Journal of Photochemistry and Photobiology B: Biology 129 (2013) 48–56

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Synthesis, characterization, DNA interaction, antioxidant and anticancer activity studies of ruthenium(II) polypyridyl complexes Guang-Bin Jiang a, Yang-Yin Xie a, Gan-Jian Lin a, Hong-Liang Huang b,⇑, Zhen-Hua Liang a, Yun-Jun Liu a,⇑ a b

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China School of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University, Guangzhou 510006, PR China

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 12 August 2013 Received in revised form 26 September 2013 Accepted 27 September 2013 Available online 15 October 2013

Two new Ru(II) polypyridyl complexes [Ru(phen)2(adppz)](ClO4)2 (1) and [Ru(dip)2(adppz)](ClO4)2 (2) have been synthesized and characterized. The DNA-binding constants were determined to be 6.54 ± 0.42  105 and 7.65 ± 0.20  105 M1 for complexes 1 and 2. DNA binding experiments indicated that complexes 1 and 2 interact with DNA through intercalative mode. Antioxidant activity shows that the complexes have significant hydroxyl radical scavenging activity. Cytotoxic activities suggest that the complex 2 exhibits higher cytotoxic activity against BEL-7402, MG-63 and SKBR-3 cells than complex 1 under identical conditions. Complexes 1 and 2 can induce apoptosis of BEl-7402 cells. We have identified several cellular mechanisms induced by 1 and 2 in BEL-7402 cells, including the level detection of ROS, activation of procaspase 3, caspase 7, the expression of antiapoptotic proteins Bcl-x, Bcl-2, proapoptotic proteins Bad, Bax, Bid and cell cycle arrest. Thus, complexes 1 and 2 inhibit growth of BEL-7402 cells through induction of apoptotic cell death, enhancement of ROS levels and S-phase and G0/G1 cell cycle arrest. Further investigations have shown that complex 2 induces apoptosis by regulating the expression of Bcl-2 family proteins. Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved.

Keywords: Ru(II) polypyridyl complex DNA-binding Cytotoxicity in vitro Reactive oxygen species Cell cycle arrest Western blot

1. Introduction Binding studies of small molecules to DNA are very important in the development of DNA molecular probes and new therapeutic reagents [1–3]. Polypyridyl ruthenium(II) complexes bind to DNA via non-covalent interactions such as electrostatic binding, groove binding and intercalation. Many Ru(II) complexes exhibit interesting properties upon binding to DNA or RNA or G-quadruplex DNA [4–13]. [Ru(bpy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ (dppz = dipyrido[3,2-a:20 ,30 -c]phenazine) show no luminescence in aqueous solution at ambient temperature, but luminesce brightly upon binding DNA intercalatively with the dppz ligand between adjacent DNA base pairs, displaying the characteristic of 00 molecular light switch00 [14–16]. Complex [Ru(phen)2(mitatp)]2+ shows large DNA-binding affinity and can cleave pBR322 DNA in visible light [17]. On the other hand, significant side effect of cisplatin has motivated extensive investigations into alternative metal-based cancer therapies. Ruthenium complex is considered to be one of the most promising drugs, a number of ruthenium complexes have been shown to display high anticancer activities. [Ru(bpy)2(dppn)]2+ exhibits high cytotoxicity toward MCF-7 cancer call line comparable to that of cisplatin [18], [Ru(phen)2(AHPIP)]2+ can effectively inhibit the cell

