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Cytotoxicity In Vitro, Apoptosis, Cellular Uptake, Cell Cycle Distribution, Mitochondrial Membrane Potential Detection, DNA Binding, and Photocleavage of Ruthenium(ii) Complexes
CSIRO PUBLISHING
Full Paper
Aust. J. Chem. 2013, 66, 555–563
http://dx.doi.org/10.1071/CH12564
Cytotoxicity In Vitro, Apoptosis, Cellular Uptake,
Cell Cycle Distribution, Mitochondrial Membrane
Potential Detection, DNA Binding, and Photocleavage
of Ruthenium(II) Complexes
Gan-Jian Lin,A Zheng-Zheng Li,A Jun-Hua Yao,B Hong-Liang Huang,C,D
Yang-Yin Xie,A and Yun-Jun LiuA,D
A
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou,
510006, China.
B
Instrument Analysis and Research Center, Sun Yat-Sen Uiversity, Guangzhou,
510275, China.
C
School of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University,
Guangzhou, 510006, China.
D
Corresponding authors. Email: hhongliang@163.com; lyjche@163.com
Four new ruthenium(II) complexes [Ru(bpy)2(NHPIP)](ClO4)2 (Ru-1), [Ru(phen)2(NHPIP)](ClO4)2 (Ru-2), [Ru(bpy)2
(AHPIP)](ClO4)2 (Ru-3), and [Ru(phen)2(AHPIP)](ClO4)2 (Ru-4) (bpy ¼ 2,20 -bipyridine; phen ¼ 1,10-phenanthroline;
NHPIP ¼ 2-(3-nitro-4-hydroxylphenyl)imidazo[4,5-f][1,10]phenanthroline; AHPIP ¼ 2-(3-amino-4-hydroxylphenyl)
imidazo[4,5-f][1,10]phenanthroline) were synthesized and characterized by elemental analysis, electrospray mass
spectrometry, and 1H NMR spectroscopy. The cytotoxicity in vitro of these complexes against BEL-7402, HeLa,
MG-63, and MCF-7 cells was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
method. Ru-4 shows the highest cytotoxic activity towards the selected cell lines among the four complexes. The
morphological apoptosis was assayed by an acridine orange/ethidium bromide staining method, and the percentages of
necrotic and apoptotic cells were determined by flow cytometry. The cellular uptake and the cell cycle arrest in BEL-7402
cell was investigated. The results showed these complexes inhibit the proliferation of BEL-7402 cells at G0/G1 phase
arrest. The detection of mitochondrial membrane potentials using the fluorescence probe JC-1 (5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide) exhibited that the mitochondrial membrane potentials decrease.
Upon irradiation, these complexes can effectively cleave pBR322 DNA.
Manuscript received: 7 November 2012.
Manuscript accepted: 7 January 2013.
Published online: 5 February 2013.
Introduction
Stable, inert, coordinatively saturated, and water-soluble ruthenium complexes are ideally suited and extremely valuable as
non-covalent probes of both structural and functional aspects of
nucleic acid chemistry.[1–4] A majority of the reported ruthenium
complexes contain two bipyridine or phenanthroline units as
ancillary ligands. Some of the complexes exhibit unique
characteristics, for example, [Ru(bpy)2(dppz)]2þ (bpy ¼ 2,20 bipyridine; dppz ¼ dipyrido[3,2-a:20 ,30 -c]phenazine) shows no
luminescence in aqueous solution at ambient temperature, but
luminesces brightly upon binding intercalatively with the dppz
ligand between adjacent DNA base pairs, displaying the characteristic of a ‘molecular light switch’. The ruthenium(II) complex
[Ru(phen)2(mdpz)]2þ (phen ¼ 1,10-phenanthroline, mdpz ¼ 7,7methylenedioxyphenyldipyrido[3,2-a:2,3-c]phenazine), as the
first true molecular light switch, enhances the stability of the triplex RNA structure.[5] These studies on DNA or RNA as binding
targets have stimulated the development of ruthenium complexes
as anticancer agents. Schatzschneider et al. reported that
Journal compilation Ó CSIRO 2013
[Ru(bpy)2(dppz)]2þ can effectively inhibit the proliferation of
HT-29 cells, and [Ru(bpy)2(dppn)]2þ can be uptaken and had a
low IC50 (half maximal inhibitory concentration) value against
MCF-7 cells.[6] The chiral RuII complex L-[Ru(phen)2
(p-MOPIP)]2þ (p-MOPIP ¼ 2-(4-methoxylphenyl)imidazo[4,5-f]
[1,10]phenanthroline) shows a high cytotoxic effect towards
HepG-2 cells.[7] [Ru(bpy)2(dmdpq)]2þ (dmdpq ¼ 2,9-dimethyldipyrido[3,2-f:20 ,30 -h]-quinoxaline) is not cytotoxic in the dark,
but when irradiated with light (. 450 nm), this complex displays
potencies superior to cisplatin against A549 cells.[8] [Ru(Hdpa)2
(dppz)]2þ exhibits cytotoxicity ,8 times larger than cisplatin
against the human cervical epidermoid carcinoma cell line
(ME 180).[9] Complexes [Ru(phen)2(DBHIP)]2þ and [Ru(phen)2
(DNPIP)]2þ (DBHIP ¼ 2-(3,5-dibromo-4-hydroxyphenyl)imidazo
[4,5-f][1,10]phenanthroline,
DNPIP ¼ 2-(2,4-dinitrophenyl)
imidazo[4,5-f][1,10]phenanthroline) can effectively suppress the
cell proliferation of BEL-7402 cells.[10,11] Based on our previous
studies,[11–13] we found that RuII polypyridyl complexes containing
amino or nitro groups as a substituent in the ligands possess
www.publish.csiro.au/journals/ajc
556
G.-J. Lin et al.
