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Cytotoxicity, apoptosis, interaction with DNA, cellular uptake, and cell cycle arrest of ruthenium(II) polypyridyl complexes containing 4,4′-dimethyl-2,2′-bipyridine as ancillary ligand
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Cytotoxicity, apoptosis, interaction
with DNA, cellular uptake, and
cell cycle arrest of ruthenium(II)
polypyridyl complexes containing 4,4′dimethyl-2,2′-bipyridine as ancillary
ligand
a
b
b
Hong-Liang Huang , Zheng-Zheng Li , Xiu-Zhen Wang , Zhenb
Hua Liang & Yun-Jun Liu
b
a
School of Life Science and Biopharmaceuticals, Guangdong
Pharmaceutical University, Guangdong, Guangzhou 510006, P.R.
China
b
School of Pharmacy, Guangdong Pharmaceutical University,
Guangdong, Guangzhou 510006, P.R. China
Accepted author version posted online: 19 Jul 2012.Published
online: 02 Aug 2012.
To cite this article: Hong-Liang Huang , Zheng-Zheng Li , Xiu-Zhen Wang , Zhen-Hua Liang & YunJun Liu (2012) Cytotoxicity, apoptosis, interaction with DNA, cellular uptake, and cell cycle arrest
of ruthenium(II) polypyridyl complexes containing 4,4′-dimethyl-2,2′-bipyridine as ancillary ligand,
Journal of Coordination Chemistry, 65:18, 3287-3298, DOI: 10.1080/00958972.2012.713945
To link to this article: http://dx.doi.org/10.1080/00958972.2012.713945
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Journal of Coordination Chemistry
Vol. 65, No. 18, 20 September 2012, 3287–3298
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Cytotoxicity, apoptosis, interaction with DNA, cellular uptake,
and cell cycle arrest of ruthenium(II) polypyridyl complexes
containing 4,49-dimethyl-2,29-bipyridine as ancillary ligand
HONG-LIANG HUANGy, ZHENG-ZHENG LIz, XIU-ZHEN WANGz,
ZHEN-HUA LIANGz and YUN-JUN LIU*z
ySchool of Life Science and Biopharmaceuticals, Guangdong Pharmaceutical University,
Guangdong, Guangzhou 510006, P.R. China
zSchool of Pharmacy, Guangdong Pharmaceutical University,
Guangdong, Guangzhou 510006, P.R. China
(Received 15 March 2012; in final form 13 June 2012)
Two new ruthenium(II) polypyridyl complexes, [Ru(dmb)2(DNPIP)](ClO4)2 (1) (DNPIP ^ 2(2,4-dinitrophenyl)imidazo[4,5-f][1,10]phenanthroline,
dmb ¼ 4,40 -dimethyl-2,20 -bipyridine)
and [Ru(dmb)2(DAPIP)](ClO4)2 (2) (DAPIP ¼ 2-(2,4-diaminophenyl)imidazo[4,5f][1,10]phenanthroline), were synthesized and characterized. The DNA-binding behaviors of these
complexes have been studied by UV-Vis absorption titration, viscosity measurements, and
photocleavage. The DNA-binding constants are 7.39 (0.16) 104 (s ¼ 2.68) and 2.73
(0.16) 104 (mol L1)1 (s ¼ 0.64) for 1 and 2, respectively. Their evaluation as cytotoxic
agents on different cancer cell lines was investigated with IC50 values of 59.5, 51.3, and
70.3 mmol L1 for 1, 4100, 87.9, and 77.9 mmol L1 for 2 against BEL-7402, HepG-2, and
MCF-7 cells, respectively. Complex 1 is more active than 2 against selected cancer cell lines. The
apoptosis induced by these complexes was studied. Cellular uptake showed that these
complexes could enter into the cytoplasm and accumulate in the nuclei. The cell cycle arrest and
antioxidant activity against hydroxyl radicals were also investigated.
