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Synthesis, DNA-binding, photocleavage, cytotoxicity, and apoptosis studies of ruthenium(II) complexes containing 3,6-dimethyldipyrido[3,2-a:2′,3′-c]phenazine
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Synthesis, DNA-binding, photocleavage,
cytotoxicity, and apoptosis studies of
ruthenium(II) complexes containing
3,6-dimethyldipyrido[3,2-a:2′,3′-
c]phenazine
Li Xu a , Nan-Jing Zhong b , Yang-Yin Xie c , Hong-Liang Huang d ,
Zhen-Hua Liang c , Zheng-Zheng Li c & Yun-Jun Liu c
a School of Chemistry and Chemical Engineering, Guangdong
Pharmaceutical University, Zhongshan 528458, PR China
b School of Food Science, Guangdong Pharmaceutical University,
Zhongshan 528458, PR China
c School of Pharmacy, Guangdong Pharmaceutical University,
Guangzhou 510006, PR China
d School of Life Science and Biopharmacology, Guangdong
Pharmaceutical University, Guangzhou 510006, PR China
Version of record first published: 14 Dec 2011.
To cite this article: Li Xu, Nan-Jing Zhong, Yang-Yin Xie, Hong-Liang Huang, Zhen-Hua Liang,
Zheng-Zheng Li & Yun-Jun Liu (2012): Synthesis, DNA-binding, photocleavage, cytotoxicity,
and apoptosis studies of ruthenium(II) complexes containing 3,6-dimethyldipyrido[3,2-a:2′,3′-
c]phenazine, Journal of Coordination Chemistry, 65:1, 55-68
To link to this article: http://dx.doi.org/10.1080/00958972.2011.640675
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Journal of Coordination Chemistry
Vol. 65, No. 1,10January 2012, 55–68
Synthesis, DNA-binding, photocleavage, cytotoxicity, and
apoptosis studies of ruthenium(II) complexes containing
3,6-dimethyldipyrido[3,2-a:29,39-c]phenazine
LI XUy, NAN-JING ZHONGz, YANG-YIN XIEx, HONG-LIANG HUANG*{,
ZHEN-HUA LIANGx, ZHENG-ZHENG LIx and YUN-JUN LIU*x
ySchool of Chemistryand Chemical Engineering,
GuangdongPharmaceutical University, Zhongshan 528458,PRChina
zSchool of FoodScience, Guangdong Pharmaceutical University,
Zhongshan 528458,PRChina
xSchool of Pharmacy, Guangdong Pharmaceutical University,
Guangzhou 510006,PRChina
{School of Life Science andBiopharmacology, Guangdong Pharmaceutical University,
Guangzhou 510006,PRChina
(Received5October2011;infinalform2November2011)
Twonewruthenium(II)polypyridylcomplexes,[Ru(bpy) (DMDPPZ)](ClO ) (1)(bpy¼2,20-
2 42
bipyridine, DMDPPZ¼3,6-dimethyldipyrido[3,2-a:20,30-c]phenazine) and [Ru(dmb)
2 (DMDPPZ)](ClO ) (2) (dmb¼4,40-dimethyl-2,20-bipyridine), have been synthesized and
42
their DNA-binding, photoinduced DNA cleavage, and cell cytotoxicity are studied. The
complexesshowgoodbindingtocalfthymusDNAintheorder:142.Bothcomplexesexhibit
efficient DNA cleavage upon irradiation via a mechanistic pathway involving formation of
singletoxygenasthereactivespecies.Thecytotoxicactivityof1and2wastestedbythe3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. These complexes
effectively inhibit the proliferation of tumor cells. The antioxidant activity against hydroxyl
radical(.OH)wasalsoexplored.
Keywords: Ruthenium complexes; Cytotoxicity; DNA-binding; Apoptosis; Antioxidant
activity
1. Introduction
Clinicaluseofcis-diamminedichloroplatinum(II)(cisplatin)andothermetalcomplexes
in treatment of human cancer has stimulated studies of interactions of nucleic acids
(DNA) with different metal complexes due to their potential applications as chemical
andstereoselectiveprobesofnucleicacidstructures,asmolecular‘‘lightswitches’’,and
as anticancer drugs or complexes with other biological functions [1–5]. Ruthenium(II)
polypyridine complexes, due to a combination of easily constructed rigid chiral
structuresspanningallthreespatialdimensionsandarichphotophysicalrepertoire,are
*Correspondingauthors.Email:honglianghuangcn@hotmail.com;lyjche@163.com
JournalofCoordinationChemistry
ISSN0095-8972print/ISSN1029-0389online(cid:2)2012Taylor&Francis
http://dx.doi.org/10.1080/00958972.2011.640675
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56 L. Xu etal.