growth of BEL-7402 [19], and [Ru(phen)2(p-MOPIP)]2+ can suppress the proliferation of HepG-2 cells with a low IC50 value of 7.2 ± 1.3 lM [20], and NAMI-A and KP109 have entered clinical trials [21,22]. In this report, two new ruthenium(II) polypyridyl complexes [Ru(phen)2(adppz)](ClO4)2 (phen = 1,10-phenanthroline, adppz = 7-aminodipyrido[3,2-a:20 ,30 -c]phenazine) (1) and [Ru(dip)2(adppz)](ClO4)2 (dip = 4,7-diphenyl-1,10-phenanthroline, Scheme 1) (2) were synthesized and characterized by elemental analysis, ES-MS and 1H NMR. The DNA-binding behaviors were investigated by electronic absorption spectra, luminescence spectra and viscosity measurements. The antioxidant activity of the complexes against hydroxyl radical was explored. The cytotoxicity in vitro of the complexes was assessed by MTT assays (MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). The morphological apoptosis of BEL-7402 cells induced by complexes 1 and 2 was studied by fluorescent microscopy. The reactive oxygen species (ROS) and cell cycle arrest were analyzed by flow cytometry. The cellular uptake was studied with DAPI staining cell nuclei method, and western blotting analysis was also performed. 2. Experimental 2.1. Materials and method

⇑ Corresponding authors. Tel.: +86 20 39352122; fax: +86 20 39352129. E-mail (Y.-J. Liu).

addresses:

[email protected]

(H.-L.

Huang),

[email protected]

All reagents and solvents were purchased commercially and used without further purification unless otherwise noted.

1011-1344/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.09.009

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Scheme 1. Synthetic route for complexes 1 and 2.

Ultrapure MilliQ water was used in all experiments. DMSO, 4-nitro-1,2-diaminobenzene and RPMI 1640 were purchased from Sigma. Cell lines of BEL-7402 (Hepatocellular), A549 (Human lung carcinoma), MG-63 (Human osteosarcoma) and SKBR-3 (Human breast cancer) were purchased from the American Type Culture Collection. RuCl3v H2O was purchased from the Kunming Institution of Precious Metals. 1,10-phenanthroline was obtained from the Guangzhou Chemical Reagent Factory. Microanalyses (C, H, and N) were obtained with a Perkin–Elmer 240Q elemental analyzer. Electrospray ionization mass spectra (ESMS) 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 [D6] as solvent and tetramethylsilane (TMS) as an internal standard at 500 MHz at room temperature. 2.2. Synthesis of complexes 1,10-phenanthroline-5,6-dione [23] were prepared according to the methods in the literature. Adppz was prepared by reducing 7nitro-dppz [24] with Pd/C and NH2NH2H2O and refluxed for 4 h. The hot solution was filtered and evaporated under reduced pressure to remove the solvent to 5 cm3. Upon cooling, a red precipitate was obtained by filtration and washed with cool ethanol. Yield: 75%. Anal. Calc for C18H11N5: C, 72.72; H, 3.73; N, 23.56%. Found: C, 72.84; H, 3.64; N, 23.66%. FAB-MS: m/z = 298 [M+1]+. 1H NMR (DMSO-d6): d 9.48 (d, 1H, J = 8.0 Hz), 9.43 (d, 1H, J = 8.0 Hz), 9.17 (d, 1H, J = 5.0 Hz), 9.10 (d, 1H, J = 6.5 Hz), 8.06 (d, 1H, J = 6.5 Hz), 7.86-7.90 (m, 2H), 7.50 (d, 1H, J = 8.5 Hz), 7.14 (d, 1H, J = 5.0 Hz), 6.55 (s, 2H). 2.2.1. Synthesis of [Ru(phen)2(adppz)](ClO4)2 (1) A mixture of cis-[Ru(phen)2Cl2]2H2O [25] (0.284 g, 0.5 mmol) and adppz (0.149 g, 0.5 mmol) in ethylene glycol (70 cm3) was refluxed under argon for 6 h to give a clear red solution and the solvent was removed to about 10 cm3 under reduced pressure. 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. Calc for C42H27N9Cl2O8Ru: C, 52.67; H, 2.84; N, 13.16%. Found: C, 52.58; H, 2.89; N, 13.34%. 1H