Keywords: Ruthenium(II) complex; Cytotoxicity; Apoptosis; Cellular uptake; Cell cycle arrest
1. Introduction
There have been extensive studies on the factors that govern the affinity and specificity
of binding of small molecules to DNA, leading to the discovery that molecules bind to
DNA by different mechanisms and exert their biological activities [1]. Binding studies of
small molecules with deoxyribonucleic acid (DNA) are important in design of new and
more efficient drugs targeted to DNA [2, 3]. Small molecular compounds bind to DNA
with non-covalent interactions, such as electrostatic binding, groove binding, and
intercalative binding. Intercalating and groove binding molecules are important tools in
molecular biology and many are clinically useful in the treatment of cancer [4, 5].
Ruthenium polypyridyl complexes bind to DNA by intercalation and some Ru(II)
*Corresponding author. Email: lyjche@163.com
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2012.713945
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H.-L. Huang et al.
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Scheme 1.
The structures of 1 and 2.
complexes act as molecular light switches [6–13]. Studies on biological activity of
ruthenium(II) complexes have received attention [14–20]. [Ru(phen)2-p-MOPIP]2þ can
induce mitochondria-mediated and caspase-dependent apoptosis in human cancer cells
[17] and [Ru(bpy)2(dppn)]2þ exhibits cytotoxic activity with a low micromolar IC50
value [21]. Based on our previous investigation [22, 23], to further understand the
relation between structure of ruthenium(II) polypyridyl complexes and biological
activities, in this report, we synthesize two new Ru(II) polypyridyl complexes,
[Ru(dmb)2(DNPIP)] (ClO4)2 (1) (dmb ¼ 4,40 -dimethyl-2,20 -bipyridine, DNPIP ¼ 2(2,4-dinitrophenyl)imidazo[4,5-f][1,10]phenanthroline)
and
[Ru(dmb)2(DAPIP)]
(ClO4)2
(2)
(DAPIP ¼ 2-(2,4-diaminophenyl)imidazo[4,5-f][1,10]phenanthroline,
scheme 1). Their DNA-binding behaviors were studied by absorption titration,
viscosity measurements, and photoactivated cleavage. The biological characteristics
of these complexes were also investigated.
2. Materials and methods
2.1. Materials
Calf thymus DNA (CT-DNA) was obtained from the Sino-American Biotechnology
Company. pBR322 DNA was obtained from Shanghai Sangon Biological Engineering
& Services Co., Ltd. Dimethyl sulfoxide (DMSO) and RPMI 1640 were purchased from
Sigma. Cell lines of hepatocellular (BEL-7402), hepatocellular (HepG-2), and breast
cancer (MCF-7) were purchased from American Type Culture Collection; agarose and
ethidium bromide (EB) were obtained from Aldrich. RuCl3 xH2O was purchased from
Kunming Institute of Precious Metals. 1,10-Phenanthroline was obtained from
Guangzhou Chemical Reagent Factory.
2.2. Synthesis and characterization of Ru(II) complexes
2.2.1. Synthesis of [Ru(dmb)2(DNPIP)](ClO4)2 (1). A mixture of cis-[Ru(dmb)2Cl2]
2H2O (0.286 g, 0.5 mmol) [24] and DNPIP (0.193 g, 0.5 mmol) [23] in ethylene glycol
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Ruthenium(II) polypyridyl complexes
3289
(20 cm3) was refluxed under argon for 8 h to give a clear red solution. Upon cooling, a
red precipitate was obtained by dropwise addition of saturated aqueous NaClO4
solution. The 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, solvent removed under reduced pressure, and a red powder was obtained.
Yield: 72%. Anal. Calcd for C43H34N10Cl2O12Ru (%): C, 48.97; H, 3.25; N, 13.28.
