Ruthenium(II) polypyridyl complexes induce BEL-7402 cell apoptosis by ROS-mediated mitochondrial pathway

Journal of Inorganic Biochemistry 141 (2014) 170–179

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Ruthenium(II) polypyridyl complexes induce BEL-7402 cell apoptosis by ROS-mediated mitochondrial pathway Guang-Bin Jiang a, Xiang Zheng b, Jun-Hua Yao c, Bing-Jie Han a, Wei Li a, Ji Wang a, Hong-Liang Huang d,⁎, Yun-Jun Liu a,⁎ a

School of Pharmacy, Guangdong Pharmaceutical University,Guangzhou, 510006, PR China The Frst Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, 510062, PR China Instrumentation Analysis and Research Center, Sun Yat-Sen University, Guangzhou, 510275, PR China d School of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China b c

a r t i c l e

i n f o

Article history: Received 24 June 2014 Received in revised form 3 September 2014 Accepted 3 September 2014 Available online 16 September 2014 Keywords: Ruthenium(II) polypyridyl complex Cytotoxicity Comet assay ROS Mitochondrial membrane potential Western blot analysis

a b s t r a c t A new ligand dmdppz and its four ruthenium(II) polypyridyl complexes [Ru(dmb)2(dmdppz)](ClO4)2 (1), [Ru(bpy)2(dmdppz)](ClO4)2 (2), [Ru(phen)2(dmdppz)](ClO4)2 (3) and [Ru(dmp)2(dmdppz)](ClO4)2 (4) (where dmb, bpy, phen, dmp and dmdppz stand for 4,4′-dimethyl-2,2′-bipyridine, 2,2′-bipyridine, 1,10phenanthroline, 2,9-dimethyl-1,10-phenanthroline and 5,8-dimethoxylpyrido[3,2-a:2′,3′-c]phenazine, respectively) have been synthesized and characterized. Their DNA binding behaviors show that the complexes bind to calf thymus DNA by intercalation. The complexes exhibit efficient photocleavage of pBR322 DNA on irradiation. The cytotoxicity of the ligand and the complexes toward HepG-2, HeLa, MG-63, A549 and BEL-7402 were assayed by MTT ((3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide)) method. The IC50 values of the complexes 1, 2, 3 and 4 toward BEL-7402 cells are 14.6, 16.8, 18.0 and 16.7 μM, respectively. Dmdppz shows no cytotoxic activity against selected cell lines. The cellular uptake, apoptosis, comet assay, reactive oxygen species (ROS), mitochondrial membrane potential and western blot analysis were investigated. These results indicate that complexes 1–4 exert their toxicity through the intrinsic ROS-mediated mitochondrial pathway, which is accompanied by the regulation of Bcl-2 family proteins. © 2014 Elsevier Inc. All rights reserved.

1. Introduction DNA as a carrier of genetic information is often a target for drugs [1]. The interaction of transition metal complexes with DNA has attracted much attention [2]. In recent years, ruthenium(II) complexes have emerged as promising antitumor agents and these complexes interact with DNA through intercalative mode [3–9]. Among these studies, ruthemium(II) polypyridyl complexes have attracted considerable attention due to a combination of their rigid chiral structures and rich photophysical character [10–12]. Ruthenium(II) complexes containing dipyridophenazine (dppz) ligand have been studied extensively because of strong DNA binding and high cytotoxic activity [13–15]. To date, two ruthenium-based complexes, NAMI-A and KP1019 have entered clinical trials [16,17]. [Ru(phen)2(AHPIP)]2+ (phen = 1,10phenanthroline) can effectively inhibit the cell growth of BEL-7402 cells [18]. [Ru(dip)2(1-Py-βC)]2+ (dip = 4,7-diphenyl-1,10-phenanthroline) shows high inhibition of cell growth on HeLa cells (IC 50 = 1.9 ± 0.2 μM) [19]. Schatzschneider reported [Ru(bpy)2(dppn)]2 + (bpy = 2,2′-bipyridine, dppn = benzo[i]dipyrido[3,2-a:2′,3′-h] ⁎ Corresponding authors. Tel.: +86 20 39352122; fax: +86 20 39352129. E-mail addresses: [email protected] (H.-L. Huang), [email protected] (Y.-J. Liu).

http://dx.doi.org/10.1016/j.jinorgbio.2014.09.001 0162-0134/© 2014 Elsevier Inc. All rights reserved.