N
[Ru(bpy)2Cl2]
CHO
N
N
H
N
N
N
Ru
N
NO2
HO
N
H
N
O HAc, NH4Ac
N
N
N
O
N
NO2
2⫹
OH
Ru-1
N
NO2
OH
N
NHPIP
N
N
H
N
N
N
N
H
N
N
N
N
H
N
N
N
Ru
[Ru(phen)2Cl2]
N
NO2
2⫹
OH
Ru-2
N
Pd/C
NH2NH2⋅H2O
[Ru(bpy)2Cl2]
N
N
N
H
N
N
N
Ru
NH2
N
NH2
2⫹
Ru-3
OH
N
OH
AHPIP
N
N
[Ru(phen)2Cl2]
Ru
N
NH2
OH
2⫹
Ru-4
N
Scheme 1. Synthetic route of ligand and complexes.
significant cytotoxicity in vitro. In order to evaluate further the
influence of nitro and amino groups as substituents on the cytotoxic
activity in vitro, in this report, two new ligands, NHPIP (2-(3-nitro4-hydroxylphenyl)imidazo[4,5-f][1,10]phenanthroline), containing a nitro group as substituent, and AHPIP (2-(3-amino-4hydroxylphenyl)imidazo[4,5-f][1,10]phenanthroline), obtained by
reducing the nitro group into an amino group, and their four
RuII polypyridyl complexes [Ru(bpy)2(NHPIP)](ClO4)2 (Ru-1),
[Ru(phen)2(NHPIP)](ClO4)2 (Ru-2), [Ru(bpy)2(AHPIP)](ClO4)2
(Ru-3), and [Ru(phen)2(AHPIP)](ClO4)2 (Ru-4, Scheme 1)
were synthesized and characterized by elemental analysis, electrospray mass spectrometry, and 1H NMR spectroscopy. Their
cytotoxicity in vitro was evaluated by MTT assay (MTT ¼ (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). The
morphological apoptosis and the percentage of necrotic and apoptotic cells of BEL-7402 induced by these complexes were also
studied by fluorescent microscopy and flow cytometry. The cellular
uptake was investigated. The cell cycle distribution induced by the
complexes was determined by flow cytometry. The mitochondrial
membrane potentials induced by Ru-1–4 were determined under
fluorescent microscopy. The DNA binding and photocleavage
were also investigated by electronic absorption titration and gel
electrophoresis. In addition, the structure–activity relationships are
also explored.
Experimental
Materials and Methods
All reagents and solvents were purchased commercially and
used without further purification unless specially noted. Ultrapure MilliQ water was used in all experiments. Dimethyl
sulfoxide (DMSO) and RPMI 1640 (RPMI ¼ Roswell Park
Memorial Institute) were purchased from Sigma. Cell lines of
BEL-7402 (human hepatocellular carcinoma cell line), HeLa
(human vercix adenocarcinoma cell line), MG-63 (human
osteosarcoma cell line), and MCF-7 (human breast cancer cell
line) were purchased from American Type Culture Collection.
RuCl3xH2O was purchased from Kunming Institution of
Precious Metals. 1,10-Phenanthroline was obtained from
Guangzhou Chemical Reagent Factory.
Physical Measurements
Microanalysis (C, H, and N) was carried out with a Perkin–
Elmer 240Q elemental analyser. Electrospray ionisation mass
spectrometry (ES-MS) was performed on a LCQ system
(Finnigan MAT, USA) using methanol as the mobile phase. Fast
atom bombardment (FAB) mass spectra were measured on a VG
ZAB-HS spectrometer in a 3-nitrobenzyl alcohol matrix. The
spray voltage, tube lens offset, capillary voltage, and capillary
temperature were set at 4.50 kV, 30.00 V, 23.00 V, and 2008C,
respectively, and the quoted m/z values are for the major peaks in
the isotope distribution. 1H NMR spectra were recorded on a
Varian-500 spectrometer with (D6)DMSO as solvent and
tetramethylsilane as an internal standard at 500 MHz at room
temperature.
Synthesis and Characterisation of Ligands and Complexes
Synthesis of Ligand NHPIP
A mixture of 1,10-phenanthroline-5,6-dione (0.315 g,
1.5 mmol),[14] 3-nitro-4-hydroxyphenylaldehyde (0.250 g,
Ruthenium(II) Complexes and Bioactivity
1.5 mmol), ammonium acetate (2.31 g, 30 mmol), and glacial
acetic acid (30 cm3) was refluxed with stirring for 2 h. The
cooled solution was diluted with water and neutralized with
concentrated aqueous ammonia. The precipitate was collected
and purified by column chromatography on silica gel (60–100
mesh) with ethanol as eluent to give the compound as a red
powder. Yield: 80 %. Anal. Calc. for C19H11N5O3: C 63.87,
H 3.10, N 19.60. Found: C 63.68, H 3.18, N 19.44 %. m/z (FAB)
358 ([M þ H]þ). dH (500 MHz, (D6)DMSO) 9.05 (dd, 2H, J 5.5,
5.0), 9.00 (dd, 2H, J 8.1, 8.1), 8.82 (d, 1H, J 8.0), 8.02 (s, 1H),
7.80 (dd, 2H, J 4.3, 4.4), 6.83 (d, 1H, J 8.0), 4.07 (s, 1H).