Found (%): C, 48.65; H, 3.14; N, 13.01. ESI-MS [CH3CN, m/z]: 855.3 ([M – 2ClO4 –
H]þ), 428.2 ([M – 2ClO4]2þ). 1H NMR (500 MHz, DMSO-d6): 8.80 (d, 1H, Hl,
J ¼ 8.0 Hz), 8.73 (s, 1H, Hj), 8.67 (d, 1H, Hc, J ¼ 4.5 Hz), 8.63 (d, 1H, Hc, J ¼ 5.0 Hz),
8.47 (d, 1H, Hm, J ¼ 6.5 Hz), 7.80 (d, 2H, Ha, J ¼ 5.0 Hz), 7.68 (d, 4H, H6,60 , J ¼ 5.5 Hz),
7.40 (t, 4H, H3,30 , J ¼ 6.0 Hz), 7.23 (t, 2H, Hb, J ¼ 6.5 Hz), 7.14–7.18 (m, 4H, H5,50 ), 1.90
(s, 6H, HCH3), 1.75 (s, 6H, HCH3). 13C NMR (DMSO-d6, ppm): 156.40 C (2), 156.25 C
(20 ), 150.41 C (6), 150.31 C (60 ), 149.35 C (a), 149.18 C (e, k), 147.86 C (i), 147.33 C (4),
144.64 C (g), 129.59 C (c, h), 128.39 C (l), 125.28 C (d, f, m), 124.94 C (3), 119.13 C
(b, 5), 118.11 C (j), 20.78 C (CH3).
(2). A
mixture
of
2.2.2. Synthesis
of
[Ru(dmb)2(DAPIP)](ClO4)2
[Ru(dmb)2(DNPIP)](ClO4)2 (1) (0.527 g, 0.5 mmol) (dissolved in minimum acetonitrile),
Pd/C (0.20 g, 10% Pd), NH2NH2 H2O (8 cm3), and ethanol (20 cm3) were refluxed
under argon for 8 h. The hot solution was filtered and evaporated under reduced
pressure to reduce the solvent to 6 cm3. Upon cooling, a red precipitate was obtained by
dropwise addition of saturated aqueous NaClO4 solution. The 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: 70%. Anal. Calcd for
C43H38N10Cl2O8Ru (%): C, 51.92; H, 3.85; N, 14.08. Found (%): C, 51.68; H, 3.71;
N, 13.96. ESI-MS [CH3CN, m/z]: 795.3 ([M – 2ClO4 – H]þ), 398.4 ([M – 2ClO4]2þ).
1
H NMR (500 MHz, DMSO-d6): 9.06 (d, 2H, Hc, J ¼ 8.0 Hz), 8.72 (d, 4H, H6,60 ,
J ¼ 8.5 Hz), 8.03 (d, 2H, Ha, J ¼ 5.0 Hz), 7.85 (t, 2H, Hb, J ¼ 5.0 Hz), 7.66 (d, 4H, H3,30 ,
J ¼ 5.5 Hz), 7.34–7.42 (m, 4H, H5,50 ), 7.15 (d, 1H, Hm, J ¼ 6.0 Hz), 6.02–6.06 (m, 1H,
Hl), 5.74 (s, 1H, Hj), 5.47 (s, 4H, HNH2), 2.07 (s, 6H, HCH3), 1.75 (s, 6H, HCH3).
13
C NMR (DMSO-d6, ppm): 156.34 C (2), 156.18 C (20 ), 155.13 C (6,60 ), 151.53 C (a),
150.56 C (4), 150.41 C (40 ), 149.69 C (e), 149.55 C (k), 149.39 C (i), 144.75 C (g), 144.19
C (d, f, m), 130.19 C (c), 128.51 C (3), 128.38 C (30 ), 125.77 C (5), 125.49 C (50 ), 124.99 C
(b), 104.00 C (h), 99.58 C (l), 98.76 C (j), 20.78 C (CH3).
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.
2.3. Methods
Doubly distilled water was used to prepare buffers. A solution of CT-DNA in the buffer
gave a ratio of UV absorbance at 260 and 280 nm of ca 1.8–1.9 : 1, indicating that the
DNA was sufficiently free of protein [25]. The DNA concentration per nucleotide was
determined by absorption spectroscopy using the molar absorption coefficient
(6600 (mol L1)1 cm1) at 260 nm [26].
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H.-L. Huang et al.
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental
analyzer. Electrospray mass spectra (ESI-MS) were recorded on an LCQ system
(Finnigan MAT, USA) using methanol as the 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 spectra were recorded on a Varian-500 spectrometer.
All chemical shifts are given relative to tetramethylsilane (TMS). UV-Vis spectra were
recorded on a Shimadzu UV-3101PC spectrophotometer at room temperature.