NMR (DMSO-d6): d 9.50 (d, 1H, J = 7.0 Hz), 9.42 (d, 1H, J = 7.0 Hz), 8.77 (dd, 4H, J = 7.5, J = 7.5 Hz), 8.39 (s, 4H), 8.26 (d, 3H, J = 5.5 Hz), 8.12 (dd, 2H, J = 8.0, J = 7.0 Hz), 8.05 (t, 4H, J = 5.5 Hz), 7.45-7.83 (m, 4H), 7.60 (d, 1H, J = 5.0 Hz), 7.18 (d, 1H, J = 7.5 Hz), 6.85 (s, 2H), ES-MS (CH3CN): m/z 757.5 ([M-2ClO4-H]+), 379.4 ([M-2ClO4]2+). 2.2.2. Synthesis of [Ru(dip)2(adppz)](ClO4)2 (2) This complex was synthesized in a manner identical to that described for complex 1, with [Ru(dip)2Cl2]2H2O [26] in place of [Ru(phen)2Cl2]2H2O. Yield: 71%. Anal. Calc for C66H43N9Cl2O8Ru: C, 62.81; H, 3.43; N, 9.99%. Found: C, 62.72; H, 3.37; N, 10.21%. 1 H NMR (DMSO-d6): d 9.50 (d, 2H, J = 7.5 Hz), 8.39 (dd, 4H, J = 5.5, J = 5.5 Hz), 8.35 (d, 2H, J = 5.5 Hz), 8.33 (s, 4H), 8.24 (d, 1H, J = 5.5 Hz), 8.18 (d, 1H, J = 8.5 Hz), 7.93-7.97 (m, 2H), 7.84 (d, 2H, J = 7.5 Hz), 7.81 (d, 2H, J = 5.5 Hz), 7.61-7.72 (m, 20H), 7.20 (s, 1H), 6.91 (s, 2H). ES-MS (CH3CN): m/z 532.0 ([M-2ClO4]2+). 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. DNA-binding studies The DNA-binding was performed at room temperature. Buffer A [5 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride, 50 mM NaCl, pH 7.0] was used for absorption titration. 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 (e260 = 6600 M1 cm1) [28]. The absorption titrations of the complex in buffer were performed using a fixed concentration (20 lM) for complex to which increments of the DNA stock solution were added. Ru-DNA solutions were allowed to incubate for 5 min before the absorption spectra were recorded. The intrinsic binding constants K, based on the absorption titration, were measured by monitoring the changes in absorption at the MLCT band with increasing concentration of DNA using the following equation [29]. 2

ðea  ef Þ=ðeb  ef Þ ¼ b  ðb  2K 2 C t ½DNA=sÞ ðb ¼ 1 þ KC t þ K½DNA=2sÞ

1=2

=2KC t

ð1Þ ð2Þ

where [DNA] is the concentration of CT DNA in base pairs, the apparent absorption coefficients ea, ef and eb correspond to Aobsed/ [Ru], the absorbance for the free ruthenium complex, and the absorbance for the ruthenium complex in fully bound form, respectively.

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K is the equilibrium binding constant, Ct is the total metal complex concentration in nucleotides and s is the binding site size. 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 complexes were calculated from the relation g = (t – t0)/t0, where t is the observed flow time of the DNA-containing solution and t0 is the flow time of buffer alone [31,32]. Data were presented as (g/g0)1/3 versus binding ratio [33], where g is the viscosity of DNA in the presence of complexes and g0 is the viscosity of DNA alone.

microscope (Nikon, Yokohama, Japan) with excitation at 350 nm and emission at 460 nm. 2.7. Cellular uptake and co-localisation studies 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. Complexes tested were then added to the wells. The plates were incubated at 37 °C in a 5% CO2 incubator for 24 h. Upon completion of the incubation, the wells were washed three times with phosphate buffered saline (PBS), after removing the culture mediums in the wells. The cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and visualized by fluorescence microscope. 2.8. Reactive oxygen species assays

