstained with acridine orange (AO) and ethidium bromide (EB) (AO: 100 µg/
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mL, EB: 100 µg/mL) or Hoechst 33,258 for 10 min. The cells were observed and imaged with a fluorescence microscope (Nikon, Yokohama, Japan) with excitation at 350 nm and emission at 460 nm.
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4.6. Annexin V-FITC apoptosis detection
HepG2 cells were seeded into 6-well plates at a density of 1 × 106 cells per well and incubated for 24 h. The different concentration of compounds were added into the
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above well for 24 h, cells were collected and washed with PBS twice, and then stained
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with fluorescein isothiocyanate (FITC)-conjugated Annexin V and then PI. Cells were quantified by a FACS Calibur flow cytometry (Beckman Dickinson & Co., Franklin Lakes, NJ).
4.7. Autophagy induced by the complexes HepG2 cells were seeded onto chamber slides in 12-well plates and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS and 18
ACCEPTED MANUSCRIPT incubated at 37 °C in 5% CO2. The medium was removed and replaced with medium (final DMSO concentration, 0.05% v/v) containing 25 µM of complexes 1-3 for 24 h. The medium was removed again, and the cells were washed with ice-cold PBS twice.
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Then the cells were stained with MDC (monodansylcadaverine) solution (50 µM) for 10 min and washed with PBS twice. The cells were observed and imaged under
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Beclin-1 protein was assayed by western blot.
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fluorescence microscope. The effect of the complexes on the expression of LC3 and
4.8. Reactive oxygen species (ROS) detection
Intracellular ROS levels were measured with a fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA). HepG2 cells were seeded into
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6-well plates at a density of 1 × 106 cells per well. After incubation for 24 h, the medium was replaced with medium containing different concentrations of compounds for 24 h. Then the cells were stained with 20 µM DCFH-DA in PBS for 30 min in the
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dark. Finally, the cells were harvested and washed twice with PBS. The data were
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obtained by flow cytometry.
4.9. Location assay of the complexes in the mitochondria HepG2 cells were placed in 24-wellmicroassay culture plates (4 × 104 cells per
well) and grown overnight at 37 oC in a 5% CO2 incubator. 12.5 µM of the complexes was added to the wells at 37 oC in a 5% CO2 incubator for 4 h and further co-incubated with Mito Tracker ® Deep Green FM (100 nM) at 37 oC for 1 h. Upon 19
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microscope.
4.10. Measurement of mitochondrial membrane potential (MMP, △Ψm)
HepG2 cells (2 × 105 per well) were treated using compounds for 24 h. JC-1 (1
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mg˖ml-1) as fluorescence probe for determination of MMP was added to stain cells at
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37 oC for 30 min. Then the cells were washed twice with PBS and the fluorescent intensity of JC-1 was determined by flow cytometry.
4.11. Cell cycle arrest studies
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HepG2 cells were seeded into six-well plates (Costar, Corning Corp., New York) at a density of 1 × 106 cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS, and incubated at 37 °C and 5% CO2.
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The medium was removed and replaced with medium (final DMSO concentration
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0.05% v/v) containing 25 µM complexes 1-3. After incubation for 24 h, the cell layer was trypsinized and washed with cold PBS and fixed with 70% ethanol. Twenty µL of RNAse (0.2 mg/mL) and 20 µL of propidium iodide (0.02 mg/mL) were added to the cell suspensions and the mixtures were incubated at 37 °C for 30 min. The samples were then analyzed with a FACS Calibur flow cytometry. The number of cells analyzed for each sample was 10,000.
20
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at the desired concentration and added into Matrigel. Twenty-five thousands of HepG2 cells in serum free media were then seeded in the top chamber of the two chamber Matrigel system. To the low compartment, RPMI and 5% FBS were added
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as chemo-attractant. Cells were allowed to invade for 24 h. After incubation,
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non-invading cells were removed from the upper surface and cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% of crystal violet. Membranes were photographed and the invading cells were counted under a light
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microscope. Mean values from three independent assays were calculated.
4.13. Western blot analysis
HepG2 cells were seeded in 3.5 cm dishes for 24 h and incubated with 25.0 µM
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of complexes 1-3 in the presence of 10% FBS. Then cells were harvested in lysis
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buffer. After sonication, the samples were centrifuged for 20 min at 13 000g. The protein concentration of the supernatant was determined by BCA assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done loading equal amount of proteins per lane. Gels were then transferred to poly(vinylidene difluoride) membranes (Millipore) and blocked with 5% non-fat milk in TBST (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) buffer for 1 h. The membranes were incubated with primary antibodies (1:5000 dilution) in 5% non-fat milk overnight at 4 21
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C, and after washed four times with TBST for a total of 30 min, then the secondary
antibodies conjugated with horseradish peroxidase at 1:5000 dilution for 1 h at room temperature and washed four times with TBST. The blots were visualized with the
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Amersham ECL Plus western blotting detection reagents according to the manufacturer's instructions. To assess the presence of comparable amount of proteins
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in each lane, the membranes were stripped finally to detect the β-actin.
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Data analysis
All data were expressed as mean ± SD. Statistical significance was evaluated using t-tests. Differences were considered to be significant when the *P value was less than
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0.05.
Acknowledgements
This work was supported by the National Nature Science Foundation of China (No.
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81403111), the Natural Science Foundation of Guangdong Province (Nos.
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2016A030313726, 2016A030313728) and Project of Innovation for Enhancing Guangdong
Pharmaceutical
University,
Provincial
Experimental
Demonstration Center of Chemistry & Chemical Engineering.
22
Teaching
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Captions for Scheme and Figures Table 1 IC50 values (µM) of the complexes against the selected cell lines. Scheme 1 The structures of complexes 1-3.
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Fig. 1 Comet assay of HepG2 cell (a) exposure to 25 µM of complexes 1 (b), 2 (c) and 3 (d) for 24 h.
Fig. 2 (A) Apoptosis in HepG2 cells (a) exposure to 25 µM of complexes 1 (b), 2 (c)
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and 3 (d) for 24 h and the cells were stained with AO/EB. (B) The percentage of
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apoptotic cell was determined by flow cytometry. HepG2 cells (a) exposure to 25 µM of complexes 1 (b), 2 (c) and 3 (d) for 24 h.