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2.4. DNA-binding studies
DNA-binding was performed at room temperature. Buffer (5 mmol L1 tris(hydroxymethyl)aminomethane (Tris) hydrochloride, 50 mmol L1 NaCl, pH 7.0) was used for
absorption titration and viscosity measurements. Viscosity measurements were carried
out using an Ubbelodhe viscometer maintained at 25.0 (0.1) C in a thermostatic bath.
DNA samples approximately 200 base pairs in average length were prepared by
sonication to minimize complexities arising from DNA flexibility [27]. Flow time was
measured with a digital stopwatch, each sample was measured three times, and an
average flow time was calculated. Relative viscosities for DNA in the presence and
absence of complexes were calculated from the relation ¼ (t t0)/t0, where t is the
observed flow time of the DNA-containing solution and t0 is the flow time of buffer
alone [28, 29]. Data are presented as (/0)1/3 versus binding ratio (r ¼ 0.04, 0.08, 0.12,
and 0.16) [30], where is the viscosity of DNA in the presence of complexes and 0 is the
viscosity of DNA alone.
The absorption titrations of the complex in buffer were performed using a fixed
concentration (20 mmol L1) for complex to which increments of DNA stock solution
were added. Ru-DNA solutions were allowed to incubate for 5 min before the
absorption spectra were recorded. The intrinsic binding constants K, based on the
absorption titration, were measured by monitoring changes in absorption at the metalto-ligand charge transfer (MLCT) band with increasing concentration of DNA using
the following equation [31],
ð"a "f Þ=ð"b "f Þ ¼ ðb ðb2 2K2 Ct ½DNA=sÞÞ1=2 =2KCt ,
ð1aÞ
ðb ¼ 1 þ KCt þ K½DNA=2sÞ,
ð1bÞ
where [DNA] is the concentration of CT-DNA in base pairs, the apparent absorption
coefficients "a, "f, and "b correspond to Aobsd/[Ru], the absorbance for the free
ruthenium complex, and the absorbance for the ruthenium complex in fully bound
form, respectively. K is the equilibrium binding constant, Ct is the total concentration of
metal complex, and s is the binding site size.
2.5. Scavenger measurements of hydroxyl radical (. OH)
The hydroxyl radical (. OH) in aqueous media was generated by the Fenton system [32].
The solution of the tested complexes was prepared with DMF (N,N-dimethylformamide). An assay mixture of 5 mL contained the following reagents: safranin
Ruthenium(II) polypyridyl complexes
3291
(28.5 mmol L1), EDTA-Fe(II) (100 mmol L1), H2O2 (44.0 mmol L1), the tested compounds (0.5–3.5 mmol L1), and a phosphate buffer (67 mmol L1, pH ¼ 7.4). The assay
mixtures were incubated at 37 C for 30 min in a water bath and the absorbance was
measured at 520 nm. All tests were run in triplicate and expressed as the mean. Ai was
the absorbance in the presence of the tested compound; A0 was the absorbance in the
absence of tested compounds; and Ac was the absorbance in the absence of tested
compound, EDTA-Fe(II), H2O2. The suppression ratio (a) was calculated on the basis
of (Ai A0)/(Ac A0) 100%.
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2.6. Cytotoxicity assay in vitro
Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide (MTT) assay procedures were used [33]. Cells were placed in 96-well microassay culture plates (8 103
cells per well) and grown overnight at 37 C in a 5% CO2 incubator. The complexes
tested were dissolved in DMSO and diluted with RPMI 1640 and then added to the
wells to achieve final concentrations ranging from 106 to 104 mol L1. Control wells
with cells were prepared by addition of culture medium (100 mL). Wells containing
culture medium without cells were used as blanks and cisplatin was used as positive
control. The plates were incubated at 37 C in a 5% CO2 incubator for 72 h. Upon
completion of the incubation, stock MTT dye solution (20 mL, 5 mg mL1) was added to
each well. After 4 h incubation, 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 on a microplate spectrophotometer
at 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 get the mean values. Three different tumor cell lines were the subject of this
study: BEL-7402, HepG-2, and MCF-7.