quinoxaline) exhibits a low micromolar IC50 (6.4 ± 1.9 μM) value against HT-29 cells which is comparable to cisplatin under identical conditions, but complex [Ru(bpy)2(dppz)]2 + shows very low cytotoxic effect on MCF-7 cells (IC50 = 90.2 ± 19.6 μM) [15]. Complex [Ru(dmp)2(adppz)]2 + (dmp = 2,9-dimethyl-1,10-phenanthroline, adppz = 7-aminodipyrido[3,2-a:2′,3′-c]phenanzine) can effectively inhibit the cell growth with very low IC50 values of 2.4 ± 0.4 and 2.2 ± 0.3 μM against A549 and SK-BR-3 cells [20]. [Ru(phen)2(dadppz)]2 + (dadppz = 7,8-diaminodipyrido[3,2-a:2′,3′-c]phenanzine) can effectively kill the BEL-7402 cells (IC50 = 11.8 ± 1.1 μM) and induces cell cycle arrest at the G0/G1 phase in BEL-7402 cells [21]. [Ru(dip)2 (dcdppz)]2+ (dcdppz = 7,8-dichlorodipyrido[3,2-a:2′,3′-c]phenanzine) shows higher cytotoxicity (IC50 = 12.3 ± 1.4 μM) on HeLa cells [22]. These data show that the derivatives of dppz (substituted with amino group or chloride atom in dppz) can enhance the cytotoxic activity of the complexes against the selected cancer cells. These characteristics prompt us to design and synthesize a new derivative (containing methoxyl group as substitutent) of dppz. To obtain much information on the anticancer activity of the ruthenium(II) complexes, in this report, a new ligand dmdppz (dmdppz = 5,8-dimethoxylpyrido[3,2-a:2′,3′-c]phenazine) and its four ruthenium(II) polypyridyl complexes [Ru(dmb)2 (dmdppz)]

G.-B. Jiang et al. / Journal of Inorganic Biochemistry 141 (2014) 170–179

(ClO 4) 2 (1)), [Ru(bpy) 2(dmdppz)](ClO 4) 2 (2), [Ru(phen) 2 (dmdppz)](ClO 4 ) 2 (3) and [Ru(dmp) 2 (dmdppz)](ClO 4 ) 2 (4) (Scheme 1) were synthesized and characterized by elemental analysis, ESI-MS, 1 H NMR. The interaction of the four ruthenium(II) polypyridyl complexes with CT DNA have been studied by electronic absorption titration, viscosity measurements and photocleavage of pBR322 DNA. The anticancer activity of the complexes toward HepG-2, A549, HeLa, MG-63 and BEL-7402 cell lines was assayed by MTT method. (MTT = (3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide)). The cellular uptake, AO/EB staining, comet assay, reactive oxygen species (ROS) and mitochondrial membrane potential in BEL-7402 cells induced by these complexes were investigated. The cell cycle distribution was analyzed by flow cytometry and the apoptotic pathway was also investigated in detail by western blot analysis. The results state clearly that the four ruthenium(II) polypyridyl complexes intercalate into DNA base pairs and the complexes have significant effect on inhibiting cell proliferation and can effectively induce apoptosis in BEL-7402 cells. 2. Experimental section 2.1. Materials and methods All reagents and solvents were purchased commercially and used without further purification unless otherwise noted. 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. Ultrapure MilliQ water was used in all experiments. DMSO and RPMI 1640 were purchased from Sigma. Cell lines of BEL-7402 (Hepatocellular), HeLa (Human cervical cancer cell line), MG-63 (Human osteosarcoma), HepG-2 (Hepatocellular) and A549 (Human lung carcinoma) were purchased from the American Type Culture Collection. RuCl 3 · 3H2 O was obtained from the Kunming Institution of Precious Metals. 1,10phenanthroline was obtained from the Guangzhou Chemical Reagent Factory. Microanalyses (C, H, and N) were performed with a Perkin-Elmer 240Q elemental analyzer. Electrospray ionization mass spectra (ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA) using methanol 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 spectra were recorded on a Varian-500 spectrometer with DMSO-d 6 as

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solvent and tetramethylsilane (TMS) as an internal standard at 500 MHz at room temperature.

2.2. Synthesis of ligand and complexes 2.2.1. Synthesis of ligand (dmdppz) A mixture of 1,10-phenanthroline-5,6-dione (0.106 g, 0.500 mmol) [23], damb (1,2-diamino-3,6-dimethoxybenzene) (0.084 g, 0.500 mmol) [24] and ethanol (40 mL) was refluxed with stirring for 2 h. The yellow precipitate was separated by filtration. The precipitate was washed with ethanol and dried under vacuo. ESI-MS (CH3CN): m/z 343 [(M + 1)]. Yield: 81%. Anal. Calc. for C20H14N4O2: C, 70.17; H, 4.12; N, 16.37%. Found: C, 70.25; H, 4.04; N, 16.25%.

2.2.2. Synthesis of [Ru(dmb)2(dmdppz)](ClO4)2 (1) A mixture of cis-[Ru(dmb)2Cl2] · 2H2O [25] (0.288 g, 0.500 mmol) and dmdppz (0.171 g, 0.500 mmol) in ethylene glycol (20 mL) was heated at 150 °C 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. The solvent was removed under reduced pressure and a red powder was obtained. Yield: 73%. Anal. Calc. for C44H38N8Cl2O10Ru: C, 52.28; H, 3.79; N, 11.09%. Found: C, 52.37; H, 3.65; N, 11.14%. ESI-MS (CH3CN): m/z 406.4 [M-2ClO4]2 +. 1H NMR (DMSO-d6): δ 9.34 (d, 2H, J = 6.5 Hz), 8.75 (d, 4H, J = 8.0 Hz), 8.22 (dd, 2H, J = 5.5, J = 5.5 Hz), 7.91 (t, 2H, J = 5.5 Hz), 7.64 (d, 2H, J = 6.0 Hz), 7.60 (d, 2H, J = 5.5 Hz), 7.48 (s, 2H), 7.43 (d, 2H, J = 5.5 Hz), 7.22 (d, 2H, J = 6.0 Hz), 4.10 (s, 6H), 2.56 (s, 6H), 2.48 (s, 6H).