Fig. 3 (A) Autophagy in HepG2 cell (a) was treated with 25 µM of complexes 1 (b), 2 (c) and 3 (d) for 24 h and the cells were stained with MDC. (B) MDC
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fluorescent intensity in the autophagy was determined by flow cytometry while HepG2 cells (a) were exposed to 25 and 50 µM of complexes 1 (b and c), 2 (d and e) and 3 (f and g) for 24 h. (C) The conversion of LC3-I to LC3-II
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and expression of Beclin-1 protein was assayed by western blot after HepG2
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cells were incubated with 25 µM of complexes 1-3 for 24 h. Fig. 4 Intracellular ROS levels in HepG2 cells (a) exposed to 25 and 50 µM of complexes 1 (b and c), 2 (d and e) and 3 (f and g) for 24 h and the DCF fluorescent intensity was determined by flow cytometry.
Fig. 5 (A) The location of the complexes in the mitochondria in HepG2 cell exposure to 12.5 µM of complexes 1-3 for 4 h. (B) The changes of mitochondrial membrane potential was studied after HepG2 cells were treated with 25 µM of 27
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complexes 1-3 for 24 h. *P < 0.05 represents significant differences compared with control.
Fig. 6 (A) Microscope images of invading A549 cells that have migrated through the
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Matrigel: the extent of inhibition of cell invasion by complexes 1 (b), 2 (c) and
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3 (d) against HepG2 (a) cells can be seen from the decrease in the numbers of invading cells. (B) The percentage of invading HepG2 cells induced by 25 and 50 µM of complexes 1-3 for 24 h. *P < 0.05 represents significant differences compared with control.
h.
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Fig. 7 The cell cycle arrest in HepG2 cells exposed to 25 µM of complexes 1-3 for 24
Fig. 8 Western blot analysis of caspase 3, Bcl-2, Bcl-x, Bak and Bax in HepG2 cells
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control.
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treated with 25 µM of complexes 1-3 for 24 h. β-actin was used as internal
Fig. 9 The molecular mechanism of the complexes induced apoptosis in HepG2 cell.
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Table 1 IC50 values (µM) of the complexes against the selected cell lines HepG2
HeLa
A549
BEL-7402
SiHa
SGC-7901
LO2
MPIP
> 200
60.2 ± 5.2
> 200
> 200
> 200
> 200
> 200
1
25.5 ± 3.5
68.0 ± 5.5
> 200
> 200
> 200
41.6 ± 6.0
> 200
2
35.6 ± 1.9
> 200
3
27.4 ± 2.3
82.1 ± 4.6
Cisplatin
22.8 ± 2.1
7.3 ± 0.8
> 200
24.6 ± 1.3
> 200
> 200
> 200
33.8 ± 4.0
13.9 ± 0.8
50.6 ± 4.9
> 200
49.6 ± 1.2
6.3 ± 1.0
11.2 ± 1.2
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ACCEPTED MANUSCRIPT Highlights Three new ruthenium (II) complexes were synthesized and characterized. The cytotoxicity in vitro, apoptosis, ROS and mitochondrial membrane potential
The comet assay and cell invasion was investigated.
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were studied.
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The protein expression induced by the complexes was assayed by western blot.
S0223-5234(17)30588-3
DOI:
10.1016/j.ejmech.2017.07.066
Reference:
EJMECH 9629
To appear in:
European Journal of Medicinal Chemistry
Received Date: 13 May 2017 Revised Date:
24 July 2017
Accepted Date: 27 July 2017
Please cite this article as: D. Wan, B. Tang, Y.-J. Wang, B.-H. Guo, H. Yin, Q.-Y. Yi, Y.-J. Liu, Synthesis and anticancer properties of ruthenium (II) complexes as potent apoptosis inducers through mitochondrial disruption, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.07.066. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract Three ruthenium (II) complexes were synthesized and characterized. The anticancer activity was evaluated by cytotoxicity in vitro, apoptosis, cell invasion, cell cycle
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arrest, ROS, mitochondrial membrane potential and western blot.
ACCEPTED MANUSCRIPT
Submitted to Eur. J. Med. Chem.
Synthesis and anticancer properties of ruthenium (II) complexes as
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potent apoptosis inducers through mitochondrial disruption
Dan Wana, Bing Tanga, Yang-Jie Wanga, Bo-Hong Guoa,*, Hui Yinc,*, Qiao-Yan
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006,
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a
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Yia, Yun-Jun Liua,b,*
PR China c
Guangdong Cosmetics Engineering & Technology Research Center, Guangzhou,
510006, PR China
Department of Microbiology and Immunology, Guangdong Pharmaceutical
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c
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University, Guangzhou 510006, PR China
*Corresponding
author.
address:
[email protected]
[email protected] (H. Yin); [email protected] (Y.J. Liu). 1
(B.H.
Guo);
ACCEPTED MANUSCRIPT Abstract
A
new
ligand
MHPIP
(MHPIP
=
2-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its three ruthenium
(II)
complexes
[Ru(N-N)2(MHPIP)](ClO4)2
(N-N
=
phen:
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1,10-phenanthroline 1; dmp = 2,9-dimethyl-1,10-phenanthroline 2; ttbpy = 4,4'-ditertiarybutyl-2,2'-bipyridine 3) were synthesized and characterized. The cytotoxic activity in vitro was studied by MTT method. The complexes 1-3 show
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moderate cytotoxic effects on the cell growth in HepG2 cells with an IC50 value of
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25.5 ± 3.5, 35.6 ± 1.9 and 27.4 ± 2.3 µM, respectively. The apoptosis was investigated with AO/EB and Annex V/PI staining methods and comet assay. The reactive oxygen species, mitochondrial membrane potential were investigated under a fluorescent microscope. Autophagy assay shows that the complexes can cause
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autophagy and up-regulate the expression of Beclin-1 protein. Additionally, the complexes inhibit the cell growth in HepG2 cells at G0/G1 phase, and the complexes can regulate the expression of caspase 3 and Bcl-2 family proteins. The studies
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demonstrate that the complexes induce apoptosis in HepG2 cells through DNA
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damage and ROS-mediated mitochondrial dysfunction pathways.