2.4. Scavenger measurements of hydroxyl radical (OH) The hydroxyl radical (OH) in aqueous media was generated by the Fenton system [34]. The solution of the tested complexes was prepared with DMF (N,N-dimethylformamide). The 5 ml of assay mixture contained following reagents: safranin (28.5 lM), EDTA-Fe(II) (100 lM), H2O2 (44.0 lM), the tested compounds (0.5–4.5 lM) and a phosphate buffer (67 mM, pH = 7.4). The assay mixtures were incubated at 37 °C for 30 min in a water bath. After which, the absorbance was measured at 520 nm. All the 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; Ac was the absorbance in the absence of tested compound, EDTA-Fe(II), H2O2. The suppression ratio (ga) was calculated on the basis of (Ai  A0)/(Ac  A0)  100%. 2.5. In vitro cytotoxicity assay Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay procedures were used. 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 test 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 lL). 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 lL, 5 mg  mL–1) was added to each well. After 4 h, buffer (100 lL) 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.

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 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 different concentrations of complex 2 (12.5 lM and 25 lM) for 24 h. The medium was removed again. The fluorescent dye (CM-H2DCFDA) was added to the medium with a final concentration of 10 lM to cover the cells. The treated cells were then washed with cold PBS–EDTA twice, collected by trypsinization and centrifugation at 1500 rpm for 5 min, and resuspended in PBS–EDTA. Fluorescence activated cell sorting flow cytometry was performed to analyze the cells with an excitation wavelength of 488 nm and emission at 525 nm. The fluorescence intensity was calculated by the determined fluorescence intensity minus the fluorescence intensity of the complexes in the corresponding concentration. 2.9. Cell cycle arrest by flow cytometry BEL-7402 or SKBR-3 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 fetal bovine serum (FBS, 10%) 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 or 2 (25 or 50 lM). After incubation for 24 h, the cell layer was trypsinized and washed with cold phosphate buffered saline (PBS) and fixed with 70% ethanol. Twenty lL of RNAse (0.2 mg/mL) and 20 lL 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 10000 [35]. 2.10. Western blotting analysis

2.6. Apoptosis assay by AO/EB and Hoechst 33258 staining BEL-7402 cells were seeded onto chamber slides in six-well plates at a density of 2  105 cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 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 and 2 (12.5–50 lM) for 24 h. The medium was removed and the cells were washed with ice-cold PBS, and fixed with formalin (4%, w/v). Cell nuclei were counterstained with AO/EB solution (100 lg mL1 AO, 100 lg mL1 EB) or Hoechst 33258 (10 lg/mL in PBS) for 10 min, then observed and imaged by fluorescence

BEL-7402 cells were seeded in 3.5-cm dishes for 24 h and incubated with different agents at 25 or 50 lM in the presence of 10% FBS. Then cells were harvested in lysis buffer. After sonication, the samples were centrifuged for 20 min at 13,000g. The protein concentration of the supernatant was determined by BCA assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done loading equal amount of proteins per lane. Gels were then transferred to poly (vinylidene difluoride) membranes (Millipore) and blocked with 5% non-fat milk in TBST buffer for 1 h. Then the membranes were incubated with primary antibodies at 1:5,000 dilutions in 5% non-fat milk overnight at 4 °C, and washed four times with TBST for a total of 30 min. After which the