Scheme1. Thestructuresof1and2.
regarded as promising candidates and several ruthenium complexes have now been
proposed as potential anticancer substances, demonstrating remarkable anticancer
activityandshowinglowergeneraltoxicity[6–10].Amongthesecomplexes,theRu(II)-
dppz complexes with bidentate ancillary ligands (co-ligands), e.g., bpy, phen, dmb,
dmp, etc., have been reported for their interesting properties [11–13]. Changing
substituent groups in the intercalative ligand can also create differences in the space
configuration and electron density distribution of Ru(II) polypyridine complexes,
resulting in different spectral properties, DNA-binding behaviors, photocleavage
properties, and even their biological activities in vitro. In this article, we report the
synthesis and characterization of two new ruthenium(II) polypyridine complexes,
[Ru(bpy) (DMDPPZ)](ClO ) (1) (bpy¼2,20-bipyridine, DMDPPZ¼3,6-dimethyldi-
2 4 2
pyrido[3,2-a:20,30-c]phenazine) and [Ru(dmb) (DMDPPZ)](ClO ) (2) (dmb¼4,40-
2 4 2
dimethyl-2,20-bipyridine, scheme 1). Their DNA-binding behaviors were studied by
electronic absorption titration, viscosity measurements, and photoactivated cleavage.
The results indicate that 1 and 2 intercalate between base pairs of DNA. The
cytotoxicities of 1 and 2 have been evaluated by MTT assay, showing that these
complexes exhibit high activity against MCF-7 (breast cancer), Hela (epithelial
carcinoma), BEL-7402 (hepatocellular carcinoma), and MG-63 (osteosarcoma) cells
in a dose-dependent manner. The results obtained from the apoptosis assay show that
these complexes can induce apoptosis of BEL-7402 cells and experiments on
antioxidant activity of these complexes against hydroxyl radical (.OH) were also
explored.
2. Experimental
2.1. Materials and methods
Calf thymus DNA (CT-DNA) was obtained from the Sino-American Biotechnology
Company.pBR322DNAwasobtainedfromShanghaiSangonBiologicalEngineering
&ServicesCo.,Ltd.Dimethylsulfoxide(DMSO)andRPMI1640werepurchasedfrom
Sigma. Cell lines of MCF-7, Hela, BEL-7402, and MG-63 were purchased from
American Type CultureCollection; agaroseand ethidium bromide(EB)were obtained
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Ruthenium(II) complexes 57
from Aldrich. RuCl (cid:2)xH O was purchased from Kunming Institution of Precious
3 2
Metals. 1,10-Phenanthroline was obtained from Guangzhou Chemical Reagent
Factory. Doubly distilled water was used to prepare buffers (5mmolL(cid:3)1
Tris(hydroxymethylaminomethane)-HCl, 50mmolL(cid:3)1 NaCl, pH¼7.2). A solution of
CT-DNA in the buffer gave a ratio of UV absorbance at 260 and 280nm of ca
1.8–1.9:1, indicating that DNA was sufficiently free of protein [14]. The DNA
concentration per nucleotide was determined by absorption spectroscopy using the
molar absorption coefficient (6600 (molL(cid:3)1)(cid:3)1cm(cid:3)1) at 260nm [15].
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental
analyzer. Electrospray mass spectra (ES-MS) were recorded on a 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 4.50kV, 30.00V,
23.00V and 200(cid:4)C, respectively; the quoted m/z values are for the major peaks in the
isotopedistribution.1HNMRspectrawererecordedonaVarian-500spectrometer.All
chemical shifts were given relative to tetramethylsilane (TMS). UV-Vis spectra were
recorded on a Perkin-Elmer LS 55 spectrofluorophotometer at room temperature.
2.2. Synthesis and characterization
Complexes 1 and 2 are mixtures of lambda and delta enantiomers.