Keywords: Ruthenium(II) complexes; DNA damage; apoptosis; autophagy; cell invasion; Bcl-2 family proteins.
1. Introduction The clinical success of cisplatin along with oxaliplatin and carboplatin as anticancer drugs has raised significant interest in the development of a wide range of transition 2
ACCEPTED MANUSCRIPT metal complexes with DNA/protein binding ability as well as potential anticancer activity [1]. However, these drugs exhibit side effects, severe toxicity and drug resistance [2,3]. Based on these reasons, many scientists are actively searching for
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other transition metal complexes as platinum alternative as antitumor candidates [4-10]. Among these metal complexes, ruthenium complexes, owing to their rich photochemical and photophysical properties and low cytotoxicity, have been paid
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great attention [11-24]. Recently, the studies of the ruthenium polypyridyl complexes
2,9-dimethyl-1,10-phenanthroline,
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on the bioactivity have made great progress. [Ru(dmp)2(dhbn)](ClO4)2 (dmp = dhbn
12,13-diphenyl-4,5,9,11,14,16-hexaazadibenzo[a,c]naphthacene)
= can
effectively
inhibit the cell growth in HepG2 cells with an IC50 value of 17.7 ± 1.1 µM. This
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complex induces apoptosis in HepG2 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway [25]. Chen et al. reported that RuPOP, a ruthenium polypyridyl complex with potent antitumor activity, was able to effectively
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inhibit growth and metastasis of MDA-MB-231 cells and synergistically enhance
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TRAIL-induced apoptosis [26]. Wang et al. reported that dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers could enter HeLa cells efficiently and localize within lysosomes [27]. [Ru(MeIm)(npip)]2+ (npip = 2-(4-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline) induces A549 cells apoptosis by acting on both mitochondrial homeostasis destruction and death receptor signal pathways. This complex may be a candidate as a chemotherapeutic agent against human tumor [28]. To obtain more information on the anticancer 3
ACCEPTED MANUSCRIPT activity of ruthenium complexes, in this article, a new ligand MHPIP (MHPIP = 2-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its three ruthenium(II)
complexes
[Ru(N-N)2(MHPIP)](ClO4)2
(N-N
=
phen:
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1,10-phenanthroline 1; dmp = 2,9-dimethyl-1,10-phenanthroline 2; ttbpy = 4,4'-ditertiarybutyl-2,2'-bipyridine 3) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1H NMR and
13
C NMR. The anticancer activity of
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the complexes was investigated by cytotoxicity in vitro, apoptosis, reactive oxygen
family proteins expression.
2. Results and discussion
The
ligand
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2.1. Synthesis and characterization
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species, mitochondrial membrane potential, cell cycle arrest, autophagy and Bcl-2
MHPIP
was
prepared
through
condensation
of
1,10-phenanthroline-5,6-dione with 1-methyl-1H-pyrazole-4-carboxaldehyde using a
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similar method to that described by Steck and Day [29]. The corresponding Ru(II)
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complexes were synthesized by direct reaction of MHPIP with the appropriate precursor complexes in ethylene glycol. The desired Ru(II) complexes were isolated as the perchlorates and purified by column chromatography. Each synthetic step involved here is straightforward and provides a relatively high yield of the desired product in pure form. In the ESI-MS spectra for the Ru(II) complexes, all of the expected signals [M-2ClO4-H]+ and [M-2ClO4]2+ were observed. The measured molecular weights were consistent with the expected values. 4
ACCEPTED MANUSCRIPT The absorption spectra of 10 µM of complexes 1-3 mainly consist of three resolved bands in range 200–600 nm (Fig. S1). The bands at 270-290 nm is attributed to intraligand (IL) π–π* transitions, the band at 340-360 nm is attributed to the π–π*
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transition and the lowest energy bands at 450-465 nm is assigned to the metal-to-ligand charge transfer (MLCT) transitions. Complexes 1 and 3 can emit in PBS solution at room temperature with a maximum appearing at 583 nm for 1 and
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608 nm for 3, respectively, whereas complex 2 can not emit at ambient temperature
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(Fig. S1b).
The stability of the complexes in PBS solution was studied by UV-Vis spectra at room temperature. As shown in Fig. S2, no obvious changes in absorbance of 10 µM complexes 1 (a), 2 (b) and 3 (c) at 0 h and 24 h were observed, which indicates that
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the complexes are stable in PBS solution.
2.2. The cytotoxic activity evaluation in vitro of the complexes
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The cytotoxic activity of the complexes 1-3 was evaluated against a set of cancer
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cell lines (HepG2, HeLa, A549, BEL-7402, SiHa, SGC-7901) and human normal hepatocyte LO2 cells. Cisplatin was used as a control, the IC50 values are listed in Table 1. As expectation, the ligand shows no cytotoxic activity against HepG2, A549, BEL-7402, SiHa, SGC-7901 and LO2 cells. The complex 3 displays cytotoxicity toward most selected cancer cell lines. Complexes 1 and 2 have no cytotoxic activity against A549 and SiHa cell lines. This may be caused by larger hydrophobicity of ancillary ligand ttbpy than those of phen and dmp. Thus, it is easy for complex 3 to 5
ACCEPTED MANUSCRIPT enter into the cells compared with complexes 1 and 2 under the same condition. The cytotoxic effect of complexes 1-3 is lower against the selected cancer cells than cisplatin under identical condition, but their cytotoxic activity is comparable to that of
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[Ru(dmp)2(AQTP)]2+ (IC50 = 32.9 ± 3.1 µM) toward HepG2 cells [13]. Interestingly, complexes 1 and 2 have no cytotoxic activity, complex 3 exhibits low cytotoxic activity against human normal hepatocyte LO2 cells. The results reveal that the ligand
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bonded to metal to form metal complexes, the cytotoxic activity can be enhanced.