2.2.3. Synthesis of [Ru(bpy)2(dmdppz)](ClO4)2 (2) This complex was synthesized in an identical manner to that described for complex 1, with cis-[Ru(bpy)2Cl2] · 2H2O [25] in place of cis-[Ru(dmb)2Cl2] · 2H2O. Yield: 72%. Anal. Calc. for C40H30N8Cl2O10Ru: C, 50.32; H, 3.17; N, 11.74%. Found: C, 50.39; H, 3.24; N, 11.56%. ESI-MS (CH3CN): m/z 378.3 [M-2ClO4]2+. 1H NMR (DMSO-d6): δ 9.41 (d, 2H, J = 8.0 Hz), 8.88 (dd, 4H, J = 8.0, J = 8.0 Hz), 8.23 (d, 4H, J = 6.5 Hz), 8.14 (t, 2H, J = 7.5 Hz), 7.95 (t, 2H, J = 7.5 Hz), 7.83 (d, 2H, J = 5.5 Hz), 7.78 (d, 2H, J = 5.5 Hz), 7.60 (t, 2H, J = 6.5 Hz), 7.49 (s, 2H), 7.41 (t, 2H, J = 6.5 Hz), 4.11 (s, 6H).

Scheme 1. The synthetic route of ligand and complexes.

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2.2.4. Synthesis of [Ru(phen)2(dmdppz)](ClO4)2 (3) This complex was synthesized in an identical manner to that described for complex 1, with cis-[Ru(phen)2Cl2] · 2H2O [25] in place of cis-[Ru(dmb)2Cl2] · 2H2O. Yield: 74%. Anal. Calc. for C44H30N8Cl2O10Ru: C, 52.70; H, 3.02; N, 11.17%. Found: C, 52.56; H, 3.11; N, 11.23%. ESI-MS (CH3CN): m/z 402.5 [M-2ClO4]2+. 1H NMR (DMSO-d6): δ 9.25 (d, 2H, J = 8.0 Hz), 8.81 (dd, 4H, J = 7.5, J = 7.5 Hz), 8.41 (s, 4H), 8.31 (d, 2H, J = 5.5 Hz), 8.15 (d, 2H, J = 5.5 Hz), 8.08 (d, 2H, J = 5.0 Hz), 7.85 (dd, 4H, J = 5.5, J = 5.0 Hz), 7.78 (dd, 2H, J = 5.0, J = 5.5 Hz), 7.48 (s, 2H), 4.10 (s, 6H).

in the presence of complexes and η0 is the viscosity of DNA solution alone. For the gel electrophoresis experiment, supercoiled pBR 322 DNA (0.1 μg) was treated with the complexes in buffer B, and the solution was then irradiated at room temperature with a UV lamp (365 nm, 10 W). The samples were analyzed by electrophoresis for 1.5 h at 80 V on a 0.8% agarose gel in TBE (89 mM Tris-borate acid, 2 mM EDTA, pH = 8.3). The gel was stained with 1 μg/mL ethidium bromide and photographed on an Alpha Innotech IS–5500 fluorescence chemiluminescence and visible imaging system.

2.2.5. Synthesis of [Ru(dmp)2(dmdppz)](ClO4)2 (4) This complex was synthesized in an identical manner to that described for complex 1, with cis-[Ru(dmp)2Cl2] · 2H2O [26] in place of cis-[Ru(dmb)2Cl2] · 2H2O. Yield: 73%. Anal. Calc. for C48H38N8Cl2O10Ru: C, 54.45; H, 3.62; N, 10.58 %. Found: C, 54.62; H, 3.68; N, 10.39%. ES-MS (CH3CN): m/z 430.2 [M-2ClO4]2+. 1H NMR (DMSO-d6): δ 9.37 (d, 2H, J = 8.0 Hz), 8.93 (d, 2H, J = 8.5 Hz), 8.46 (dd, 4H, J = 8.0, J = 8.0 Hz), 8.26 (d, 2H, J = 8.5 Hz), 7.99 (d, 2H, J = 7.5 Hz), 7.60 (dd, 2H, J = 6.0, J = 5.5 Hz), 7.51 (dd, 2H, J = 5.5, J = 5.5 Hz), 7.44 (s, 2H), 7.41 (d, 2H, J = 8.5 Hz), 4.07 (s, 6H), 1.93 (s, 6H), 1.84 (s, 6H). 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.