2.2.1. Synthesisof[Ru(bpy) (DMDPPZ)](ClO ) (1). Amixtureofcis-[Ru(bpy) Cl ](cid:2)
2 4 2 2 2
2H O(0.288g,0.5mmol)[16]andDMDPPZ[17](0.155g,0.5mmol)inethyleneglycol
2
(20cm3) was refluxed under argon for 8h to give a clear red solution. Upon cooling, a
red precipitate was obtained by dropwise addition of saturated aqueous NaClO
4
solution. The crude product was purified by column chromatography on neutral
alumina with a mixture of CH CN-toluene (3:1, v/v) as eluent. The mainly red band
3
was collected, solvent was removed under reduced pressure, and a red powder was
obtained.Yield: 70%.Anal.CalcdforC H N Cl O Ru:C, 52.07;H,3.28;N, 12.14.
40 30 8 2 8
Found (%): C, 52.02; H, 3.32; N, 12.10. ES-MS [CH CN, m/z]: 822.9 ([M–ClO ]þ), 3 4
723.1([M–2ClO –H]þ),362.3([M–2ClO ]2þ).1HNMR(500MHz,DMSO-d ):(cid:2)¼9.63
4 4 6
(d, 2H, J¼8.0Hz), 8.82 (d, 2H, J¼8.5Hz), 8.77 (d, 2H, J¼8.0Hz), 8.48
(dd,2H,J¼7.8Hz), 8.13 (m, 6H), 7.95 (d, 2H, J¼8.0Hz), 7.88 (dd, 2H, J¼7.7Hz),
7.76 (dd, 2H, J¼7.6Hz), 7.41 (dd, 4H, J¼7.8Hz), 2.48 (s,6H).
2.2.2. Synthesis of [Ru(dmb) (DMDPPZ)](ClO ) (2). This complex was synthesized
2 4 2
using the same procedure described for 1. Yield: 68%. Anal. Calcd for
C H N Cl O Ru: C, 53.99; H, 3.91; N, 11.45. Found (%): C, 53.95; H, 3.98;
44 38 8 2 8
N, 11.41. ES-MS [CH CN, m/z]: 879.3 ([M–ClO ]þ), 778.9 ([M–2ClO –H]þ), 390.1
3 4 4
([M–2ClO ]2þ). 1H NMR (500MHz, DMSO-d ): (cid:2)¼9.59 (d, 2H, J¼8.5Hz), 8.67 (d,
4 6
4H, J¼8.0Hz), 8.48 (d, 2H, J¼8.2Hz), 8.16 (d, 2H, J¼8.6Hz), 7.92 (d, 2H,
J¼8.2Hz), 7.68 (d, 2H, J¼7.6Hz), 7.57 (d, 2H, J¼7.8Hz), 7.23 (d, 4H, J¼8.0Hz),
2.49 (s,6H), 1.92 (s,12H).
Caution: Perchlorate salts of metal compounds with organic ligands are potentially
explosive,andonlysmallamountsofthematerialshouldbepreparedandhandledwith
great care.
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58 L. Xu etal.
2.3. DNA-binding and photoactivated cleavage
The DNA-binding and photoactivated cleavage experiments were performed at room
temperature. Buffer A [5mmolL(cid:3)1 tris(hydroxymethyl)aminomethane (Tris) hydro-
chloride, 50mmolL(cid:3)1 NaCl, pH 7.0] was used for absorption titration, luminescence
titration, and viscosity measurements. Buffer B (50mmolL(cid:3)1 Tris-HCl, 18mmolL(cid:3)1
NaCl, pH 7.2) was used for DNA photocleavage experiments.
The absorption titrations of the complex in buffer were performed using a fixed
concentration (10mmolL(cid:3)1) for complex to which increments of DNA stock solution
were added. Ru-DNA solutions were incubated for 5min before absorption spectra
were recorded. The intrinsic binding constants K, based on the absorption titration,
were measured according to the literature [18].
Thermal denaturation studies were carried out with a Perkin-Elmer Lambda 35
spectrophotometer equipped with a Peltier temperature-controlling programmer
((cid:5)0.1(cid:4)C). The melting temp (T ) was taken as the mid-point of the hyperchromic
m
transition. The melting curves were obtained by measuring the absorbance at 260nm
for solutions of CT-DNA (100mmolL(cid:3)1) in the absence and presence of RuII complex
(10mmolL(cid:3)1)asafunctionoftemperature.Thetemperaturewasscannedfrom30(cid:4)Cto
95(cid:4)Cat1(cid:4)Cmin(cid:3)1.Thedataarepresentedas(A(cid:3)A )/(A (cid:3)A )versusT,whereA,A ,
0 f 0 0
and A are the observed, the initial, and the final absorbances at 260nm, respectively.
f
ViscositymeasurementswerecarriedoutusinganUbbelodheviscometermaintained
at25.0((cid:5)0.1)(cid:4)Cinathermostaticbath.DNAsamplesapproximately200basepairsin
averagelengthwerepreparedbysonicationtominimizecomplexitiesarisingfromDNA
flexibility [19]. The relative viscosity of CT-DNA solution was measured according to
the literature [20–22].