Synthesis of Ligand NAPIP
NHPIP (0.179 g, 0.5 mmol) was completely dissolved in
ethanol (30 cm3) with stirring for 1 h. Pd/C (0.20 g, 10 % Pd)
and NH2NH2H2O (8 cm3) were then added and the solution
refluxed for 6 h. The hot solution was filtered and evaporated to
remove the solvent under reduced pressure. The red compound
obtained was washed with cool ethanol and dried at 508C under
vacuum. Yield: 72 %. Anal. Calc. for C19H13N5O: C 69.72,
H 4.00, N 21.39. Found: C 69.61, H 4.12, N 21.26 %. m/z (FAB)
328 ([M þ 1]þ). dH (500 MHz, (D6)DMSO) 9.00 (dd, 2H, J 5.0,
4.5), 8.89 (d, 2H, J 8.2), 7.78–7.82 (m, 2H), 7.57 (d, 1H, J 7.0),
7.35 (d, 1H, J 5.2), 6.82 (d, 1H, J 8.5), 4.81 (s, 2H, HNH2), 3.36
(s, 1H, HOH).
Synthesis of [Ru(bpy)2(NHPIP)](ClO4)2 (Ru-1)
A
mixture
of
cis-[Ru(bpy)2Cl2]2H2O
(0.260 g,
0.5 mmol)[15] and NHPIP (0.193 g, 0.5 mmol) in ethylene glycol
(20 cm3) was heated at 1208C under argon for 8 h to give a clear
red solution. Upon cooling, a red precipitate was obtained by
dropwise addition of a saturated aqueous NaClO4 solution. The
crude product was purified by column chromatography on
neutral alumina with a mixture of MeCN/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:
71 %. Anal. Calc. for C39H27N9Cl2O11Ru: C 48.31, H 2.81,
N 13.00. Found: C 48.18, H 2.89, N 12.88 %. m/z (ES, CH3CN)
769.4 ([M 2ClO4 H]þ), 385.5 ([M 2ClO4]2þ). dH
(500 MHz, (D6)DMSO) 8.91 (d, 2H, J 8.5), 8.87 (d, 4H,
J 9.0), 8.78 (s, 1H), 8.21 (t, 2H, J 7.0), 8.10 (t, 4H, J 7.5), 7.96
(d, 1H, J 4.5), 7.84 (d, 4H, J 5.0), 7.57–7.61 (m, 4H), 7.35 (t, 2H,
J 7.0), 6.54 (d, 1H, J 8.5), 3.37 (s, 1H).
Synthesis of [Ru(phen)2(NHPIP)](ClO4)2 (Ru-2)
This complex was synthesized by an identical method as
described for Ru-1, with cis-[Ru(phen)2Cl2]2H2O[15] in place
of cis-[Ru(bpy)2Cl2]2H2O. Yield: 70 %. Anal. Calc. for
C43H27N9Cl2O11Ru: C 50.75, H 2.67, N 12.39. Found:
C 50.85, H 2.76, N 12.27 %. m/z (ES, CH3CN) 817.6 ([M
2ClO4 H]þ), 409.3 ([M 2ClO4]2þ). dH (500 MHz, (D6)
DMSO) 9.07 (s, 1H), 8.77 (d, 2H, J 8.5), 8.75 (d, 4H, J 7.5),
8.39 (s, 4H), 8.14 (d, 2H, J 5.0), 8.09 (d, 2H, J 5.0), 8.00 (d, 1H,
J 8.0), 7.94 (d, 2H, J 5.0), 7.75–7.79 (m, 6H), 6.61 (d, 1H, J 8.0),
3.38 (s, 1H).
Synthesis of [Ru(bpy)2(AHPIP)](ClO4)2 (Ru-3)
This complex was synthesized by an identical method as
described for Ru-1, with AHPIP in place of NHPIP. Yield: 71 %.
Anal. Calc. for C39H29N9Cl2O9Ru: C 49.85, H 3.11, N 13.42.
Found: C 49.74, H 3.03, N 13.52 %. m/z (ES, CH3CN) 739.5
([M 2ClO4 H]þ), 370.4 ([M 2ClO4]2þ). dH (500 MHz,
557
(D6)DMSO) 8.86 (dd, 4H, J 8.0, 8.0), 8.20 (t, 2H, J 6.8), 8.08
(t, 4H, J 7.5), 8.02 (d, 2H, J 5.5), 7.89–7.94 (m, 4H), 7.86 (d, 2H,
J 7.6), 7.58 (d, 4H, J 7.5), 7.33 (d, 1H, J 7.6), 7.02 (d, 1H, J 8.0),
6.55 (d, 1H, J 8.0), 4.85 (s, 2H), 3.36 (s, 1H).
Synthesis of [Ru(phen)2(AHPIP)](ClO4)2 (Ru-4)
This complex was synthesized by an identical method as
described for Ru-2, with AHPIP in place of NHPIP. Yield: 70 %.
Anal. Calc. for C43H29N9Cl2O9Ru: C 52.29, H 2.96, N 12.76.