2.4. Cytotoxicity assay in vitro

2.3. DNA-binding and photoactivated cleavage The DNA-binding and photoactivated cleavage experiments were performed at room temperature. Buffer A [5 mM Tris–HCl, 50 mM NaCl, pH 7.0] was used for absorption titration, and viscosity measurements. Buffer B (50 mM Tris–HCl, 18 mM NaCl, pH 7.2) was used for DNA photocleavage experiments. Solutions of CT DNA in buffer A gave a ratio of UV–vis absorbance of 1.8–1.9:1 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein [27]. The concentration of DNA was determined spectrophotometrically (ε260 = 6600 M−1 cm−3) [28]. The absorption titrations of the complex in buffer were performed using a fixed concentration (20 μM) for complex to which increments of the DNA stock solution were added. The intrinsic binding constant K, based on the absorption titration, was measured by monitoring the changes in absorption at the MLCT (metal-to-ligand charge transfer) band with increasing concentration of DNA using the following equation [29].
i ½DNA=ðε a −ε f Þ ¼ ½DNA=ðεb −ε f Þ þ 1=½K b ðε b −ε f Þ

Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay procedures were used [34]. Cells were placed in 96-well microassay culture plates (8 × 10 3 cells per well) and grown overnight at 37 °C in a 5% CO 2 incubator. The tested compounds were then added to the wells to achieve final concentrations ranging from 10− 6 to 10− 4 M. Control wells were prepared by addition of culture medium (100 μL). The plates were incubated at 37 °C in a 5% CO2 incubator for 48 h. Upon completion of the incubation, stock MTT dye solution (20 μL, 5 mg · mL−1) was added to each well. After 4 h, buffer (100 μL) 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 calculated by plotting the percentage viability versus concentration on a logarithmic graph and reading off the concentration at which 50% of cells remained viable relative to the control. Each experiment was repeated at least three times to obtain the mean values. 2.5. Apoptosis assessment by AO/EB staining Apoptosis studies were performed with a staining method utilizing acridine orange (AO) and ethidium bromide (EB). According to the difference in membrane integrity between necrotic and apoptosis. AO can pass through cell membrane, but EB cannot. Under fluorescence microscope, living cells appear green. Necrotic cells stain red but have a nuclear morpholopy resembling that of viable cells. Apoptosis cells appear green, and morphological changes such as cell blebbing and formation of apoptotic bodies will be observed. BEL-7402 cells was incubated in the absence and presence of complexes 1–4 at concentration of 12.5 μM at 37 °C and 5% CO2 for 48 h, then each cell culture was stained with AO/EB solution (100 μg/ml AO, 100 μg/mL EB). Samples were observed under a fluorescence microscope. 2.6. Comet assay

where [DNA] is the concentration of DNA in base pairs, εa, εf and εb correspond to the apparent absorption coefficient Aobsd / [Ru], the extinction coefficient for the free ruthenium complex and the extinction coefficient for the ruthenium complex in the fully bound form, respectively. In plots of [DNA] / (εa − εf) versus [DNA], Kb is given by the ratio of slope to the intercept. Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a constant temperature 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 [30]. Flow time was measured with a digital stopwatch, and each sample was measured three times, and an average flow time was calculated. Relative viscosities for DNA in the presence and absence of complex were calculated from the relation η = (t − t0)/t0, where t is the observed flow time of the DNAcontaining solution and t0 is the flow time of buffer alone [31,32]. The change in the viscosity was presented as (η/η0) 1/3 versus binding ratio [Ru]/[DNA] [33], where η is the viscosity of DNA solution

DNA damage was investigated by means of comet assay. BEL-7402 cells in culture medium were incubated with 12.5 and 25 μM of complexes 1–4 for 24 h at 37 °C. The control cells were also incubated in the same time. The cells were harvested by a trypsinization process at 24 h. A total of 100 μL of 0.5% normal agarose in PBS was dropped gently onto a fully frosted microslide, covered immediately with a coverslip, and then placed at 4 °C for 10 min. The coverslip was removed after the gel had set. 50 μL of the cell suspension (200 cells/μL) was mixed with 50 μL of 1% low melting agarose preserved at 37 °C. A total of 100 μL of this mixture was applied quickly on top of the gel, coated over the microslide, covered immediately with a coverslip, and then placed at 4 °C for 10 min. The coverslip was again removed after the gel had set. A third coating of 50 μL of 0.5% low melting agarose was placed on the gel and allowed to set at 4 °C for 15 min. After solidification of the agarose, the coverslips were removed, and the slides were immersed in an ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 90 mM sodium sarcosinate, NaOH, pH 10, 1% Triton X-100 and 10% DMSO) and placed in a refrigerator at 4 °C for 2 h. All

G.-B. Jiang et al. / Journal of Inorganic Biochemistry 141 (2014) 170–179 Table 1 IC50 values of dmdppz and complexes 1, 2, 3 and 4 toward the selected cell lines. Complexes