G.-B. Jiang et al. / Journal of Photochemistry and Photobiology B: Biology 129 (2013) 48–56

secondary antibodies conjugated with horseradish peroxidase at 1:5,000 dilution for 1 h at room temperature and then washed 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 b-actin. 3. Results and discussion 3.1. Electronic absorption titration As shown in Fig. 1, the electronic absorption spectra of complexes 1 and 2 mainly consist of two or three resolved bands in the range of 200–600 nm. The bands below 300 nm are attributed to intraligand (IL) p–p⁄ transitions, the bands at the range of 300– 400 nm are attributed to p–p⁄ transitions, and the lowest energy bands at 440–460 nm are assigned to the metal-to-ligand charge transfer (MLCT) transition. As increasing the concentrations of calf thymus DNA (CT DNA), the MLCT transition bands of complexes 1 at 448 nm and 2 at 442 nm exhibit hypochromism of 27.5% and 22.1%, and bathochromism of 3 and 4 nm, 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 the base pairs of DNA. The DNA-binding constants were determined by monitoring the change in absorption at MLCT bands with increasing concentrations of DNA. The values of Kb are 6.54 ± 0.42  105 (s = 1.41) and 7.65 ± 0.20  105 M1 (s = 1.72) for complexes 1 and 2, respectively. These values are comparable with that of [Ru(dip)2(dadppz)]2+ (3.0 ± 0.2  105 M1) [36], but smaller than that of complex [Ru(bpy)2(dppz)]2+ (4.9  106 M1) [37]. 3.2. Luminescence spectra studies Fig. 2 shows that complexes 1 and 2 can emit luminescence in Tris buffer at ambient temperature, with a maximum appearing at 599 nm and 603 nm for 1 and 2. Upon addition of DNA, the emission intensities of complexes 1 and 2 grow to 1.35 and 1.13 times larger than the original, respectively. The enhancement of emission intensity is indicative of binding of the complexes to the hydrophobic pocket of DNA and be protected by DNA efficiently. 3.3. Viscosity measurements It is well known that a classical intercalation of a ligand into DNA is known to cause a significant increase in the viscosity of a

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DNA solution due to an increase in the separation of the base pairs at the intercalative site and, hence, an increase in the overall DNA molecular length. A partial and/or non-classical intercalation of ligand could bend (or kink) the DNA helix, reduces its effective length and, concomitantly, its viscosity [31,38]. The change in the relative viscosity of CT DNA on addition of complexes 1 and 2 is shown in Fig. 3. On increasing the amounts of complexes 1 and 2, the relative viscosity of CT DNA solution increase steadily. The increased degree of viscosity, which may depend on its affinity to DNA, followed the order of complex 2 > complex 1. The large enhancement in the relative viscosity values of complex 2 revealed that complex 2 is a better intercalator than complex 1, which are consistent with their DNA-binding constants. These results suggest that complexes 1 and 2 intercalate between the base pairs of DNA. 3.4. Antioxidant activity studies The hydroxyl radical is one of the most reactive products of reactive oxygen species (ROS). Among all free radicals, the hydroxyl radical is by far the most potent and therefore the most dangerous oxygen metabolite, which would result in cell membrane disintegration, membrane protein damage, DNA mutation and further initiate or propagate the development of many diseases. Elimination of this radical is one of the major aims of antioxidant administration [39]. As shown in Fig. 4 and Table 2, the inhibitory effect of the ligand adppz and its two complexes on OH was concentration-dependent and suppression ratio increased with increasing of the sample concentration in the range of 0.5– 4.0 lM. The suppression ratio against OH valued from 3.8% to 62.4% for adppz, 10.2% to 72.2% for complex 1, and 3.6% to 81.7% for complex 2, respectively. Obviously, complex 2 shows higher antioxidant activity than complex 1 under identical conditions. Additionally, it is clear that the hydroxyl radical scavenging activity can be enhanced when the ligand bonds Ru(II) metal center to form complexes. 3.5. Cytotoxicity in vitro assay The cytotoxicity in vitro was evaluated by MTT assay. The cell viability is depicted in Fig. 5. The inhibitory concentration 50 (IC50), defined as the concentration required to reduce the size of the cell population by 50%, and the IC50 values of complexes 1 and 2 against BEL-7402, A549, MG-63 and SKBR-3 cells are listed in Table 1. The cell viability was found to be concentration-dependent, namely, the cell viability decreased with increasing complex concentration. See from Table 1, complex 2 shows higher cytotoxic activity than complex 1 toward BEL-7402, MG-63 and SKBR-3 cells,

Fig. 1. Absorption spectra of complexes in Tris–HCl buffer upon addition of CT DNA in the presence of complexes 1 (a) and 2 (b). [Ru] = 20 lM. Arrow shows the absorbance change upon the increase of DNA concentration. Plots of (ea  ef)/(eb  ef) versus [DNA] for the titration of DNA with Ru(II) complexes.