Found: C 52.40, H 3.07, N 12.63 %. m/z (ES, CH3CN) 787.4
([M 2ClO4 H]þ), 394.5 ([M 2ClO4]2þ). dH (500 MHz,
(D6)DMSO) 8.76 (d, 4H, J 8.5), 8.38 (s, 4H), 8.11 (d, 2H,
J 6.0), 8.07 (d, 2H, J 6.5), 7.94 (d, 2H, J 5.0), 7.74–7.77 (m, 6H),
7.67 (d, 2H, J 8.5), 7.64 (d, 1H, J 8.0), 7.12 (d, 1H, J 6.0), 6.85
(d, 1H, J 8.0), 4.83 (s, 2H), 3.35 (s, 1H).
Caution: Perchlorate salts of metal compounds with organic
ligands are potentially explosive, and only small amounts of the
material should be prepared and handled with great care.
Cytotoxicity Assay In Vitro
MTT assay procedures were used.[16] Cells were placed in
96-well microassay culture plates (8 103 cells per well) and
grown overnight at 378C in a 5 % CO2 incubator. Complexes
tested were then added to the wells to achieve final concentrations ranging from 106 to 104 M. Control wells were prepared
by addition of culture medium (100 mL). The plates were incubated at 378C in a 5 % CO2 incubator for 24 h. Upon completion
of the incubation, stock MTT dye solution (20 mL, 5 mg mL1)
was added to each well. After 4 h, buffer (100 mL) containing
N,N-dimethylformamide (50 %) and sodium dodecyl sulfate
(20 %) was added to solubilize the MTT formazan. The optical
density of each well was then measured with a microplate
spectrophotometer at a wavelength of 490 nm. The IC50 values
were determined by plotting the percentage viability versus
concentration on a logarithmic graph and reading off the concentration at which 50 % of cells remain viable relative to the
control. Each experiment was repeated at least three times to
obtain the mean values. Four different tumour cell lines were the
subject of this study: BEL-7402 (human hepatocellular carcinoma cell line), HeLa (human vercix adenocarcinoma cell line),
MG-63 (human osteosarcoma cell line), and MCF-7 (human
breast cancer cell line).
Apoptosis Studies with Acridine Orange (AO)/Ethidium
Bromide (EB) Staining Method
Apoptotic studies were performed with a staining method
utilising AO and EB.[17] A monolayer of BEL-7402 cells was
incubated in the absence or presence of complexes Ru-1 and
Ru-2 at concentration of 25 mM at 378C and 5 % CO2 for 24 h.
Each cell culture was then stained with AO/EB solution
(100 mg mL1 AO, 100 mg mL1 EB). Samples were observed
under a fluorescence microscope.
Apoptosis Assay by Flow Cytometry
After chemical treatment, 1 106 cells were harvested,
washed with phosphate-buffered saline (PBS), fixed with 70 %
ethanol, and finally, maintained at 48C for at least 12 h. The
pellets were stained with a fluorescent probe solution containing 50 mg mL1 propidium iodide (PI) and 1 mg mL1
annexin in PBS on ice in the dark for 15 min. The fluorescence
emission was then measured at 530 and 575 nm (or equivalent)
using 488 nm excitation by a FACS Calibur flow cytometer
558
(Beckman Dickinson & Co., Franklin Lakes, NJ). A minimum
of 10 000 cells were analysed.
Cellular Uptake Studies
Cells were placed in 24-well microassay culture plates (4 104
cells per well) and grown overnight at 378C in a 5 % CO2
incubator. Complexes tested were then added to the wells. The
plates were incubated at 378C in a 5 % CO2 incubator for 48 h.
Upon completion of the incubation, the wells were washed three
times with PBS, after removing the culture medium in the wells.
The cells were visualized by fluorescence microscopy.
Cell Cycle Arrest by Cytometric Analysis
BEL-7402 cells were seeded into six-well plates (Costar,
Corning Corp, New York) at a density of 2 105 cells per well
and incubated for 24 h. The cells were cultured in RPMI 1640
supplemented with fetal bovine serum (FBS, 10 %) and incubated at 378C and 5 % CO2. The medium was removed and
replaced with medium (final DMSO concentration, 1 % v/v)
containing complexes Ru-1–4 (25 and 50 mM). After incubation
for 24 h, the cell layer was trypsinized, washed with cold PBS,
and fixed with 70 % ethanol. RNAse (20 mL, 0.2 mg mL1) and
20 mL of PI (0.02 mg mL1) were added to the cell suspensions
and they were incubated at 378C for 30 min. The samples were
then analysed with a FACSCalibur flow cytometer (Becton
Dickinson & Co., Franklin Lakes, NJ). The number of cells
analysed for each sample was 10 000.[18]
Mitochondrial Membrane Potential Assay
Cells were treated for 24 h with complexes in 12-well plates
and were then washed three times with cold PBS. The cells
were then detached with trypsin-EDTA solution. Collected cells
were incubated for 20 min with 1 mg mL1 of JC-1 (5,50 ,6,60 tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide)
in culture medium at 378C in the dark. Cells were immediately
centrifuged to remove the supernatant. Cell pellets were suspended in PBS and then photographed under a microscope.
DNA Binding and Photoactivated Cleavage
The DNA binding and photoactivated cleavage experiments
were performed at room temperature. Buffer A (5 mM tris
(hydroxymethyl)aminomethane (Tris) hydrochloride, 50 mM
NaCl, pH 7.0) was used for absorption titrations, luminescence
titrations, and viscosity measurements. Buffer B (50 mM TrisHCl, 18 mM NaCl, pH 7.2) was used for DNA photocleavage
experiments. The absorption titrations of the complexes in
buffer were performed using a fixed concentration (20 mM) for
complexes to which increments of the DNA stock solution were
added. Ru–DNA solutions were allowed to incubate for 5 min
before the absorption spectra were recorded.