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Because all complexes 1-3 show moderate cytotoxic effect on the cell growth in HepG2, this cell was selected to undergo the following experiments.
2.3. DNA damage studies
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DNA fragmentation is a hallmark of apoptosis, mitotic catastrophe or both [30]. The DNA damage was investigated by single cell gel electrophoresis (comet assay) in an agarose gel matrix. As shown in Fig. 1, in the control (a), HepG2 cells do not show
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any comet like appearance. After HepG2 cells were treated with 25 µM of the
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complexes 1 (b), 2 (c) and 3 (d) for 24 h, the cell nuclei were stained with EB, the HepG2 cell demonstrates statistically significant and well-formed comets, and the length of the comet tail represents the extent of DNA damage. These results indicate that the three complexes indeed induce DNA fragmentation, which is further evidence of apoptosis.
2.4. Apoptosis assay with AO/EB and Annex V/PI double staining methods 6
ACCEPTED MANUSCRIPT The comet assay suggests that the complexes can induce apoptosis in HepG2 cells. Apoptosis and necrosis, two major types of cell death, are delineated in response to a death stimulus. Disruption or inappropriate regulation of apoptotic and necrosis
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processes can result in several diseases including cancer [31,32]. The morphological changes of the cells was assayed with acridine orange (AO)/ethidium bromide (EB) stained cells. As shown in Fig. 2A, in the control (a), the living cells show bright
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green with intact cell nuclei. After the treatment of HepG2 cells with 25 µM of
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complexes 1 (b), 2 (c) and 3 (d) for 24 h, the apoptotic cells with apoptotic features such as cell blebbing, nuclear shrinkage and chromatin condensation were observed. To quantitatively compare the apoptotic effect of the complexes, Annex V/PI double staining was used to evaluate the percentage of apoptotic cells. As shown in Fig. 2B,
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HepG2 cells were treated with 25 µM of complexes 1 (c), 2 (e) and 3 (g) for 24 h, the percentage of apoptotic (Q3) cells was increased to 5.44% for 1, 3.18% for 2 and 16.5% for 3 in comparison with the control (1.58%). The apoptotic effects follow the order
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cell.
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of 3 > 1 > 2. The results indicate that the complexes can induce apoptosis in HepG2
2.5. Autophagy induced by the complexes Autophagy is a lysosomal degradation pathway and it is essential for
homeostasis under normal conditions. Autophagy is not only a survival response to growth factor or nutrient deprivation but also a way for tumor cell suppression [33,34]. To investigate the autophagic effect of the complexes on HepG2 cells, the cells were 7
ACCEPTED MANUSCRIPT treated with complexes 1-3 for 24 h, then the cells were stained with monodansylcadaverine (MDC) [35]. As shown in Fig. 3A, in the control (a), no obvious green fluorescent points were observed. After the treatment of HepG2 cell
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with 25 µM of complexes 1, 2 and 3 for 24 h, a number of bright green fluorescent points were observed, which indicates that the autophagic vacuoles were formed. The MDC fluorescent intensity is proportional to the autophagic effects. As shown in Fig.
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3B, exposure of HepG2 cell to 25.0 µM of complexes 1-3 led to an increase of MDC
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fluorescent intensity of 170.81 times for 1, 2.72 times for 2 and 3.83 times for 3 than that of control, respectively. The increase of MDC fluorescent intensity demonstrates the increasing number of autophagic cells. Moreover, the autophagic effect shows a concentration-dependent manner. In addition, the conversion of LC3-I to LC3-II is a
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hallmark of autophagy, and Beclin-1 is necessary to form autophagosomes in autophagy. As seen in Fig. 3C, the conversion of LC3-I to LC3-II occurred and there were significant increases in the expression of Beclin-1 protein in HepG2 cells treated
EP
with 25 µM of the complexes 1-3. Taken the above results together, the results
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indicate that the complexes can induce autophagy in HepG2 cells.
2.6. Intracellular reactive oxygen species levels determination It has been reported that apoptosis can be triggered by increasing intracellular
ROS levels [36], and Ru(II) polypyridyl complexes can markedly increase the intracellular ROS levels [4, 37-39]. To study whether the cell death induced by the complexes is dependent on the ROS levels, HepG2 cells were exposed to the 8
ACCEPTED MANUSCRIPT complexes for 24 h, and then the ROS levels were determined using a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as fluorescent probe [40,41]. This dye is cleaved by intracellular esterases into its non-fluorescent form (DCFH).
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Then the non-fluorescent substrate is oxidized by intracellular free radicals to produce a fluorescent product, dichlorofluorescein (DCF). The DCF fluorescent intensity was determined by flow cytometry. As shown in Fig. 4, after the treatment of HepG2 cells
SC
(a) with 25 µM of complexes 1 (b), 2 (d) and 3 (f) for 24 h, the DCF fluorescent
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intensity increases 6.22 times for 1, 1.39 times for 2 and 1.33 times for 3 than that of control. Exposure HepG2 cells to 50 µM of complexes 1 (c), 2 (e) and 3 (g) for 24 h, the DCF fluorescent intensity increases 13.62, 1.45 and 1.54 times than the original, respectively. The intracellular ROS levels depend on the balance of generation and
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consumption of ROS induced by the complexes. Complex 1 induces the highest enhancement in the ROS levels among the three complexes. This may be caused by higher generation and lower consumption of intracellular ROS levels of complex 1
EP
than those of complexes 2 and 3. These data reveal that the complexes can increase
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the intracellular ROS levels.