IC50 (μM) HepG-2

HeLa

MG-63

A549

BEL-7402

dmdppz 1 2 3 4 Cisplatin

N200 N100 N200 N200 44.3 ± 4.7 11.5 ± 1.2

N100 95.2 N100 60.4 ± 4.2 14.3 ± 1.4 7.2 ± 1.4

N200 19.7 ± 1.2 N100 18.8 ± 1.3 24.8 ± 1.2 6.5 ± 0.5

N200 93.9 N100 N100 21.1 ± 2.1 6.8 ± 0.8

N200 14.6 ± 16.8 ± 18.0 ± 16.7 ± 11.6 ±

1.1 1.3 1.2 1.1 1.3

of the above operations were performed under low lighting conditions to avoid additional DNA damage. The slides, after removal from the lysis solution, were placed horizontally in an electrophoresis chamber. The reservoirs were filled with an electrophoresis buffer (300 mM NaOH, 1.2 mM EDTA) until the slides were just immersed in it, and the DNA was allowed to unwind for 30 min in electrophoresis solution. Then the electrophoresis was carried out at 25 V and 300 mA for 20 min. After electrophoresis, the slides were removed, washed thrice in a neutralization buffer (400 mM Tris, HCl, pH 7.5). Nuclear DNA was stained with 20 μL of EtBr (20 μg / mL) in the dark for 20 min. The slides were washed in chilled distilled water for 10 min to neutralize the excess alkali, air-dried and scored for comets by fluorescence microscopy. 2.7. Reactive oxygen species (ROS) levels studies BEL-7402 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 medium supplemented with 10% of fetal bovine serum (FBS) and incubated at 37 °C and 5% CO2. The medium was removed and replaced with medium (final DMSO concentration 0.05% v/v) containing complexes 1–4 (12.5 μM) for 24 h. The medium was removed again. The fluorescent dye 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) was added to the medium with a final concentration of 10 μM to cover the cells. The treated

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cells were then washed with cold PBS-EDTA twice (PBS = phosphate buffered saline), collected by trypsinization and centrifugation at 1500 rpm for 5 min, and resuspended in PBS-EDTA. Fluorescence was imaged with fluorescent microscope at an excitation wavelength of 488 nm and emission at 525 nm. 2.8. The change of mitochondrial membrane potential assay BEL-7402 cells were treated with complexes 1–4 for 24 h 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 μg/mL of 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolcarbocyanine iodide (JC-1) in culture medium at 37 °C in the dark. Cells were immediately centrifuged to remove the supernatant. Cell pellets were suspended in PBS and then imaged under fluorescence microscope. 2.9. Cell cycle arrest studies BEL-7402 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. The medium was removed and replaced with medium (final DMSO concentration 0.05% v/v) containing complexes 1–4 (12.5 μM). 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 FACSCalibur flow cytometry. The number of cells analyzed for each sample was 10,000 [35]. 2.10. Western blot analysis BEL-7402 cells were seeded in 3.5 cm dishes for 24 h and incubated with different concentrations of the complex in the presence of 10% FBS.

Fig. 1. Apoptosis in BEL-7402 cells (a) exposure to 12.5 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h. L, A and N stand for living, apoptotic and necrotic cells, respectively.

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Fig. 2. Comet assay of EB-stained control (a) and 25 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) treated BEL-7402 cancer cells at 24 h incubation. The red (white/gray in the gray-scale print version) well-formed comets were observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Then cells were harvested in lysis buffer. After sonication, the samples were centrifuged for 20 min at 13,000 g. The protein concentration of the supernatant was determined by BCA (bicinchoninic acid) 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

at 1:5000 dilutions in 5% non-fat milk overnight at 4 °C, and after being washed for 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 for four times with TBST. The blots were visualized with the Amersham ECL Plus western blotting detection reagents according to the manufacturer's instructions. To assess the presence of comparable amount of proteins in each lane, the membranes were stripped finally to detect the β-actin.

Fig. 3. Intracellular ROS was detected in BEL-7402 cells (a) exposure to Rosup (b) and 25 μM of complexes 1 (c), 2 (d) 3 (e) and 4 (f) for 24 h. Rosup was used as positive control and the green (white/gray in the gray-scale print version) spots were observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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different cytotoxic activity toward HepG-2, HeLa, MG-63, A549 and BEL-7402 cells. 3.2. Apoptosis assay

3. Results and discussion

In order to observe the morphological changes, BEL-7402 cells were stained with acridine orange (AO) and ethidium bromide (EB). Since AO is a crucial dye that stains nuclear DNA across an intact cell membrane and EB only stains cells that have lost membrane integrity. The living cells will be uniformly stained green, apoptotic cells are stained green and contain apoptotic characteristics such as cell blebbing, nuclear shrinkage and chromatin condensation, necrotic cells are stained as red and can be found by the AO/EB double staining. After BEL-7402 cells were exposed to 12.5 μM of complexes 1–4 for 24 h, the morphological changes are shown in Fig. 1. In the control, the living cells of BEL-7402 were stained bright green in spots. The treatment of BEL7402 cells with complexes 1–4, green apoptotic cells with apoptotic characteristics such as nuclear shrinkage and chromatin condensation, as well as red necrotic cells, were observed. These observations indicate that complexes 1–4 can induce apoptosis in BEL-7402 cells. Other ruthenium(II) complexes with similar apoptotic features were also reported [36–38].