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Fig. 2. Emission spectra of complexes 1 (a) and 2 (b) in Tris–HCl buffer in the absence and presence of CT DNA. Arrow shows the intensity change upon increasing DNA concentrations.

different anticancer activity against different tumor cell lines. Furthermore, the two complexes all show lower cytotoxic effect than cisplatin on the selected cell lines. 3.6. Apoptosis studies by AO/EB and Hoechst 33258 staining methods

Fig. 3. Effect of increasing amounts of complexes 1 and 2 on the relative viscosity of CT DNA at 25 (±0.1)°C. [DNA] = 250 lM.

Fig. 4. Scavenging effect of ligand adppz and complexes 1 and 2 on hydroxyl radicals. Experiments were performed in triplicate.

Table 1 The IC50 values of complexes 1 and 2 against the selected cell lines. Complex

1 2 Cisplatin

To observe the morphological characteristics of apoptotic nuclei, BEL-7402 cells were stained with acridine orange (AO) and ethidium bromide (EB). The AO/EB staining is sensitive to DNA and was used to assess the changes in nuclear morphology. Apoptotic and necrotic cells can be distinguished from one another using fluorescence microscopy. The apoptotic cells usually show apoptotic features such as nuclear shrinkage and chromatin condensation. In the control, the living cells were stained bright green in spots (Fig. 6a) and exhibit homogeneous nuclei staining. After BEL-7402 cells were exposed to 25 lM of complexes 1 (6b) and 2 (6c) for 24 h, green apoptotic cells were stained by acridine orange and display typical apoptotic changes (e.g., staining bright, condensed chromatin, and fragmented nuclei), and red necrotic cells stained by ethidium bromide were also observed. Similar results for other Ru(II) complexes were also found [40–43]. The apoptosis was also performed with Hoechst 33258 staining method. After the treatment of BEL-7402 cells (6d) with 12.5 lM of complex 2 for 24 h (6e), the green apoptotic cells with apoptotic features such as nuclear shrinkage were observed. The results show that complexes 1 and 2 can induce apoptosis of BEL-7402 cells. In addition, the cell apoptosis of BEL-7402 cells was analyzed by flow cytometry. The percentage of living, necrotic and apoptotic cells is shown in Fig. 7. In the control (a), the percentage of apoptotic cells is 0.00%. BEL-7402 cells exposure to 25 (b) or 50 lM (c) of complex 1, the percentage of apoptotic cells is 7.32% and 12.14%, respectively. The apoptotic effect induced by complex 1 is concentration dependent. Unexpectedly, complex 2 show very low apoptotic effect on BEL-7402 cells at 25 (d) and 50 lM (e). Comparing the apoptotic effect, complex 1 shows more effective apoptotic activity than complex 2 under identical conditions. These results also indicate that the apoptotic effect of complexes 1 and 2 on BEL-7402 cells is not consistent with their cytotoxic activity. 3.7. Cellular uptake and co-localisation studies

IC50 (lM) BEL-7402

A549

MG-63

SKBR-3

23.5 ± 2.3 12.7 ± 1.1 11.5 ± 1.2

16.7 ± 1.3 30.2 ± 2.6 7.3 ± 1.4

40.3 ± 3.2 15.9 ± 1.4 6.6 ± 0.5

81.0 ± 4.4 21.2 ± 1.8 6.7 ± 0.8

but less cytotoxicity against A549 cells than complex 2 under identical conditions. This indicates that different complex displays