Results and Discussion
Synthesis and Characterization
The ligand NHPIP was synthesized by condensing 1,10phenanthroline-5,6-dione, 3-nitro-4-hydroxyphenylaldehyde,
and ammonium acetate in glacial acetic acid. The ligand AHPIP
was prepared by reducing NHPIP in ethanol with Pd/C and
NH2NH2H2O. The complexes Ru-1–4 were prepared in high
yield by direct reaction of NHPIP or AHPIP with appropriate
precursor complexes in ethylene glycol. The desired RuII
complexes were isolated as the perchlorates and purified by
G.-J. Lin et al.
column chromatography. In the ES-MS of the complexes, the
signals of [M 2ClO4 H]þ and [M 2ClO4]2þ were
observed. The ES-MS results are given in the Experimental
section. The observed molecular weights are consistent with
the expected values.
Cytotoxicity Assay In Vitro
The cytotoxicity of complexes Ru-1–4 towards the above cell
lines was assayed by cell survival using the MTT method.
Cisplatin was used as a positive control. The cell viability is
depicted in Fig. 1, and the IC50 values are listed in Table 1.
Unexpectedly, Ru-1 shows no cytotoxicity towards BEL7402, HeLa, and MG-63 cell lines. Ru-2 and Ru-4 show
higher activity than Ru-1 and Ru-3, respectively. Comparing
the IC50 values, Ru-4 shows the highest cytotoxic activity to
BEL-7402 cells with the lowest IC50 value (13.6 1.2 mM)
among the four complexes. The cytotoxic potency of Ru-4
on BEL-7402 cells is higher than that of complex [Ru
(bpy)2(hnip)]2þ (IC50 ¼ 39.2 mM, hnip ¼ 2-(2-hydroxy-1naphthyl)imidazo[4,5-f][1,10]phenanthroline),[19] and is similar
to those of [Ru(phen)2(BHIP)]2þ (IC50 ¼ 20.6 mM, BHIP ¼ 2-(3bromo-4-hydroxyphenyl)imizado[4,5-f][1,10]phenanthroline),[20]
[Ru(phen)2(dcdppz)]2þ
(IC50 ¼ 16.9 mM,
dcdppz ¼ h,jdichlorodipyrido[3,2-a:20 ,30 -c]phenanzine),[21] and cisplatin.
However, Ru-1, Ru-2, and Ru-3 are less cytotoxic than cisplatin
against the selected cell lines. In addition, Fig. 1 shows that cell
viability was found to be concentration dependent, and the cell
viability decreased on increasing the concentrations of complexes Ru-1–4. Ru-4 shows the highest antitumour activity
among the four complexes. The different antitumour activity of
the four complexes, on the one hand, is due to the different
ancillary ligands. On going from bpy to phen, the plane area and
hydrophobicity increase, leading to a higher antitumour activity
for Ru-4. On the other hand, an amino group as substituent may
enhance the antitumour activity.
Apoptosis Studies by the AO/EB Staining Method
AO can pass the cell membrane of living or early apoptotic cells,
while staining by EB indicates loss of membrane integrity.
Under a fluorescence microscope, living cells appear green,
necrotic cells stain red but have a nuclear morphology resembling that of viable cells. In the control (Fig. 2a), the living cells
are stained bright green in spots. After treatment of BEL-7402
cells with 25 mM of Ru-1 and Ru-2 for 24 h, the green apoptotic
cells with apoptotic features such as nuclear shrinkage, chromatin condensation, as well as red necrotic cells, were observed
(Fig. 2b and c). Similar results were also found for complexes
Ru-3 and Ru-4.
Apoptosis Assay by Flow Cytometry
Induction of apoptosis is one of the considerations in drug
development, most of the cytotoxic anticancer drugs in current
use have been shown to induce apoptosis in susceptible cells.[22]
The morphological apoptosis studies shows that these complexes can effectively induce BEL-7402 cell apoptosis. In order
to determine the percentage of apoptotic and necrotic cells, the
apoptotic assay was investigated by flow cytometry. After
exposure of Ru-2, Ru-3, and Ru-4 (25 mM) to BEL-7402 cells
for 24 h, the percentage of living, necrotic, and apoptotic cells is
shown in Fig. 3. The percentage of apoptotic cells was 25.42,
15.85, and 40.47 % for Ru-2, Ru-3, and Ru-4, respectively,
Ruthenium(II) Complexes and Bioactivity
559
(b) 120
Ru-1
Ru-2
Ru-3
Ru-4
Cell viability [%]
100
80
60
40
80
60
40
20
20
0
0
0
3.12
6.25
25
12.5
50
100
Ru-1
Ru-2
Ru-3
Ru-4
100
Cell viability [%]
(a) 120
0
200
3.12
Concentration [μM]
12.5
25
50
100
200
Concentration [μM]
(c) 120
(d) 120
80
60
40
80
60
40
20
20
0
0
0
6.25
12.5
25
50
100
200
Concentration [μM]
Ru-1
Ru-2
Ru-3
Ru-4
100
Cell viability [%]
Ru-1
Ru-2
Ru-3
Ru-4
100
Cell viability [%]
6.25
0
3.12
6.25
12.5
25
50
100
200
Concentration [μM]
Fig. 1. Cell viability of (a) BEL-7402, (b) HeLa, (c) MCF-7, and (d) MG-63 cell proliferation in vitro induced by Ru-1, Ru-2, Ru-3, and Ru-4 (see main text
for definition of cell types and full complex identities). Each point is the mean standard error obtained from three independent experiments.