3.1. In vitro cytotoxicity assay

3.3. Comet assay

The cytotoxicity of the complexes 1–4 toward HepG-2, HeLa, MG-63, A549 and BEL-7402 cell lines was evaluated using MTT assay. The IC50 values of complexes 1–4 against the five tumor cell lines are listed in Table 1. As can be seen from Table 1, unexpectedly, ligand dmdppz is found to show no cytotoxic activity toward the selected cell lines. Among these complexes, complexes 1 and 3 can effectively inhibit the cell growth of MG-63 and BEL-7402 cells, complex 2 displays high cytotoxic activity only against BEL-7402 cells, whereas complex 4 shows effective cytotoxic activity against all the five cell lines. The difference in cytotoxic activity of the complexes 1–4 toward the same cell line may be caused by different ancillary ligands, different ancillary ligands induce different function of complexes 1–4. Comparing the IC50 values, the cytotoxicity of these complexes is lower than cisplatin under identical conditions. These results demonstrate that complexes 1–4 display

DNA fragmentation is a hallmark of apoptosis, mitotic catastrophe or both [39]. Single cell gel electrophoresis (comet assay) in an agarose gel matrix was used to study DNA fragmentation. As shown in Fig. 2, in the control, BEL-7402 cells fail to show a comet like appearance. Treatment of BEL-7402 cells with 25 μM of complexes 1–4 shows statistically significant and well-formed comets, and the length of the comet tail represents the extent of DNA damage. These results clearly indicate that the four complexes indeed induce DNA fragmentation, which is further evidence of apoptosis.

Fig. 4. Effects on ROS generation induced by different concentrations of complexes 1 (orange, 2nd and 3rd columns from left), 2 (red, 4th and 5th columns from left), 3 (green, 6th and 7th columns from left) and 4 (blue, two far right columns) in BEL-7402 cells. Data were calculated from three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. ROS levels detection Many potential anticancer and chemopreventive agents induce apoptosis through ROS generation [40]. DCFH-DA was used as fluorescent

Fig. 5. Assay of BEL-7402 cells mitochondrial membrane potential with JC-1 as fluorescence probe staining method. BEL-7402 cells (a) exposed to 12.5 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h.

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complexes 1, 2, 3 and 4 follows the order of 3 N 4 N 1 N 2 at 25 μM. Moreover, the level of ROS shows a concentration-dependent manner. 3.5. Mitochondrial membrane potential assay

Fig. 6. Assay of BEL-7402 cells mitochondrial membrane potential with JC-1 as fluorescence probe staining method. BEL-7402 cells exposed to 12.5 and 25 μM of complexes 1 (orange, 2nd and 3rd columns from left), 2 (red, 4th and 5th columns from left), 3 (green, 6th and 7th columns from left) and 4 (blue, two far right columns) for 24 h. *p b 0.05 represents significant differences compared with the control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

probe to detect ROS change in BEL-7402 cells [19], DFCH-DA is cleaved by intracellular esterases into its non-fluorescent form (DCFH). The non-fluorescent substrate is oxidized by intracellular free radicals to produce a fluorescent product, namely, dichlorofluorescein (DCF). As shown in Fig. 3, in the control (a), no fluorescence image is found. After BEL-7402 cells were exposed to 25 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h, the fluorescence images are observed. These results demonstrate that ROS in BEL-7402 cell lines can be generated and complexes 1–4 can increase the levels of ROS. To quantitatively compare the levels of the ROS induced by 1–4, we also measured the fluorescent intensity of DCF. As shown in Fig. 4, BEL-7402 cells were treated with 25 μM of complexes 1–4, the fluorescent intensities are 144.7, 121.7, 179.3 and 161.2, respectively. Compared the complexes 1–4 with the control, the fluorescent intensities of DCF grow 2.19, 1.84, 2.72 and 2.44 times than the original. ROS generation induced by

Mitochondria play a vital role in apoptosis triggered by chemical reagent. Chemical-induced apoptosis mediated by the mitochondria/ apoptotic cascades is often associated with the collapse of the mitochondrial membrane potential as a result of leakage of proapoptotic factors. The lipophilic fluorescent probe 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to determine the change in the mitochondrial membrane potential. As shown in Fig. 5a, JC-1 exhibits red fluorescence in the control corresponding to high mitochondrial membrane potential. After BEL-7402 cells were exposed to 12.5 μM of complexes 1, 2, 3 and 4 for 24 h, a significant increase of the green fluorescence is observed (5b–5e). The changes from red to green fluorescence suggest that complexes 1–4 induce the decrease of mitochondrial membrane potential. In order to quantitatively determine the change of the mitochondrial membrane potential induced by 1–4, the ratio of red/green fluorescent intensity is calculated by determining the red and green fluorescent intensities with microplate analyzer (Fig. 6). In the control, the ratio of red/green is 2.62. BEL-7402 cells were treated with 25 μM of 1–4 for 24 h, the ratios of red/green are 1.09, 0.45, 0.50 and 0.48, respectively. The decrease in the ratio of red/green suggests that the red fluorescent intensity reduces and the green fluorescent intensity increases. Fig. 6 also shows the decrease in mitochondrial membrane potential is dose-dependent. These results show that the complexes can induce the decrease of mitochondrial membrane potential. 3.6. Cell cycle arrest studies To understand further the mechanism of cell death, the cell cycle arrest in BEL-7402 cells was investigated using flow cytometry in propidium-iodide-stained cells after BEL-7402 cells were exposed to