2.7. Location assay of the complexes and evaluation of the mitochondrial membrane potential changes
Mitochondrial changes, including loss of mitochondrial membrane potential (∆Ψm), are key events that take place during drug-induced apoptosis [42]. The maintenance of mitochondrial membrane potential (∆Ψm) is important for 9
ACCEPTED MANUSCRIPT mitochondrial integrity and bioenergetic function [43]. To study the location of the complexes in the mitochondria, Mito Tracker ® Deep Green FM (ThermoFisher, 100 nM) was used as red fluorescent probe. As shown in Fig. 5A, in the control, the
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mitochondria were stained in green. After the treatment of HepG2 cells with 12.5 µM of complexes 1-3 for 4 h, the complexes emit red fluorescence. The merge of the red and green indicates that the complexes entered into the mitochondria. The
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mitochondrial membrane potential induced by the complexes was investigated using
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JC-1 as a fluorescent probe. As shown in Fig. 5B, in the control (a), JC-1 emits red fluorescence corresponding to high mitochondrial membrane potential. However, HepG2 cells were incubated with cccp (b, positive control) and 25.0 µM of complexes 1 (c), 2 (d) and 3 (e) for 24 h, JC-1 emits bright green fluorescence, which
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corresponds to low mitochondrial membrane potential. To compare the effect of the complexes on the changes of mitochondrial membrane potential, the ratio of the green/red fluorescent intensity was determined by flow cytometry. As shown in Fig.
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5C, in the control, the ratio of green/red fluorescence is 2.26. Treatment of the cells
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with 50.0 µM of complexes 1-3, the ratios of green/red fluorescence are 3.69, 3.64 and 5.51, respectively. The increases of the ratio demonstrate that the green fluorescent intensities increase and the red fluorescent intensities reduce. The efficiency of the complexes on mitochondrial membrane potential exhibits a concentration-dependent manner and follows the order of 3 > 2 > 1. The results show that the complexes can induce a decrease in the mitochondrial membrane potential.
10
ACCEPTED MANUSCRIPT 2.8. Cell invasion studies Metastasis, the main cause of death of cancer patients, is the spread of tumour cells from a primary tumour source to other organs, and remains as one of the ultimate
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challenges in cancer treatment [44]. Thus, it is urgent to find an effective drug to inhibit cell invasion. The effects of the complexes on the cell invasion were studied by Matrigel invasion assay. As shown in Fig. 6A and 6B, HepG2 cells were treated
SC
with 50 µM of complexes 1, 2 and 3 for 24 h, the percentage of inhibiting the cell
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invasion reaches 86.5%, 81.9% and 60.9%, respectively. Complex 1 displays the highest inhibiting efficiency among the three complexes. Furthermore, the complexes reveal a concentration-dependent manner to inhibit the cell invasion.
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2.9. The cell cycle distribution analysis
Cell cycle and control of apoptosis are the significant regulatory mechanisms of cell growth, development, and differentiation. Cell cycle checkpoints guarantee the
EP
maintenance of genomic integrity by preventing DNA damage and incomplete DNA
AC C
cells from further cell division [45]. The effect of the complexes on cell cycle progression of HepG2 cells was examined by flow cytometry. As shown in Fig. 7, in the control, the percentage in the cells at G0/G1 is 54.9%. The treatment of HepG2 cells led to an increase of 4.1% for 1, 10.7% for 2 and 5.3% for 3 in the cells at G0/G1 phase, which was accompanied by corresponding reduction of percentage in the cells at G2/M phase. The data demonstrate that complexes 1, 2 and 3 inhibit cell proliferation of HepG2 cells at G0/G1 phase. From these results we can conclude that 11
ACCEPTED MANUSCRIPT growth inhibition of HepG2 cells by the complexes involves a profound cell cycle arrest in G0/G1 phase, indicating its potential of interaction with DNA.
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2.10. The effect of the complexes on the expression of Bcl-2 family proteins Caspase 3 is one of the key executioners of apoptosis. The activation of caspase 3 is generally regarded as one of the most obvious characteristics in the apoptosis
SC
stage for many cells [46]. The effect of the complexes on the expression of caspase is
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shown in Fig. 8, treatment of HepG2 cells with 25 µM of complexes 1-3 for 24 h resulted in an increase in the expression levels of caspase 3. As expectation, the complexes treated the HepG2 cells for 24 h and down-regulated the expression of anti-apoptotic proteins Bcl-2 and Bcl-x. The activation of pro-apoptotic proteins is
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helpful for inducing apoptosis. HepG2 cells were incubated with 25 µM of complexes 1-3, the expression levels of Bak and Bax were up-regulated. Taken above together, we conclude that the complexes induce apoptosis in HepG2 cell through the following
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two pathways (Fig. 9): (I) the complexes enhance ROS levels, and induce a decrease
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in the mitochondrial membrane potential. The changes of mitochondrial membrane potential activate the expression of caspase 3. Then the activation of caspase 3 stimulates the cell apoptosis. (II) The complexes can cause DNA damage, and then the complexes inhibit the cell growth at G0/G1 phase. Finally, the complexes induce cell apoptosis. This work will be helpful for design and synthesis of new ruthenium(II) complexes as potent anticancer drugs.
12
ACCEPTED MANUSCRIPT
3. Conclusions Three complexes [Ru(phen)2(MHPIP)](ClO4)2 1, [Ru(dmp)2(MHPIP)](ClO4)2 2 and [Ru(ttbpy)2(MHPIP)](ClO4)2 3 were synthesized and characterized. The
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cytotoxicity in vitro shows that the complexes display moderate cytotoxic activity toward HepG2 cells. Complexes 1-3 can induce apoptosis, enhance the intracellular ROS levels and induce a decrease in the mitochondrial membrane potential. MDC
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staining demonstrates that the complexes can cause autophagy and up-regulate the
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expression of Beclin-1 protein. The cell cycle distribution indicates that the complexes inhibit the cell growth in HepG2 cells at G0/G1 phase. The cell invasion shows that the complexes can effectively inhibit the cell invasion. In addition, the complexes can activate caspase 3, down-regulate the expression of Bcl-2 and Bcl-x
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proteins, and up-regulate the expression levels of proapoptotic proteins Bak and Bax. In summary, the complexes induce apoptosis through DNA damage and
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ROS-mediated mitochondrial dysfunction pathways.