Table 1. The IC50 (half maximal inhibitory concentration) values of
Ru-1]4 against selected cell lines
See main text for definition of cell types and full complex identities
IC50 [mM]
Complexes
Ru-1
Ru-2
Ru-3
Ru-4
Cisplatin
BEL-7402
HeLa
MCF-7
MG-63
.100
43.9 4.4
44.5 4.1
13.6 1.2
19.4 2.3
.100
78.7 6.2
82.3 5.8
18.6 1.8
18.1 1.6
57.7 4.7
46.3 3.8
18.9 1.6
24.7 2.6
.100
67.7 5.5
65.6 5.1
34.3 3.1
6.6 0.4
which is consistent with the IC50 values. Complex Ru-4 showed
the highest apoptotic effect among the three complexes.
Cellular Uptake Studies
In the functional study, the efficacy of Ru-1–4 was evaluated on
human hepatocellular carcinoma BEL-7402 cells. The uptake of
these complexes by BEL-7402 cells was studied using a fluorometric method. Ru-1–4 (25 mM) was added to the wells
(4 104 cells per well) and incubated at 378C in a 5 % CO2
incubator for 24 h. The wells were then washed three times with
PBS, and after discarding the culture medium, the cells were
observed under a fluorescent microscope. As shown in Fig. 4,
after treatment of BEL-7402 cells with the four complexes,
bright red fluorescent spots in the images were observed. The
results show that these complexes can be uptaken by cells, and
they can enter into the cytoplasm and accumulate in the nuclei.
Similar results were observed for other ruthenium(II) polypyridyl complexes.[23,24]
Effect of Ru-1–4 on the Cell Cycle Distribution
BEL-7402 cells were treated with 25 or 50 mM of Ru-1–4 for
24 h, and the distribution of cells in various compartments
during the cell cycle was analysed by flow cytometry in
PI-stained cells. The cell cycle distribution is shown in Fig. 5.
When the concentration of these complexes reached 50 mM, an
obvious enhancement (10.48, 7.76, 5.15, and 7.04 % for Ru-1,
Ru-2, Ru-3, and Ru-4) in the percentage of cells at the G0/G1
phase was observed, accompanied by a corresponding reduction
(10.39, 2.62, 4.51, and 2.99 % for Ru-1, Ru-2, Ru-3, and Ru-4)
in the percentage of cells in the S phase (Fig. 5b). In addition, at a
concentration of 25 mM, these complexes also induced an
increase at the G0/G1 phase (Fig. 5a). These results suggest
the antiproliferative mechanism induced by the complexes on
BEL-7402 cells is G0/G1 phase arrest.
560
G.-J. Lin et al.
(a)
(b)
(c)
nec
liv
ap
ap
nec
Fig. 2. BEL-7402 (human hepatocellular carcinoma cell line) cells were stained by acridine orange (AO)/ethidium bromide (EB) and observed under
fluorescence microscopy. (a) Control, (b) exposure to 25 mM of Ru-1, and (c) exposure to Ru-2 incubated at 378C and 5 % CO2 for 24 h. Liv: living cells,
ap: apoptotic cells; nec: necrotic cells.
(a)
104
104
(b)
102
FL2-H
103
Propidium Iodide
FL2-H
Propidium Iodide
103
101
101
100
100
100
101
102
103
104
100
102
FL1-H
Annexin V-FITC
Annexin V-FITC
(d)
103
104
103
104
104
103
102
FL2-H
103
Propidium Iodide
FL2-H
101
FL1-H
(c) 104
Propidium Iodide
102
102
101
101
100
100
100
101
102
103
104
100
101
102
FL1-H
FL1-H
Annexin V-FITC
Annexin V-FITC
Fig. 3. The percentage of living (L), necrotic (N), and apoptotic (A) ruthenium complex-treated BEL-7402 (human hepatocellular carcinoma cell line)
cells as analysed by FACS calibur flow cytometry. (a) Control. After 24 h exposure to 25 mM of (b) Ru-2, (c) Ru-3, and (d) Ru-4. (See main text for full
complex identities.)
Ruthenium(II) Complexes and Bioactivity
561
(a)
(b)
(c)
(d)
Fig. 4. BEL-7402 (human hepatocellular carcinoma cell line) cells incubated with 25 mM of (a) Ru-1, (b) Ru-2, (c) Ru-3, and (d) Ru-4 for 24 h, imaged by
fluorescence microscopy. (See main text for full complex identities.)
Ru-2
100
Control
69.49
71.38
Ru-1
(b)
72.10
67.63
72.10
71.38
80
Control
64.34
71.99
100
64.34
74.82
(a)
Ru-1
80
Ru-2
Ru-3
Ru-3
60
6.40
10.90
7.49
11.54
11.45
20
21.13
21.50
19.61
Ru-4
40
13.73
6.31
13.38
7.27
7.49
11.54
20
18.99
21.59
40
20.74
21.13
24.12
Ru-4
24.12
60
0
0
G0/G1
S
G2/M
G0/G1
S
G2/M
Fig. 5. Cell cycle distribution of BEL-7402 (human hepatocellular carcinoma cell line) cells with (a) 25 and (b) 50 mM of complexes Ru-1–4 incubated for
24 h. (See main text for full complex identities and description of cell cycle phases.)