2.7. Apoptosis assessment by acridine orange/EB staining
Apoptosis studies were performed with a staining method utilizing acridine orange
(AO) and EB [34]. AO can pass through cell membrane, but EB cannot. Under
fluorescence microscope, live cells appear green. Necrotic cells stain red but have a
nuclear morphology resembling that of viable cells. Apoptotic cells appear green, and
morphological changes such as cell blebbing and formation of apoptotic bodies will be
observed. A monolayer of BEL-7402 cells was incubated in the absence and presence of
2 at 50 mmol L1 at 37 C and 5% CO2 for 48 h. Then each cell culture was stained with
AO/EB solution (100 mg mL1 AO, 100 mg mL1 EB). Samples were observed under a
fluorescence microscope.
2.8. Cellular uptake study
Cells were placed in 24-well microassay culture plates (4 104 cells per well) and grown
overnight at 37 C in a 5% CO2 incubator. Complex 2 was then added to the wells. The
plates were incubated at 37 C in a 5% CO2 incubator for 24 h. Then the wells were
3292
H.-L. Huang et al.
washed three times with phosphate buffered saline (PBS), after removing the culture
medium, the cells were visualized by fluorescence microscopy.
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2.9. Cell cycle arrest
HepG-2 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 (10% FBS) and incubated at 37 C and 5%
CO2. The medium was removed and replaced with medium (final DMSO concentration,
1% v/v) containing 1 (50 mmol L1). After incubation for 24 h, the cell layer was
trypsinized, washed with cold PBS, and fixed with 70% ethanol. 20 mL of RNAse
(0.2 mg mL1) and 20 mL of propidium iodide (PI, 0.02 mg mL1) were added to the cell
suspensions and incubated at 37 C for 30 min. Then the samples were analyzed by an
FACS Calibur flow cytometer (Becton Dickinson & Co., Franklin Lakes, NJ). The
number of cells analyzed was 10,000 [35].
3. Results and discussion
3.1. Synthesis and characterization
DNPIP was synthesized by refluxing a mixture of 1,10-phenanthroline-5,6-dione and
2,4-dinitrobenzaldehyde following a similar method described by Steck and Day [36].
[Ru(dmb)2(DNPIP)](ClO4)2 (1) was prepared by direct reaction of DNPIP with
[Ru(dmb)2Cl2] 2H2O in ethylene glycol in relatively high yield. [Ru(dmb)2(DAPIP)]
(ClO4)2 (2), was synthesized by reducing 1 in the presence of Pd/C and NH2NH2 H2O
in ethanol. The desired Ru(II) complexes were isolated as the perchlorates and purified
by column chromatography. In 1H NMR spectra, the chemical shift of Hj in DNPIP is
9.06 ppm [23], a shift of 0.33 in 1 and 3.32 ppm in 2 for Hj was observed (figure S1). In
the ES-MS of 1 and 2, all of the expected signals [M – 2ClO4 – H]þ and [M – 2ClO4]2þ
were observed (figure S2). The measured molecular masses were consistent with
expected values.
Electronic absorbance spectra of the complexes in DMSO were characterized by an
intense ligand-centered transition in the UV and an MLCT transition in the visible
region. In the UV region, intense, fairly sharp bands at 288 nm for 1 and 289 nm for 2
are assigned as intraligand –* transitions. Low-energy absorptions at 480 and 456 nm
for 1 and 2, respectively, are attributed to the MLCT transition by comparison with
spectra of other ruthenium(II) complexes [37–39] (figure S3).
3.2. Electronic absorption spectra titration
Generally, the ligand-centered ! * and MLCT absorptions shift to longer
wavelengths (bathochromism) and decrease in intensity (hypochromism) with increasing concentration of CT-DNA [40]. Figure 1 shows the absorption spectra of 1 and 2 in
the presence of increasing concentrations of DNA. With increasing concentrations of
CT-DNA, the MLCT bands of 1 at 467 and 2 at 466 nm exhibit hypochromism of 41.2
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Ruthenium(II) polypyridyl complexes
3293
Figure 1. Absorption spectra of 1 (A) and 2 (B) in Tris-HCl buffer upon addition of CTDNA. [Ru] ¼ 20 mmol L1. The arrow shows the absorbance change upon increase of DNA concentration.