Fig. 7. Cell cycle distribution of BEL-7402 cells (a) exposure to 12.5 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h.

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12.5 μM of the complexes 1, 2, 3 and 4 for 24 h. As shown in Fig. 7, in the control, the percentage in cells at S phase is 20.75%. Treatment of BEL7402 cells with complexes 1, 2, 3 and 4, the percentages in the cells at S phase are 38.05%, 38.96%, 30.84% and 36.56%, respectively. An obvious increase of 17.30% for 1, 18.21% for 2, 10.09% for 3 and 15.81% for 4 in the percentage at S phase was observed, accompanied by a corresponding reduction of 15.45% for 1, 18.25% for 2, 11.16% for 3 and 7.67% for 4 in the percentage of cells in the G0/G1 phase. These data indicate that the antiproliferative mechanism induced by complexes 1–4 on the BEL-7402 cells was a S phase arrest. In addition, the increase in the percentage of apoptotic cells suggests that complexes 1–4 can induce apoptosis of BEL-7402 cells. 3.7. The assay of the expression of caspase, antiapoptotic and proapoptotic proteins Caspase-3 and -7 are executioners of apoptosis as processing of their substrates lead to morphological changes associated with apoptosis, including DNA degradation, chromatin condensation, and membrane blebbing [41]. The activation of caspase-3, -7 and procaspase-7 was assayed by western blot analysis. After the treatment of BEL-7402 cells with 12.5 and 25 μM of complex 1, the expression levels of caspase-3, procaspase-7 and caspase-7 increase (Fig. 8a and b). The Bcl-2 family of proteins constitutes a critical intracellular checkpoint in the intrinsic pathway of apoptosis [42]. Many studies on ruthenium(II) complexes inducing cell apoptosis shows that Bcl-2 family members are important key regulators of mitochondrial function during apoptosis [42–44]. To investigate the effect of the complex 1 on the expression levels of the proteins, BEL-7402 cells were treated with different concentration of complex 1 for 24 h. As shown in Fig. 8a and c, the levels of antiapoptotic protein Bcl-2 and the proapoptotic protein Bad were down-regulated. However, the levels of the Bax were up-regulated. Interestingly, the ratio of Bax/Bcl-2 protein was increased. The increase of Bax/Bcl-2 ratio indicates that mitochondria are permeabilized to release pro-apoptotic proteins [45]. These results indicate that the complex 1 induce apoptosis in BEL-7402 cells through activation of caspase-3, caspase-7, down-regulation of Bcl-2, up-regulation of Bax and ROS-mediated mitochondrial dysfunction pathways. 3.8. Absorption spectroscopic studies Electronic absorption spectroscopy is the commonest means to study the interaction of metal complexes with DNA [46]. The spectral profile of the complexes 1–4 in the increasing concentration of CT DNA is shown in Fig. 9. As the DNA concentration increased, the metal-to-ligand charge transfer (MLCT) bands of 1 at 446 nm, 2 at 444 nm, 3 at 441 nm and 4 at 451 nm exhibit hypochromism of about 16.3%, 22.2%, 20.3% and 18.8%, respectively. These spectral characteristics obviously suggest that these complexes interact with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and base pairs of DNA. To further evaluate the binding strength of the complexes with DNA, the intrinsic binding constant Kb was determined by monitoring the changes in the absorbance at the MLCT band. The K b values of 1, 2, 3 and 4 are 3.75 × 104 M−1, 3.98 × 104 M−1, 6.89 × 104 M−1 and 9.68 × 103 M−1, respectively. The Kb values of the complexes 1–4 follow the order of 3 N 2 N 1 N 4. These values are comparable to that of [Ru(dmp)2 (APIP)] 2 + (2.3 × 104 M− 1 ) [47] and [Ru(dmb) 2 (BFIP)]2 + (3.2 × 104 M− 1) [48], but less than that of complexes [Ru(phen)2(dppz)]2+ (5.1 × 106 M−1) [49] and [Ru(dmb)2(dppz)]2+ (4.5 × 106 M−1) [50]. Complex 4 shows the least binding strength to double-helical DNA. Substitution in the 2- and 9-positions of the ancillary phen ligand may cause severe steric constraints near the core of Ru(II) when the complex intercalates into the DNA base pairs. The Me groups may come into close proximity of base pairs at the