For the gel electrophoresis experiment, supercoiled pBR322 DNA (0.1mg) was
treated with the Ru(II) complexes in buffer B, and the solution was then irradiated at
room temperature with a UV lamp (365nm,10W). The samples were analyzed by
electrophoresisfor1.5hat80Vona0.8%agarosegelinTBE(89mmolL(cid:3)1Tris-borate
acid, 2mmolL(cid:3)1 EDTA, pH¼8.3). The gel was stained with 1mgmL(cid:3)1 EB and
photographed on an Alpha Innotech IS–5500 fluorescence chemiluminescence and
visible imaging system.
2.4. Continuous variation analysis
Binding stoichiometries were obtained for 1 and 2 with CT-DNA using the method of
continuous variation [23]. The concentrations of both complex and DNA were varied,
while the sum of the reactant concentrations was kept constant at 50mmolL(cid:3)1. The
fluorescence intensities of these mixtures were measured at 25(cid:4)C using an excitation
wavelengthof448and447nm.Theintensityinfluorescencewasplottedversusthemole
fraction (cid:3) of complex to generate a Job’s plot.
2.5. Cell culture and cytotoxicity assay in vitro
Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide (MTT) assay pro-
cedures were used [24]. Cells were placed in 96-well microassay culture plates (8(cid:6)103
cellsperwell)andgrownovernightat37(cid:4)Cina5%CO incubator.Compoundstested
2
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Ruthenium(II) complexes 59
were then added to the wells to achieve final concentrations ranging from 10(cid:3)6 to
10(cid:3)4molL(cid:3)1.Controlwellswerepreparedbyadditionofculturemedium(100mL).The
culture medium and cisplatin were used as negative and positive controls, respectively.
Theplateswereincubatedat37(cid:4)Cina5%CO incubatorfor48h.Uponcompletionof
2
the incubation, stock MTT dye solution (20mL, 5mgmL(cid:3)1) was added to each well.
After 4h incubation, buffer (100mL) containing DMF (50%) and sodium dodecyl
sulfate (20%) was added to solubilize the MTT formazan. The optical density of each
wellwasthenmeasuredonamicroplatespectrophotometerat490nm.TheIC values
50
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
themeanvalues.Fourdifferenttumorcelllineswerethesubjectsofthisstudy:MCF-7,
Hela, BEL-7402, and MG-63 (purchased from American Type Culture Collection).
2.6. Apoptosis studies
Apoptosisstudieswereperformedwithastainingmethodutilizingacridineorange(AO)
and EB [25]. According to the difference in membrane integrity between necrotic and
apoptosis, AO can pass through cell membrane, but EB cannot. Under fluorescence
microscope, live cells appear green. Necrotic cells stain red but have a nuclear
morphologyresemblingthatofviablecells.Apoptosiscellsappeargreen,andmorpho-
logicalchangessuchascellblebbingandformationofapoptoticbodiesareobserved.
A monolayer of BEL-7402 cells was incubated in the absence and presence of 1 at
25mmolL(cid:3)1 at 37(cid:4)C and 5% CO for 24h. After 24h, the cells were stained with
2
AO/EB solution (100mgmL(cid:3)1 AO, 100mgmL(cid:3)1 EB). Then the samples were observed
under a fluorescence microscope.