Plots of ("a "f)/("b "f) vs. [DNA] for titration of DNA with Ru(II) complexes.
and 21.79%, and bathochromism of 3 and 2 nm, respectively. These spectral
characteristics suggest that the complexes interact with DNA through a mode that
involves a stacking interaction between the aromatic chromophore and the base pairs of
DNA. In order to elucidate the DNA-binding strength of 1 and 2 with DNA, the DNAbinding constants Kb were determined by monitoring the changes in absorbance of the
MLCT band with increasing concentrations of CT-DNA. The values of Kb were
7.4 (0.2) 104 (s ¼ 2.68) and 2.7 (0.2) 104 (mol L1)1 (s ¼ 0.64) for 1 and 2,
respectively. The value of Kb for 1 is larger than that of 2, caused by the electronwithdrawing substituent (–NO2 in DNPIP) on the intercalative ligand improving the
DNA-binding affinity, and the electron-pushing substituent (–NH2 in DAPIP)
decreasing the DNA affinity.
3.3. Viscosity measurements
Viscosity measurements of DNA are regarded as the least ambiguous and the most
critical test of a DNA-binding model in solution and provide strong arguments for
intercalative DNA-binding mode [28, 29]. Intercalators unwind the double helix when
they insert between DNA base pairs, producing DNA that is somewhat elongated
relative to canonical B-form DNA. Lengthening of the helical axis gives measurable
increases in viscosities [41] that are not observed for groove binding or electrostatic
association. The effects of 1 and 2 on the relative viscosity of rod-like DNA are shown
in figure 2. With increasing concentrations of 1 and 2, the relative viscosity of the DNA
solution increased steadily. The increasing relative viscosity of DNA for 1 is larger than
that for 2, attributed to the different DNA-binding affinity of the two complexes.
Considering the DNA-binding affinities and the changes in viscosity, 1 and 2 interact
with CT-DNA by groove binding.
3.4. Antioxidant activity against hydroxyl radical
Increasing spectrum of diseases as well as aging has been a subject of antioxidant
supplementation. Among all reactive oxygen species the hydroxyl radical (. OH) is by
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3294
H.-L. Huang et al.
Figure 2. Effect of increasing amounts of 1 () and 2 (g) on the relative viscosity of CT-DNA at 25
(0.1) C. [DNA] ¼ 0.25 mmol L1.
Figure 3.
Table 1.
Scavenging effect of 1 and 2 on hydroxyl radicals. Experiments were performed in triplicate.
The scavenging ratios (%) of ligand and complexes against . OH.
Average inhibition (%) for . OH
Compound
1
2
0.5
1.0
1.5
2.0
2.5
3.0
3.5 (mmol L1)
1.6 0.6
1.8 0.7
7.3 1.4
2.1 1.1
16.2 2.1
9.6 1.7
31.3 2.2
18.5 2.0
48.4 3.1
22.9 3.2
50.1 3.2
38.9 3.4
67.2 3.3
42.2 3.6
far the most potent and dangerous oxygen metabolite; elimination of this radical is one
of the main aims of antioxidant administration [42]. The antioxidant activities of 1 and
2 are shown in figure 3 and table 1. The suppression ratio against . OH varied from 1.11
to 67.19 for 1 and 1.82 to 42.19 for 2. The inhibitory effect of these complexes on . OH
was concentration-dependent and the suppression ratio increased with increase in
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Ruthenium(II) polypyridyl complexes
Table 2.
The IC50 values of 1 and 2 on the selected cell lines.
IC50 (mmol L1)
Compound
BEL-7402
HepG-2
MCF-7
1
2
Cisplatin
59.5 3.5
4100
19.8 2.6
51.3 5.0
87.9 3.3
25.5 3.2
70.3 3.4
77.9 2.4
9.7 2.6
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sample concentrations from 0.5 to 3.5 mmol L1. Comparing the suppression ratio of 1
and 2, the antioxidant activity of 1 is higher than that of 2 under the same conditions.