Fig. 8. (a) Western blot analysis of Caspase 3, Procaspase 7, Caspase 7, Bcl-2, Bad and Bax in BEL-7402 cells treated with different concentrations of complex 1 for 24 h. β-actin was used as internal control. Panels (b) and (c): Percentage expression levels of Caspase 3 (left column), Procaspase 7 (middle column) and Caspase 7 (right column) in panel (b); and of Bcl-2 (left column), Bad (middle column) and Bax (right column) in panel (c). The percentage values are those relative to the control.

intercalation sites. These steric clashes then prevent the complex from intercalating effectively, which causes a decrease of the intrinsic constant. Such clashes would not be present with the ancillary dmb, bpy and phen ligands. However, this complex can effectively inhibit the cell growth toward the selected cell lines. This result shows that the cytotoxic activity of the complexes against the tumor cell lines is not consistent with their DNAbinding affinity.

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Fig. 9. Apsorption spectra of complexes in Tris–HCl buffer upon addition of CT DNA in the absence and presence of complexes 1 (a), 2 (b), 3 (c) and 4 (d). [Ru] = 20 μM. Arrows show the absorbance change upon the increase of DNA concentrations. Plots of [DNA] / (εa − εf) versus [DNA] for the titration of DNA with Ru(II) complexes.

3.9. Viscosity measurements In order to clarify the mode of Ru(II) complexes with CT DNA, the viscosity of DNA solution was measured. Hydrodynamic measurements (i.e., viscosity and sedimentation), being sensitive to length changes, are regarded as the least ambiguous and most critical tests for a classical intercalation model in solution in the absence of crystallographic structural data [31,32]. A classical intercalation of a ligand into DNA is known to an increase in the separation of the base pairs at the intercalation site and, hence, an increase in the overall DNA molecular length. The changes in the viscosity of CT DNA solution induced by complexes 1–4 are shown in Fig. 10. On increasing the amount of 1–4, the relative viscosity

of DNA solution increases steadily. The changes in the viscosity follow the order of 3 N 2 N 1 N 4. This is consistent with their DNA-binding affinity. These results demonstrate that all the complexes interact with CT DNA through intercalative mode. 3.10. Photocleavage of pBR322 DNA by Ru(II) complexes The cleavage reactions on plasmid DNA induced by ruthenium(II) complexes were performed and monitored by agarose gel electrophoresis. When circular plasmid DNA is subjected to electrophoresis, relatively fast migration is observed for the intact supercoiled form (Form I). If scission occurs on one strand (nicking), the supercoiled form will relax to generate a slower-moving open circular form (Form II). If both strands are cleaved, a linear form (Form III) that migrates between Form I and Form II will be generated [51]. Fig. 11 shows that no DNA cleavage was observed in the control (DNA alone) and dark (incubation of the plasmid with the complexes in darkness). However, the amounts of Form I gradually decrease and Form II gradually increase when the pBR322 DNA incubated with different concentration of complexes 1–4 upon irradiation at 365 nm for 30 min. The results indicate that complexes 1–4 can effectively cleave pBR322 DNA and the scission occurs on one strand. 4. Conclusions

Fig. 10. Effect of increasing amounts of complexes 1, 2, 3 and 4 on the relative viscosity of CT DNA at 25 (±0.1) °C. [DNA] = 100 μM.

Four ruthenium(II) polypyridyl complexes [Ru(dmb)2(dmdppz)] (ClO4)2 (1), [Ru(bpy)2(dmdppz)](ClO4)2 (2), [Ru(phen)2(dmdppz)] (ClO4)2 (3) and [Ru(dmp)2(dmdppz)](ClO4)2 (4) have been synthesized and characterized. The DNA-binding studies suggest that all the complexes interact with CT DNA through intercalative mode. Cytotoxicity assays show that the four Ru(II) complexes have high tumor activity

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Fig. 11. Photoactivated cleavage of pBR322 DNA in the presence of different concentration of complexes 1–4 upon irradiation at 365 nm for 30 min.

against BEL-7402. AO/EB stained-cells and comet assay demonstrate that complexes 1–4 can effectively induce the apoptosis of BEL-7402 cells. The cell cycle arrest studies suggest that the antiproliferative mechanism in BEL-7402 cells induced by complexes 1–4 was S phase. Additionally, the complexes can increase the levels of ROS, and induce the decrease of the mitochondrial membrane potential. These results indicate that complexes 1–4 induce apoptosis of BEL-7402 cells through the intrinsic ROS-mediated mitochondrial pathway, which is accompanied by the regulation of Bcl-2 family proteins. The results will be of value for further understanding the DNA binding and designing new ruthenium(II) complexes as potent antitumor agents. Acknowledgements This work was supported by the National Nature Science Foundation of China (No. 31070858), High-Level Personnel Project of Guangdong Province in 2013 and the Joint Nature Science Fund of the Department of Science and Technology and the First Affiliated Hospital of Guangdong Pharmaceutical University (No. GYFYLH201315) References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

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