2.7. Antioxidant activity
The hydroxyl radical (.OH) in aqueous medium was generated by the Fenton
system [26]. The solution of the tested complexes was prepared with DMF. The assay
mixture(5mL)containedsafranin(28.5mmolL(cid:3)1),EDTA-Fe(II)(100mmolL(cid:3)1),H O
2 2
(44.0mmolL(cid:3)1), the tested compounds (2.5–17.5mmolL(cid:3)1), and a phosphate buffer
(67mmolL(cid:3)1, pH¼7.4). The assay mixtures were incubated at 37(cid:4)C for 30min in a
water bath and then the absorbance was measured at 520nm. All tests were run in
triplicateandexpressedasthemean.A wastheabsorbanceinthepresenceofthetested
i
compound; A was the absorbance in the absence of tested compounds; A was the
0 c
absorbance in the absence of tested compound, EDTA-Fe(II), and H O . The
2 2
suppression ratio ((cid:4) ) was calculated on the basis of (A (cid:3)A )/(A (cid:3)A )(cid:6)100%.
a i 0 c 0
3. Results and discussion
3.1. Electronic absorption titration
In order to assess the DNA-binding behaviors of 1 and 2, absorption titrations were
carried out. As shown in figure 1, as the CT-DNA concentration is increased, the
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60 L. Xu etal.
Figure 1. Absorptionspectra inTris-HClbuffer uponaddition ofCT-DNAinthe presenceof(a)1and
(b)2.[Ru]¼10mmolL(cid:3)1.ArrowshowstheabsorbancechangeuponincreasingDNAconcentration.Plotsof
(" (cid:3)")/(" (cid:3)")vs.[DNA]fortitrationofDNAwithRu(II)complexes.
a f b f
MLCT bands of 1 at 448nm and 2 at 447nm exhibit hypochromism of 12.08% and
9.37% and bathochromism of 1nm and 2nm, respectively. However, much more
pronouncedhypochromismof29.82%for1at283nmand18.63%for2at282nmwere
observed. Although these results are different from observations on the interaction of
DNA with some mononuclear Ru(II) complexes [27, 28], which gave simultaneous
decreases in absorption for both UV and visible (MLCT) bands, considering the
spectral overlap with the MLCT transitions, these spectral characteristics obviously
suggest that 1 and 2 interact with DNA most likely through intercalation of bridging
planar aromatic ring into thebase pairsof DNA. Hiort etal.[29] deducedthat forthe
[Ru(phen) dppz]2þ-DNA system, dppz intercalates into the DNA base pairs because
2
the hypochromism of the intraligand transition of dppz is greater than that of MLCT.
The values of K for 1 and 2 are 6.6 ((cid:5)0.7)(cid:6)105 (molL(cid:3)1)(cid:3)1 (s¼2.83) and 1.6
((cid:5)0.2)(cid:6)105 (molL(cid:3)1)(cid:3)1 (s¼1.44), respectively. The values are larger than those of
[Ru(dmp) (APIP)]2þ (APIP¼2-(2-aminophenyl)imidazo[4,5-f][1,10]phenanthroline,
2
2.3(cid:6)104 (molL(cid:3)1)(cid:3)1) [30] and [Ru(dmb) (BFIP)]2þ (BFIP¼2-benzo[b]furan-2-yl-
2
1H-imidazo[4,5-f][1,10]phenanthroline, 3.2(cid:6)104 (molL(cid:3)1)(cid:3)1) [31], and comparable to
thoseofDNAintercalators[Ru(tpy)(ptp)]2þ(tpy¼2,20:60,200-terpyridine,ptp¼3-(1,10-
phenanthrolin-2-yl)-as-triazino[5,6-f]phenanthrene,1.62(cid:6)105 (molL(cid:3)1)(cid:3)1) [32],
[Ru(bpy) (taptp)]2þ (taptp¼4,5,9,18-tetraazaphenanthreno[9,10-b]triphenylene, 1.7(cid:6)
2
105 (molL(cid:3)1)(cid:3)1) [33], [Ru(MeIm) (tip)]2þ (MeIm¼1-methylimidazole, tip¼2-(thio-
4
phene-2group)-1H-imidazo[4,5-f][1,10]phenanthroline,7.20(cid:6)105(molL(cid:3)1)(cid:3)1)[34],but
is not as strong as that of their parent complexes [Ru(bpy) (dppz)]2þ (4.9(cid:6)106
2
(molL(cid:3)1)(cid:3)1) [35] and [Ru(dmb) (dppz)]2þ (4.5(cid:6)106 (molL(cid:3)1)(cid:3)1) [36].
2
3.2. Luminescence studies and continuous variation analysis
Emission intensities of 1 and 2 from their MLCT excited states upon excitation at 448
and 447nm are found to depend on DNA concentration. For each titration of
CT-DNA, luminescence enhancements occur within minutes of DNA addition,
indicating that association rates are relatively rapid. As shown in figure 2, as the
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Ruthenium(II) complexes 61
Figure2. Emissionspectraof(a)1and(b)2inTris-HClbufferintheabsenceandpresenceofCT-DNA.