3.5. Cytotoxicity assay
The cytotoxicities of 1 and 2 were evaluated on BEL-7402 (hepatocellular), HepG-2
(hepatocellular), and MCF-7 (breast cancer) by cell survival after 72 h of exposure using
the MTT assay. Cisplatin was used as positive control. The concentrations varied from
6.25 to 400 mmol L1 and IC50 values calculated after 72 h of incubation with 1 and 2 are
listed in table 2. The cell viability was concentration-dependent, and increasing the
concentrations of 1 and 2 caused a decrease in cell viability. Comparing the IC50 values
(table 2), 1 shows higher activity than 2 against selected tumor cell lines, but their
cytotoxic activities are far lower than that of cisplatin.
3.6. Apoptosis activity studies
In order to gain some insight into cell death type induced by 2, the apoptosis assays
were performed on BEL-7402 cells with AO and EB staining. Cells sensing an
inflicted aggression by a chemical compound undergo two major forms of death,
necrosis or apoptosis, and each with very distinct characteristics. The control and
treatment of BEL-7402 cells with 2 are shown in figure 4. In the absence of 2, the
living cells were stained bright green in spots (figure 4A). However, after treatment
with 2, the green apoptotic cells containing apoptotic bodies, as well as red necrotic
cells, were also observed (figure 4B). The results show 2 can effectively induce
apoptosis.
3.7. Cellular uptake studies
In the functional study, cellular uptake of 2 (50 mmol L1) by BEL-7402 cells was
studied using fluorescence microscopy. In control experiments, BEL-7402 cells do not
show luminescence (data not presented). After treatment of BEL-7402 cells with 2,
bright red fluorescence spots in the images were observed (figure 5). The result showed
that 2 can be uptaken by BEL-7402 cells, enter into the cytoplasm, and accumulate in
the nuclei.
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H.-L. Huang et al.
Figure 4. BEL-7402 cells were stained by AO/EB and observed under fluorescence microscopy. BEL-7402
cells without treatment (A) and in the presence of 2 (B) incubated at 37 C and 5% CO2 for 48 h. Cells in a, b,
and c are living, apoptotic, and necrotic cells, respectively.
Figure 5. BEL-7402 cells incubated with 2 (50 mmol L1) for 24 h imaged by fluorescence microscopy.
Note that the cytoplasm is extensively stained with the Ru(II) complexes.
3.8. Flow cytometric analysis
A better understanding of the mechanism of drug-induced cytotoxicity is important in
the design of more effective chemotherapeutic agents. Inhibition of cancer cell
proliferation by cytotoxic drugs could result from induction of apoptosis or of cell cycle
arrest [43]. The effect of 1 on cell cycle of HepG-2 cells was investigated by flow
cytometry in PI (propidium iodide) stained cells after Ru(II) complex treatment for
24 h. Representative DNA distribution histograms of HepG-2 cells in the absence and
presence of 1 are shown in figure 6. Treatment of HepG-2 cells with 1 caused an
increase (8.55%) in the percentage of cells at the S-phase, accompanied by
corresponding reduction (10.08%) in the G0/G1-phase. The results indicate that 1
can inhibit the cell division in the S-phase on HepG-2 cells.
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Figure 6. Cell cycle status of HepG-2 cells after treatment with 1 (50 mmol L1) for 24 h: (A) control; and
(B) (1 þ HepG-2).
4. Conclusion
Complexes 1 and 2 bind to CT-DNA by DNA groove binding. Complex 1 shows higher
cytotoxicity than 2 on selected tumor cell lines, consistent with the DNA-binding
affinities of the two complexes. Complex 2 can effectively induce apoptosis after
treatment of BEL-7402 cells. The results obtained from cellular uptake showed that 2
can enter into the cytoplasm and accumulate in the nuclei. Flow cytometric analysis
suggested that 1 can inhibit the S-phase transition on HepG-2 cells.
Acknowledgments
This work was supported by the National Nature Science Foundation of China (Nos
31070858, 30800227) and Guangdong Pharmaceutical University.
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