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4. Experimental
4.1. Materials and methods All reagents and solvents were purchased commercially and used without further
purification unless otherwise indicated. Ultrapure MilliQ water was used in all experiments. DMSO, 4,4'-ditertiary-butyl-2,2′-bipyridine and RPMI 1640 were purchased from Sigma. 1,10-phenanthroline was obtained from the Guangzhou Chemical Reagent Factory. Cell lines of HepG2 (human hepatocellular carcinoma), 13
ACCEPTED MANUSCRIPT HeLa (human (hepatocellular),
cervical cancer), SiHa
A549
(human
cervical
(human lungcarcinoma), carcinone),
SGC-7901
BEL-7402 (gastric
adenocarcinoma) and human normal hepatocyte LO2 cells were purchased from the
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American Type Culture Collection. RuCl3·3H2O was purchased from the Kunming Institution of Precious Metals. Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental analyzer. Electrospray ionization mass spectra
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(ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA) using acetonitrile
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as mobile phase. The spray voltage, tube lens offset, capillary voltage and capillary temperature were set at 4.50 kV, 30.00 V, 23.00 V and 200 °C, respectively, and the quoted m/z values are for the major peaks in the isotope distribution. 1H NMR and 13C NMR spectra were recorded on a Varian-500 spectrometer with DMSO-d6 as solvent
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and tetramethylsilane (TMS) as an internal standard at 500 MHz at room temperature.
4.2. Stability of the complexes in buffer
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For stability studies, all the complexes were first dissolved in a minimum amount
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of DMSO (0.5% of the final volume) and then diluted with PBS to a required concentration. The stability was analyzed by monitoring the electronic spectra of the complexes over 24 h.
4.3. Synthesis of ligand and complexes 4.3.1. Preparation of ligand MHPIP A mixture of 1,10-phenthroline-5,6-dione (0.210 g, 1.00 mmol) [47], 14
ACCEPTED MANUSCRIPT 1-methyl-1H-pyrazole-4-carboxaldehyde (0.165 g, 1.5 mmol), ammonium acetate (2.31 g, 30 mmol) and glacial acetic acid (20 mL) was refluxed with stirring for 2 h. The cooled solution was diluted with water and neutralized with concentrated aqueous
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ammonia. The brown precipitate was collected and purified by column chromatography on silica gel (60–100 mesh) with ethanol as eluent to give the compound as a brown yellow powder. Yield: 0.360 g, 80%. Anal. Calc for C17H12N6:
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C, 67.99; H, 4.03; N, 27.98%. Found: C, 67.81; H, 4.20; N, 27.87%. IR (KBr, cm−1):
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3117, 2811, 1614, 1581, 1567, 1505, 1432, 1399, 1357, 1182, 1118, 1073, 1018, 861, 803, 738. FAB-MS: m/z = 301 [M + 1].
4.3.2. Synthesis of complex [Ru(phen)2(MHPIP)](ClO4)2 (1)
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A mixture of [Ru(phen)2Cl2]·2H2O [48] (0.280 g , 0.5 mmol) and MHPIP (0.150 g, 0.5 mmol) in ethylene glycol (20 mL) was heated at 150 oC under argon for 8 h to give a clear red solution. After cooling to room temperature, a red precipitate
EP
was obtained by the addition of an excess of saturated aqueous NaClO4 solution. The
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crude product was purified by column chromatography on neutral alumina with a mixture of CH3CN-toluene (3:1, v/v) as eluent. The red band was collected. The solvent was removed under reduced pressure and a red powder was obtained. Yield: 0.336 g, 70%. Anal. Anal. Calc for C41H28N10Cl2O8Ru: C, 51.26; H, 2.94; N, 14.58%. Found: C, 51.15; H, 3.08; N, 14.41%. IR (KBr, cm−1): 3390, 3049, 1615, 1496, 1476, 1426, 1364, 1307, 1197, 1143, 1121, 1086, 1013, 846, 721, 625. 1H NMR: 8.95 (d, 2H, J = 8.0 Hz), 8.77 (dd, 4H, J = 8.5, J = 8.5 Hz), 8.49 (s, 1H), 8.39 (s, 4H), 8.18 (s, 1H), 15
ACCEPTED MANUSCRIPT 8.12 (dd, 2H, J = 5.5, J = 5.5 Hz), 8.08 (dd, 2H, J = 5.5, J = 5.0 Hz), 7.98 (dd, 2H, J = 5.5, J = 5.5 Hz), 7.78-7.75 (m, 6H), 3.98 (s, 3H).
13
C NMR (DMSO-d6, 125 Hz):
152.77, 152.62, 150.03, 147.83, 147.24, 147.16, 145.06, 137.43, 136.78, 130.44,
4.3.3. Synthesis of [Ru(dmp)2(MHPIP)](ClO4)2 (2)
SC
([M-2ClO4]2+).
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130.38, 130.17, 128.05, 126.32, 126.02, 112.91, 40.33. ESI-MS (CH3CN): m/z 381.1
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This complex was synthesized in a manner identical to that described for 1, with [Ru(dmp)2Cl2]·2H2O [49] in place of [Ru(phen)2Cl2]·2H2O. Yield: 0.366 g, 72%. Anal. Calc for C45H36N10Cl2O8Ru: C, 53.16; H, 3.57; N, 13.78%. Found: C, 53.26; H, 3.46; N, 13.91%. IR (KBr, cm−1): 3379, 3085, 1615, 1596, 1509, 1439, 1412, 1370,
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1349, 1196, 1013, 843, 726, 557. 1H NMR: 13.92 (s, 1H), 8.91 (d, 2H, J = 8.5 Hz), 8.79 (d, 2H, J = 8.0 Hz), 8.45 (s, 1H), 8.41 (d, 4H, J = 8.5 Hz), 8.24 (d, 2H, J = 8.5 Hz), 8.10 (s, 1H), 7.97 (d, 2H, J = 8.0 Hz), 7.52-7.46 (m, 2H), 7.36 (t, 2H, J = 5.5 Hz),
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7.22 (d, 2H, J = 6.0 Hz), 3.97 (s, 3H), 1.93 (s, 6H), 1.71 (s, 6H). 13C NMR (DMSO-d6,
AC C
125 Hz): 167.94, 166.37, 158.26, 148.88, 147.79, 138.10, 137.36, 136.67, 130.34, 129.51, 128.91, 128.21, 127.52, 127.39, 127.11, 126.65, 126.53, 125.54, 125.32, 123.57, 112.66, 40.28, 25.55, 24.85, 24.51. ESI-MS (CH3CN): m/z 408.8 ([M-2ClO4]2+).