ArrowshowstheintensitychangeuponincreasingDNAconcentration.
Figure3. Jobplotusingluminescencedataforcomplexes1(a)and2(b)withCTDNAinTris-HClbuffer,
pH¼7.0.
concentrationofCT-DNAincreased,theemissionintensitiesof1(at602nm)and2(at
610nm) were about 1.82 and 2.06 times larger than the original. The enhancement of
emissionintensityisanindicationofbindingofthecomplextothehydrophobicpocket
ofDNA,andthecomplexcanbeprotectedefficientlybythehydrophobicenvironment
inside the DNA helix.
Binding stoichiometry with CT-DNA was then investigated through the lumines-
cence-basedJobplot(figure3).Onemajorinflectionpointfor1and2wasobservedat
(cid:3)¼0.52and0.40,respectively.Thesedatawereconsistentwitha1:1and1.5:1[DNA]/
[complex] binding mode. Compared with that obtained from electronic titration, the
bindingsizeobtainedfromcontinuousvariationanalysisisdifferentfromthatobtained
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62 L. Xu etal.
Figure4. ThermaldenaturationofCT-DNAintheabsence(g)andpresenceofcomplexes1(.)and2(m).
[Ru]¼10mM,[DNA]¼100mM.
fromelectronictitration.Thisdifferencebetweenthetwosetsofbindingsizescouldbe
caused by the different spectroscopy and calculation method.
3.3. DNA thermal denaturation studies
Thermal behavior of DNA in the presence of complexes can give insight into their
conformational changes when temperature is raised, and offer information about the
interaction strength of complexes with DNA. When the temperature in the solution
increases, the double-stranded DNA gradually dissociates to single strands [37] and
generates a hyperchromic effect on the absorption spectra of DNA bases
((cid:5) ¼260nm). In order to identify this transition process, the melting temperature
max
T ,whichisdefinedasthetemperaturewherehalfofthetotalbasepairsisunbonded,
m
is usually introduced. According to previous literatures [35, 36], the intercalation of
natural or synthesized organics and metallointercalators generally results in a
considerable increase in melting temperature (T ). The melting curves of CT-DNA
m
in the absence and presence of the complex are presented in figure 4. The thermal
denaturation experiment carried out for DNA in the absence of the Ru(II) complexes
revealedaT of60.7(cid:5)0.1(cid:4)Cunderourexperimentalconditions.Theobservedmelting
m
temperature inthepresenceof1and2was79.2(cid:5)0.2(cid:4)Cand72.3(cid:5)0.2(cid:4)C,respectively,
at a concentration ratio [Ru]/[DNA]¼1:10. The large increases in T of two Ru(II)
m
complexes (the DT is 18.5(cid:4)C and 11.6(cid:4)C for 1 and 2, respectively) are comparable to
m
that observed for classical intercalators [38, 39].
3.4. Viscosity measurements
To investigate further the DNA-binding mode of 1 and 2, viscosity measurements on
solutions of CT-DNA incubated with the complexes were performed. Partial and/or
nonclassical intercalation of ligand could bend (or kink) the DNA helix, reducing its
effective lengthand,concomitantly, itsviscosity; aclassical intercalation ofligandinto
DNAcausesasignificantincreaseintheviscosityofDNAsolutionduetoanincreasein
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Ruthenium(II) complexes 63
Figure 5. Effect of increasing amounts of 1 (g) and 2 ((cid:7)) on the relative viscosity of CT-DNA at
25((cid:5)0.1)(cid:4)C.[DNA]¼0.30mmolL(cid:3)1.
Figure6. PhotoactivatedcleavageofpBR322DNAintheabsenceandpresenceofdifferentconcentrations
of1and2afterirradiationat365nmfor30min.
the separation of the base pairs at the intercalation site and, hence, an increase in the
overall DNA molecular length [40]. When 1 and 2 are treated with CT-DNA
(0.30mmolL(cid:3)1) and the concentrations of ruthenium complexes are increased from a
ratio of R¼0–0.16 (R¼[Ru]/[DNA]), the relative viscosity of DNA increases steadily
(figure 5) in the order 142. These results showed that 1 and 2 interact with DNA
through intercalation. The large increase in the relative viscosity revealed that 1 is a
better intercalator than 2, which is consistent with our foregoing hypothesis.