After BEL-7402 cells were exposed to 12.5 lM of complexes 1 and 2 for 24 h, the cells were stained with DAPI and observed under fluorescence microscope. As shown in Fig. 8a and b, the blue channel displays DAPI stained nuclei with an excitation wavelength of 340 nm, the red channel shows the luminescence of complexes 1 and 2 with an excitation wavelength of 460 nm, and the overlay represents cellular association of 1 and 2. It is clear that complexes 1 and 2 can be successfully uptaken by BEL-7402 cells.

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G.-B. Jiang et al. / Journal of Photochemistry and Photobiology B: Biology 129 (2013) 48–56 Table 2 The data of ligand and complexes against hydroxyl radical. Complex

adppz 1 2

Average inhibition (%) for OH (lM) 0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

3.8 ± 1.2 10.2 ± 1.3 3.6 ± 1.1

11.3 ± 2.2 16.8 ± 1.6 26.4 ± 2.5

23.0 ± 2.8 29.4 ± 2.5 41.3 ± 4.6

29.4 ± 2.3 37.3 ± 2.9 68.2 ± 4.3

43.9 ± 3.5 45.7 ± 4.2 81.7 ± 5.6

51.7 ± 4.2 53.3 ± 4.8

56.8 ± 3.9 63.5 ± 3.7

62.4 ± 4.6 72.2 ± 4.7

Fig. 5. Cell viability of complexes 1 and 2 on BEL-7402 (a), A549 (b), MG-63 (c) and SKBR-3 (d) cells proliferation in vitro. Each point is the mean ± standard error obtained from three independent experiments.

Fig. 6. AO/EB staining BEL-7402 cells (a) exposure to 12.5 lM of complexes 1 (b) and 2 (c) and Hoechst 33258 staining of BEL-7402 cells (d) exposure to 12.5 lM of complex 2 (e) for 24 h. Liv, apo and nec stand for living, apoptotic and necrotic cells, respectively.

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Fig. 7. Apoptosis of BEL-7402 cells (a) exposure to complex 1 (black): 25 lM (b), 50 lM (c) and complex 2(black): 25 lM (d) and 50 lM (e) for 24 h.

Fig. 8. Images of BEL-7402 cells exposure to 25 lM of complexes 1 (a), 2 (b) and stained with DAPI at 37 °C for 24 h.

Complexes 1 and 2 gradually penetrate into the interior of the nucleus and show diffuse cytoplasmic and nuclear fluorescence. These results indicate that the complexes can enter into the cytoplasm and accumulate in the nuclei. 3.8. Reactive oxygen species (ROS) levels ROS, such as superoxide anion, radicals, hydrogen and organic peroxides, are generated by all aerobic cells as by-products of a

number of metabolite reactions and in response to various stimuli [44]. Many potential anticancer and chemopreventive agents induce apoptosis through ROS generation [45]. 20 ,70 -dichlorodihydrofluorescein diacetate was used to evaluate the intracellular ROS level in the form of cellular peroxides. This cell-permeant dye is hydrolyzed by intracellular esterases into its nonfluorescent form (DCFH). The nonfluorescent substrate is oxidized by intracellular free radicals to produce a fluorescent product, namely dichlorofluorescein (DCF). To investigate whether the cell death induced by