Mitochondrial Membrane Potential Detection
JC-1 was used as a fluorescence probe in detecting the change of
mitochondrial membrane potential induced by complexes Ru-1
and Ru-2. At high mitochondrial membrane potential, JC-1
forms aggregates, which have a red fluorescence emission peak.
At low mitochondrial membrane potential, JC-1 forms monomers, which emit a green fluorescence peak. As shown in Fig. 6,
in the control, JC-1 exhibits a red fluorescence (JC-1 aggregates)
562
G.-J. Lin et al.
(b)
(a)
(c)
Fig. 6. Assay of BEL-7402 (human hepatocellular carcinoma cell line) cells’ mitochondrial membrane potential with JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 tetraethylbenzimidazolcarbocyanine iodide) as fluorescence probe. (a) Control. After 24 h exposure to 12.5 mM of (b) Ru-1 and (c) Ru-2. (See main text for full
complex identities.)
(a)
(b) 2.0
2.0
1.5
Absorbance
Absorbance
1.5
1.0
1.0
0.5
0.5
0
0
300
400
500
300
600
(c)
500
600
500
600
(d)
2.0
Absorbance
1.2
Absorbance
400
Wavelength [nm]
Wavelength [nm]
0.8
1.5
1.0
0.4
0.5
0
0
300
400
500
600
Wavelength [nm]
300
400
Wavelength [nm]
Fig. 7. Absorption spectra of complexes in Tris-HCl buffer upon addition of calf thymus-DNA in the presence of complexes (a) Ru-1, (b) Ru-2, (c) Ru-3, and
(d) Ru-4. [Ru] ¼ 20 mM. Arrow shows the absorbance change upon the increase of DNA concentration. (See main text for full complex identities.)
with high mitochondrial membrane potential. After BEL-7402
cells are exposed to 12.5 mM Ru-1 and Ru-2 for 24 h, JC-1
shows a green fluorescence (JC-1 monomers) corresponding to
low mitochondrial membrane potential. The change from red to
green fluorescence suggests the decrease of mitochondrial
membrane potential in the presence of Ru-1 and Ru-2. These
results indicated that Ru-1 and Ru-2 induced apoptosis in
BEL-7402 cells through the mitochondrial signal transduction
pathway.
Electronic Absorption Spectra Studies
The absorption spectra of complexes Ru-1–4 mainly consist of
two or three resolved bands in the range 200–600 nm. The
lowest energy bands at 460 nm for Ru-1, 453 nm for Ru-2,
Ruthenium(II) Complexes and Bioactivity
Ru-1 [μM]
DNA 5(dark) 5
10
15
563
Ru-2 [μM]
20
5(dark) 5
10
15
Ru-3 [μM]
20
DNA 5(dark) 5
10
15
Ru-4 [μM]
20
25
5(dark) 5
10
15
20
Form II
Form I
Fig. 8. Photoactivated cleavage of pBR322 DNA in the presence of different concentrations of complexes upon irradiation at 365 nm for 30 min. (See main
text for full complex identities and form of DNA.)
459 nm for Ru-3, and 451 nm for Ru-4 are assigned to the metalto-ligand charge transfer (MLCT) transition. The bands in the
range of 300–400 nm are attributed to p–p* transitions. The
bands below 300 nm are attributed to intraligand (IL) p–p*
transitions. Fig. 7 shows the absorption spectra of complexes
Ru-1 to Ru-4 in the presence of increasing concentration of
DNA. As the concentration of calf thymus DNA (CT-DNA)
increases, the MLCT transition band of complexes Ru-1–4
exhibit hypochromism of 29.85, 29.84, 24.70, and 21.92 %,
respectively. Due to the intercalative mode involving a stacking
interaction between the aromatic chromophore and the DNA
base pairs, a large change in absorbance can be observed while a
complex binds to DNA through intercalative mode of binding.
DNA Photocleavage
When circular plasmid DNA is subject to electrophoresis, relatively fast migration will be observed for the intact supercoiled
form (Form I). If scission occurs on one strand (nicked), the
supercoiled form will relax to generate a slower-moving open
circular form (Form II).[25] The cleavage of plasmid DNA in the
absence or presence of complexes Ru-1–4 was monitored by
agarose gel electrophoresis. The DNA cleaving efficiencies of
these complexes are shown in Fig. 8. No obvious DNA cleavage
was observed for controls in which the complex was absent, or
for incubation of the plasmid with the RuII complex in the dark.
In the presence of different concentrations of the complexes, the
amount of Form I of pBR322 DNA diminishes, whereas that of
Form II increases. These results indicated that these complexes
can effectively cleave pBR322 DNA upon irradiation.
Conclusions
Four new ruthenium(II) polypyridyl complexes Ru-1–4 were
synthesized and characterized. AO/EB staining methods showed
that these complexes can effectively induce apoptosis of BEL7402 cells. Apoptotic assay by flow cytometry exhibited that the
number of apoptotic cells increased with increasing concentration of complexes. The cellular uptake studies showed that the
complexes can be successfully uptaken and complexes entered
into the cytoplasm. The cell cycle distribution demonstrated that
the antiproliferative mechanism induced by the four complexes
on BEL-7402 cells was G0/G1 phase arrest. In addition, Ru-1
and Ru-2 induced a decrease of the mitochondrial membrane
potential. These results indicated that the complexes induced
apoptosis of BEL-7402 cells through the mitochondrial signal
transduction pathway. Upon irradiation, these ruthenium(II)
complexes can effectively cleave pBR 322 DNA.