3.5. Photoactivated cleavage of pBR322 DNA
When circular plasmid DNA is subjectto electrophoresis, relatively fast migration will
be observed for the intact supercoil form (Form I). If scission occurs on one strand
(nicking), the supercoil relaxes to generate a slower moving open circular form (Form
II). If both strands are cleaved, a linear form (Form III) is generated, which migrates
betweenFormsIandIIDNA[41].ThecleavagereactionsonplasmidDNAinducedby
ruthenium(II)complexeswereperformedandmonitoredbyagarosegelelectrophoresis.
Figure 6 shows gel electrophoresis separation of pBR322 DNA after incubation with
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64 L. Xu etal.
Figure7. PhotocleavageofsupercoiledpBR322DNAby1and2(10mmolL(cid:3)1)intheabsenceandpresence
of different inhibitors [100mmolL(cid:3)1 mannitol, 200mmolL(cid:3)1 DMSO, 1000UmL–1 SOD, 1.2mmolL(cid:3)1
histidine,10mmolL(cid:3)1NaN]afterirradiationat365nmfor30min.
3
different concentrations of Ru(II) complexes and irradiation at 365nm for 30min. No
obvious DNA cleavage was observed for control in which complex was absent, or
incubation of the plasmid with the Ru(II) complex in the dark. Upon increasing
concentrations of1and2,theamountofFormI(supercoiledform)ofpBR322DNA
diminishes gradually, whereas that of Form II (circular form) increases. These results
indicate that scission occurs on one strand (nicked). Under the same experimental
conditions, 1 exhibits more effective DNA cleavage than 2. The different cleaving
efficiency is consistent with DNA-binding affinity of two Ru(II) complexes.
In order to establish the reactive species responsible for photoactivated cleavage of
the plasmid, the influence of different potentially inhibiting agents was investigated.
Figure 7 shows that DNA cleavage of the plasmid by 1 and 2 was not inhibited in the
presence of hydroxyl radical (.OH) scavengers such as mannitol [42] and DMSO [43],
which indicated that hydroxyl radical was not likely to be the cleaving agent. In the
presence of superoxide dismutase (SOD), a facile superoxide anion radical
(O.(cid:3)
)
2
quencher, thecleavagewasimproved.TheDNAcleavageoftheplasmidwasinhibited
inthepresenceofsingletoxygen(1O )scavengerhistidineandNaN [44,45],suggesting
2 3
that 1O is likely to be the reactive species responsible for cleavage. Enhancement by
2
SOD and inhibition by singlet oxygen scavengers have been observed by other
ruthenium intercalators [46–48].
3.6. Cytotoxicity assay in vitro
The cytotoxicity in vitro assay for complexes was assessed using the method of MTT
reduction. Cisplatin was used as a positive control. After treatment of MCF-7, Hela,
BEL-7402, and MG-63 cell lines for 72h with 1 and 2 in the range of concentration
(3.13!200mmolL(cid:3)1), the inhibitory percentage against growth of cancer cells was
determined. The cell viabilities (%) obtained with continuous exposure for 72h are
depicted in figure 8. The cytotoxicity was concentration-dependent. Cell viability
decreased with increasing concentrations of 1 and 2. The IC values were calculated
50
andarelistedintable1.TheIC valuesare16.4,21.2,32.6,and34.8for1,20.5,37.2,
50
16.8, and 33.2 for 2 toward MCF-7, Hela, BEL-7402, and MG-63 cells, respectively.
Comparing 1 and 2, 1 is more cytotoxic against the cell lines of MCF-7 and Hela.
Furthermore, all these complexes showed relatively lower cytotoxicity than cisplatin.
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Ruthenium(II) complexes 65
Figure 8. Cell viability of 1 and 2 on tumor MCF-7 (a), Hela (b), BEL-7402 (c), and MG-63 (d) cell
proliferationinvitro.Eachdatapointisthemean(cid:5)standarderrorobtainedfromatleastthreeindependent
experiments.
Table 1. TheIC valuesfor1and2againstselectedcelllines. 50
IC (mmolL(cid:3)1)
50
Complex MCF-7 Hela BEL-7402 MG-63
1 16.4 21.2 32.6 34.8
2 20.5 37.2 16.8 33.2
Cisplatin 12.2 10.5 13.4 (cid:3)
3.7. Apoptosis studies
Cell death was divided into two types, necrosis (accidental cell death) and apoptosis
(programmed cell death) [49]. Necrosis causes inflammation while apoptosis does not.