4.3.4. Synthesis of [Ru(ttbpy)2(MHPIP)](ClO4)2 (3) This complex was synthesized in a manner identical to that described for 1, with 16
ACCEPTED MANUSCRIPT [Ru(ttbpy)2Cl2]·2H2O [48] in place of [Ru(phen)2Cl2]·2H2O. Yield: 0.409 g, 71%. Anal. Calc for C53H60N10Cl2O8Ru: C, 55.98; H, 5.32; N, 12.32%. Found: C, 55.76; H, 5.43; N, 12.24%. IR (KBr, cm−1): 3368, 3087, 2961, 1615, 1598, 1539, 1482, 1414,
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1368, 1252, 1197, 1014, 839, 726, 558. 1H NMR: 8.96 (d, 2H, J = 7.5 Hz), 8.86 (dd, 4H, J = 8.0, J = 8.0 Hz), 8.48 (s, 1H), 8.17 (s, 1H), 7.98 (d, 2H, J = 5.5 Hz), 7.92 (t, 2H, J = 5.0 Hz), 7.66 (d, 2H, J = 6.5 Hz), 7.71 (dd, 2H, J = 6.5, J = 6.0 Hz), 7.45 (d,
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2H, J = 6.0 Hz), 7.32 (dd, 2H, J = 6.0, J = 6.0 Hz), 4.00 (s, 3H), 1.43 (s, 18H), 1.34 (s,
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18H). 13C NMR (DMSO-d6, 125 Hz): 161.91, 161.73, 156.57, 156.39, 150.83, 150.71, 147.84, 137.44, 130.43, 124.84, 124.48, 121.79, 112.82, 40.21, 35.56, 35.43, 30.11, 30.00. ESI-MS (CH3CN): 937.0 ([M-2ClO4-H]+), 469.0 ([M-2ClO4]2+).
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4.4. Cytotoxicity in vitro assay
The cytotoxicity in vitro was determined by MTT assay [50]. Cancer cells (8 × 103 cells per well) were seeded in 96-well for 24 h. Cells was incubated with the
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tested compounds to achieve final concentrations ranging from 10-6 to 10-4 M. Control
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wells were prepared by addition of culture medium (100 µL) and cisplatin was used as a positive control. After 48 h incubation, 10 µL of MTT dye solution (5 mg/mL−1) was added to each well. After incubation at 37 oC for 4 h, buffer (100 µL) containing dimethylformamide (50%) and sodium dodecyl sulfate (20%) was added to transform MTT to a purple formazan dye. The optical density of each well was then measured for three times to obtain the mean values. The IC50 values were analyzed by software of SPSS. 17
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4.5. Apoptosis studies with AO/EB staining method HepG2 cells (2 × 105) were exposed to 25 µM of the complexes and cultured in
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RPMI (Roswell Park Memorial Institute) 1640 with 10% of fetal bovine serum (FBS) and incubated at 37 °C in 5% CO2 for 24 h. The cells were washed with ice-cold phosphate buffer saline (PBS), and fixed with formalin (4%, w/v). Cell nuclei were
SC
counterstained with acridine orange (AO) and ethidium bromide (EB) (AO: 100 µg/
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mL, EB: 100 µg/mL) or Hoechst 33,258 for 10 min. The cells were observed and imaged with a fluorescence microscope (Nikon, Yokohama, Japan) with excitation at 350 nm and emission at 460 nm.
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4.6. Annexin V-FITC apoptosis detection
HepG2 cells were seeded into 6-well plates at a density of 1 × 106 cells per well and incubated for 24 h. The different concentration of compounds were added into the
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above well for 24 h, cells were collected and washed with PBS twice, and then stained
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with fluorescein isothiocyanate (FITC)-conjugated Annexin V and then PI. Cells were quantified by a FACS Calibur flow cytometry (Beckman Dickinson & Co., Franklin Lakes, NJ).
4.7. Autophagy induced by the complexes HepG2 cells were seeded onto chamber slides in 12-well plates and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS and 18
ACCEPTED MANUSCRIPT incubated at 37 °C in 5% CO2. The medium was removed and replaced with medium (final DMSO concentration, 0.05% v/v) containing 25 µM of complexes 1-3 for 24 h. The medium was removed again, and the cells were washed with ice-cold PBS twice.
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Then the cells were stained with MDC (monodansylcadaverine) solution (50 µM) for 10 min and washed with PBS twice. The cells were observed and imaged under
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Beclin-1 protein was assayed by western blot.
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fluorescence microscope. The effect of the complexes on the expression of LC3 and
4.8. Reactive oxygen species (ROS) detection
Intracellular ROS levels were measured with a fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA). HepG2 cells were seeded into
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6-well plates at a density of 1 × 106 cells per well. After incubation for 24 h, the medium was replaced with medium containing different concentrations of compounds for 24 h. Then the cells were stained with 20 µM DCFH-DA in PBS for 30 min in the
EP
dark. Finally, the cells were harvested and washed twice with PBS. The data were
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obtained by flow cytometry.
4.9. Location assay of the complexes in the mitochondria HepG2 cells were placed in 24-wellmicroassay culture plates (4 × 104 cells per
well) and grown overnight at 37 oC in a 5% CO2 incubator. 12.5 µM of the complexes was added to the wells at 37 oC in a 5% CO2 incubator for 4 h and further co-incubated with Mito Tracker ® Deep Green FM (100 nM) at 37 oC for 1 h. Upon 19
ACCEPTED MANUSCRIPT completion of the incubation, the wells were washed three times with ice-cold PBS. After discarding the culture medium, the cells were imaged under a fluorescence
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microscope.
4.10. Measurement of mitochondrial membrane potential (MMP, △Ψm)
HepG2 cells (2 × 105 per well) were treated using compounds for 24 h. JC-1 (1
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mg˖ml-1) as fluorescence probe for determination of MMP was added to stain cells at
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37 oC for 30 min. Then the cells were washed twice with PBS and the fluorescent intensity of JC-1 was determined by flow cytometry.