Disclosure Statement
No competing financial interest exists.
Acknowledgements
This work was supported by the National Nature Science Foundation of
China (No 31070858) and Guangdong Pharmaceutical University for
financial support.
References
[1] K. E. Erkkila, D. T. Odom, J. K. Barton, Chem. Rev. 1999, 99, 2777.
doi:10.1021/CR9804341
[2] P. Bhattacharya, J. K. Barton, J. Am. Chem. Soc. 2001, 123, 8649.
doi:10.1021/JA010996T
[3] S. Delaney, J. Yoo, E. D. A. Stemp, J. K. Barton, Proc. Natl. Acad. Sci.
USA 2004, 101, 10511. doi:10.1073/PNAS.0403791101
[4] B. H. Yun, J. O. Kim, B. N. Lee, P. Lincoln, N. Norden, J. M. Kim, S.
K. Kim, J. Phys. Chem. B 2003, 107, 9858. doi:10.1021/JP027828N
[5] L. F. Tan, J. Liu, J. L. Shen, X. H. Liu, L. L. Zeng, L. H. Jin, Inorg.
Chem. 2012, 51, 4417. doi:10.1021/IC300093H
[6] U. Schatzschneider, J. Niesel, I. Ott, R. Gust, H. Alborzinia, S. Wölfl,
ChemMedChem 2008, 3, 1104. doi:10.1002/CMDC.200800039
[7] D. D. Sun, Y. N. Liu, D. Liu, R. Zhang, X. C. Yang, J. Liu, Chem.–Eur.
J. 2012, 18, 4285. doi:10.1002/CHEM.201103156
[8] B. S. Howerton, D. K. Heidary, E. C. Glazer, J. Am. Chem. Soc. 2012,
134, 8324. doi:10.1021/JA3009677
[9] V. Rajendiran, M. Murali, E. Suresh, M. Palaniandavar, V. S. Periasamy, M. A. Akbarsha, Dalton Trans. 2008, 38, 2157. doi:10.1039/
B715077F
[10] Y. J. Liu, C. H. Zeng, Z. H. Liang, J. H. Yao, H. L. Huang, Z. Z. Li, F.
H. Wu, Eur. J. Med. Chem. 2010, 45, 3087. doi:10.1016/J.EJMECH.
2010.03.042
[11] H. L. Huang, Z. Z. Li, Z. H. Liang, J. H. Yao, Y. J. Liu, Eur. J. Med.
Chem. 2011, 46, 3282. doi:10.1016/J.EJMECH.2011.04.049
[12] Y. J. Liu, Z. H. Liang, Z. Z. Li, J. H. Yao, H. L. Huang, J. Organomet.
Chem. 2011, 696, 2728. doi:10.1016/J.JORGANCHEM.2011.04.020
[13] Y. J. Liu, Z. Z. Li, Z. H. Liang, J. H. Yao, H. L. Huang, DNA Cell Biol.
2011, 30, 839. doi:10.1089/DNA.2011.1243
[14] W. Paw, R. Eisenberg, Inorg. Chem. 1997, 36, 2287. doi:10.1021/
IC9610851
[15] B. P. Sullivan, D. J. Salmon, T. J. Meyer, Inorg. Chem. 1978, 17, 3334.
doi:10.1021/IC50190A006
[16] T. Mosmann, J. Immunol. Methods 1983, 65, 55. doi:10.1016/00221759(83)90303-4
[17] Cells: A Laboratory Manual (Eds D. L. Spector, R. D. Goldman, L. A.
Leinwand) 1998, Vol. 1, Ch. 15 (Cold Spring Harbour Laboratory
Press: New York, NY).
[18] K. K. Lo, T. K. Lee, J. S. Lau, W. L. Poon, S. H. Cheng, Inorg. Chem.
2008, 47, 200. doi:10.1021/IC701735Q
[19] L. F. Tan, F. C. Song, X. Q. Zou, X. L. Ling, DNA Cell Biol. 2011, 30,
277. doi:10.1089/DNA.2010.1137
[20] Q. F. Guo, S. H. Liu, Q. H. Liu, H. H. Xu, J. H. Zhao, H. F. Wu, X. Y.
Li, J. W. Wang, DNA Cell Biol. 2012, 31, 1205. doi:10.1089/DNA.
2011.1490
[21] J. A. Hickman, Cancer Metastasis Rev. 1992, 11, 121. doi:10.1007/
BF00048059
[22] H. L. Huang, Z. Z. Li, Z. H. Liang, Y. J. Liu, Eur. J. Inorg. Chem. 2011,
36, 5538. doi:10.1002/EJIC.201100848
[23] C. T. Poon, P. S. Chan, C. Man, F. L. Jiang, R. N. S. Wong, N. K. Mak,
D. W. J. Kwong, S. W. Tsao, W. K. Wong, J. Inorg. Biochem. 2010,
104, 62. doi:10.1016/J.JINORGBIO.2009.10.004
[24] O. Zava, S. M. Zakeeruddin, C. Danelon, H. Vogel, M. Grätzel,
P. J. Dyson, ChemBioChem 2009, 10, 1796. doi:10.1002/CBIC.
200900013
[25] J. K. Barton, A. L. Raphael, J. Am. Chem. Soc. 1984, 106, 2466.
doi:10.1021/JA00320A058