Inductionoftumorcellapoptosishasbeenusedasanimportantindicatortodetectthe
ability of chemotherapeutic drugs to inhibit tumor growth [50]. The type of cell death
inducedby1and2wasinvestigatedbytheapoptosisassayAO/EBstaining.TheAO/EB
staining assay can detect the difference in membrane integrity between necrotic and
apoptotic cells [25]. AO is a vital dye and can stain both live and dead cells. EB stains
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66 L. Xu etal.
Figure9. BEL-7402cellswerestainedbyAO/EBandobservedunderfluorescencemicroscopy.BEL-7402
cellswithouttreatment(a)andinthepresenceof1(b,25mmolL(cid:3)1)incubatedat37(cid:4)Cand5%CO for24h;
2
cellsinaandbarelivingandapoptoticcells,respectively.
only cells that have lost their membrane integrity. Under the Fuorescence microscope,
live cells appear green. Necrotic cells stain red, but have a nuclear morphology
resemblingviablecells.Apoptoticcellsappeargreenandmorphologicalchangessuchas
cellblebbingandformationofapoptoticbodiesareobserved.Intheabsenceof1,living
BEL-7402cellswerestainedbrightgreeninspots(figure9a).However,aftertreatment
with1,greenapoptoticcellscontainingapoptoticbodieswerealsoobserved(figure9b).
Similarresultswerealsoobservedfor2.Theresultssuggestthat1and2caneffectively
inducetheapoptosisofBEL-7402cells.
3.8. Antioxidant activity
Oxidative damage to DNA has been suggested to contribute to aging and various
diseases including cancer and chronic inflammation [51]. Among all reactive oxygen
species, the hydroxyl radical (.OH) is by far the most potent and therefore the most
dangerous oxygen metabolite, elimination of this radical is a major aim of antioxidant
administration [52]. The hydroxyl radical (.OH) in aqueous media is generated by the
Fenton system. The antioxidant activity of 1 and 2 was investigated. The inhibitory
effect is depicted in figure 10 and the suppression ratio is listed in table 2. The average
suppression ratio valued from 1.83% to 75.92% for 1, 0.86% to 85% for 2. The
antioxidant activity against hydroxyl radical of 1 and 2 is comparable under the same
experimental conditions. It is clear that 1 and 2 have high antioxidant activity. Similar
resultswereobservedforotherruthenium(II)complexes[53].Theinformationobtained
fromthisworkcouldhelpindevelopingnewantioxidantsandtherapeuticreagentsfor
some diseases.
4. Conclusion
Twonewruthenium(II)polypyridinecomplexes,[Ru(bpy) (DMDPPZ)](ClO ) (1)and
2 4 2
[Ru(dmb) (DMDPPZ)](ClO ) (2), have been synthesized and characterized. The
2 4 2
DNA-binding of these complexes with CT-DNA indicate that the two complexes
intercalate between DNA base pairs. Both complexes cleave plasmid DNA when
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Ruthenium(II) complexes 67
Figure10. Scavengingeffectof1and2onhydroxylradicals.Experimentswereperformedintriplicate.
Table 2. Thescavengingratios(%)ofcomplexesagainst.OH.
Averageinhibition(%)for.OH(mmolL(cid:3)1)
Complex 2.5 5 7.5 10 12.5 15 17.5
1 1.83 12.57 39.27 52.36 62.30 70.16 75.92
2 0.86 1.84 11.58 45.00 74.74 79.21 85.00
irradiated at 365nm for 30min. The studies of mechanism on photocleavage
demonstrate that superoxide anion radical (O.(cid:3) ) and singlet oxygen (1O ) may play
2 2
important roles. The data obtained from continuous variation analysis were consistent
with a 1:1 and 1.5:1 [DNA]/[complex] binding mode for 1 and 2, respectively.
Cytotoxicityassayinvitroshowedthat1and2displayedmoderateantitumoractivities
against selected tumor cell lines and can induce apoptosis of BEL-7402 cells.
Antioxidant activity experiments showed good antioxidant activity against hydroxyl
radical (.OH). The results should be of value in further understanding DNA-binding
and antitumor activity by Ru(II) complexes, as well as laying the foundation for
discovery of new antitumor agents.
Acknowledgments
This work was supported by the National Nature Science Foundation of China (Nos.
30800227 and 31070858) and Guangdong Pharmaceutical University.
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