4.11. Cell cycle arrest studies
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HepG2 cells were seeded into six-well plates (Costar, Corning Corp., New York) at a density of 1 × 106 cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS, and incubated at 37 °C and 5% CO2.
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The medium was removed and replaced with medium (final DMSO concentration
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0.05% v/v) containing 25 µM complexes 1-3. After incubation for 24 h, the cell layer was trypsinized and washed with cold PBS and fixed with 70% ethanol. Twenty µL of RNAse (0.2 mg/mL) and 20 µL of propidium iodide (0.02 mg/mL) were added to the cell suspensions and the mixtures were incubated at 37 °C for 30 min. The samples were then analyzed with a FACS Calibur flow cytometry. The number of cells analyzed for each sample was 10,000.
20
ACCEPTED MANUSCRIPT 4.12. Anti-metastasis effect The BD BioCoat™ Matrigel™ invasion chamber (BD Biosciences) was used according to the manufacturer's instructions. Compounds were dissolved in cell media
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at the desired concentration and added into Matrigel. Twenty-five thousands of HepG2 cells in serum free media were then seeded in the top chamber of the two chamber Matrigel system. To the low compartment, RPMI and 5% FBS were added
SC
as chemo-attractant. Cells were allowed to invade for 24 h. After incubation,
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non-invading cells were removed from the upper surface and cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% of crystal violet. Membranes were photographed and the invading cells were counted under a light
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microscope. Mean values from three independent assays were calculated.
4.13. Western blot analysis
HepG2 cells were seeded in 3.5 cm dishes for 24 h and incubated with 25.0 µM
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of complexes 1-3 in the presence of 10% FBS. Then cells were harvested in lysis
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buffer. After sonication, the samples were centrifuged for 20 min at 13 000g. The protein concentration of the supernatant was determined by BCA assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done loading equal amount of proteins per lane. Gels were then transferred to poly(vinylidene difluoride) membranes (Millipore) and blocked with 5% non-fat milk in TBST (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) buffer for 1 h. The membranes were incubated with primary antibodies (1:5000 dilution) in 5% non-fat milk overnight at 4 21
ACCEPTED MANUSCRIPT o
C, and after washed four times with TBST for a total of 30 min, then the secondary
antibodies conjugated with horseradish peroxidase at 1:5000 dilution for 1 h at room temperature and washed four times with TBST. The blots were visualized with the
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All data were expressed as mean ± SD. Statistical significance was evaluated using t-tests. Differences were considered to be significant when the *P value was less than
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Acknowledgements
This work was supported by the National Nature Science Foundation of China (No.
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2016A030313726, 2016A030313728) and Project of Innovation for Enhancing Guangdong
Pharmaceutical
University,
Provincial
Experimental
Demonstration Center of Chemistry & Chemical Engineering.
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Captions for Scheme and Figures Table 1 IC50 values (µM) of the complexes against the selected cell lines. Scheme 1 The structures of complexes 1-3.
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Fig. 1 Comet assay of HepG2 cell (a) exposure to 25 µM of complexes 1 (b), 2 (c) and 3 (d) for 24 h.
Fig. 2 (A) Apoptosis in HepG2 cells (a) exposure to 25 µM of complexes 1 (b), 2 (c)
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and 3 (d) for 24 h and the cells were stained with AO/EB. (B) The percentage of
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apoptotic cell was determined by flow cytometry. HepG2 cells (a) exposure to 25 µM of complexes 1 (b), 2 (c) and 3 (d) for 24 h.
Fig. 3 (A) Autophagy in HepG2 cell (a) was treated with 25 µM of complexes 1 (b), 2 (c) and 3 (d) for 24 h and the cells were stained with MDC. (B) MDC
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fluorescent intensity in the autophagy was determined by flow cytometry while HepG2 cells (a) were exposed to 25 and 50 µM of complexes 1 (b and c), 2 (d and e) and 3 (f and g) for 24 h. (C) The conversion of LC3-I to LC3-II
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Fig. 5 (A) The location of the complexes in the mitochondria in HepG2 cell exposure to 12.5 µM of complexes 1-3 for 4 h. (B) The changes of mitochondrial membrane potential was studied after HepG2 cells were treated with 25 µM of 27
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complexes 1-3 for 24 h. *P < 0.05 represents significant differences compared with control.
Fig. 6 (A) Microscope images of invading A549 cells that have migrated through the
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3 (d) against HepG2 (a) cells can be seen from the decrease in the numbers of invading cells. (B) The percentage of invading HepG2 cells induced by 25 and 50 µM of complexes 1-3 for 24 h. *P < 0.05 represents significant differences compared with control.
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Fig. 8 Western blot analysis of caspase 3, Bcl-2, Bcl-x, Bak and Bax in HepG2 cells
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Fig. 9 The molecular mechanism of the complexes induced apoptosis in HepG2 cell.
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Table 1 IC50 values (µM) of the complexes against the selected cell lines HepG2
HeLa
A549
BEL-7402
SiHa
SGC-7901
LO2
MPIP
> 200
60.2 ± 5.2
> 200
> 200
> 200
> 200
> 200
1
25.5 ± 3.5
68.0 ± 5.5
> 200
> 200
> 200
41.6 ± 6.0
> 200
2
35.6 ± 1.9
> 200
3
27.4 ± 2.3
82.1 ± 4.6
Cisplatin
22.8 ± 2.1
7.3 ± 0.8
> 200
24.6 ± 1.3
> 200
> 200
> 200
33.8 ± 4.0
13.9 ± 0.8
50.6 ± 4.9
> 200
49.6 ± 1.2
6.3 ± 1.0
11.2 ± 1.2
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ACCEPTED MANUSCRIPT Highlights Three new ruthenium (II) complexes were synthesized and characterized. The cytotoxicity in vitro, apoptosis, ROS and mitochondrial membrane potential
The comet assay and cell invasion was investigated.
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The protein expression induced by the complexes was assayed by western blot.