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the complex is dependent on ROS levels, BEL-7402 cells were exposed to 12.5 or 25 lM of complex 2 for 24 h. As shown in Fig. 9, in the control, the fluorescence intensity of DCF is 96.5, in the presence of 12.5 or 25 lM of 2, the fluorescence intensities of DCF are 420.5 and 1146.9. The DCF fluorescence intensities grow to 4.36 and 11.88 times larger than the original. These results suggest that complex 2 can enhance the level of intracellular ROS. 3.9. Cell cycle arrest assay The effect of complexes 1 and 2 on cell cycle of BEL-7402 and SKBR-3 cells was studied by flow cytometry in PI (propidium iodide) stained cells. As shown in Fig. 10, after the treatment of BEL-7402 cells with 25 lM of 1 and 2 for 24 h, an obvious increase of 6.85% for 1 at S-phase and 4.01% for 2 at G0/G1 phase was observed, accompanied by a corresponding reduction of 5.19% for 1 at G0/G1 and 3.50% for 2 at G2/M. These data suggest that the antiproliferative mechanism induced by complexes 1 and 2 on BEL7402 cells is S-phase and G0/G1 phase arrest, respectively. While SKBR-3 cells were exposed to 25 lM of complexes 1 and 2 for 24 h, the increase of 7.28% for 1 at G0/G1 phase and 8.33% for 2 at G2/M phase was observed, which indicates the antiproliferative mechanism of 1 and 2 on SKBR-3 cells is G0/G1 and G2/M phase, respectively. These results show that different complex displays different anticancer mechanism against different tumor cells. 3.10. Expression of caspase, antiapoptotic and proapoptotic proteins Since complexes 1 and 2 induced cell growth arrest and apoptosis, we examined some aspects of the cellular mechanisms that may account for these processes. Bcl-2 family proteins play an

Fig. 11. Western blotting analysis of procaspase 3, caspase 7, Bcl-x, Bcl-2, Bad, Bax and Bid in BEL-7402 cells treated with different concentrations of complex 2 for 24 h. b-actin was used as internal control.

important role in the intrinsic apoptosis, consisting of anti-apoptotic members, such as Mcl-1, Bcl-2, and Bcl-x, and pro-apoptotic members, including Bax, Bad, Bak, Bid, Bim. The effect of 2 on expression of Bcl-2, Bcl-x, Bad, Bax and Bid proteins in the BEL7402 cell line treated with metal complex for 24 h by western blotting. As shown in Fig. 11, the level of procaspase 3 was downregulated, whereas the level of expression of caspase 7 was upregulated. The level of expression of antiapoptotic protein Bcl-x decreased. Unexpectedly, treatment with 2 led to an increase in the expression of antiapoptotic Bcl-2 protein when the cells were exposed to 12.5–50 lM of complex 2; when treatment of proapoptotic proteins Bad, Bax and Bid with different concentrations of 2, the levels of expression of these proteins increased. Additionally, Fig. 11 also shows that the levels of expression of these proteins treated with different concentrations of 2 are concentrationdependent. 4. Conclusion

Fig. 9. DCF fluorescence intensity on reactive oxygen species (ROS) generation in BEL-7402 cells exposed to the different concentrations of complex 2 for 24 h.

Two new Ru(II) complexes [Ru(phen)2(adppz)](ClO4)2 1 and [Ru(dip)2(adppz)](ClO4)2 2 were synthesized and characterized. The DNA-binding behaviors show that the two complexes interact with CT DNA through intercalative mode. The antioxidant activity demonstrates that complexes 1 and 2 may be potential drugs to eliminate the hydroxyl radical. The cytotoxicity in vitro studies indicate that complexes 1 and 2 can inhibit the tumor cell proliferation. The apoptotic assay exhibits that the complex 1 shows more effective apoptotic effect on BEL-7402 cells than complex 2 under identical conditions. The cellular uptake suggests that these

Fig. 10. Cell cycle distribution of BEL-7402 (a) and SKBR-3 (b) cells exposure to 25 lM of complexes 1 and 2 for 24 h.

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complexes can enter into the cytoplasm and accumulate in the nuclei. The flow cytometric analysis shows that complexes 1 and 2 induce cell cycle arrest of BEL-7402 cells at S-phase and G0/G1 phase, and induce cell cycle arrest of SKBR-3 cells at G0/G1 phase and G2/ M phase, respectively. Western blotting experiments indicate that complex 2 downregulates the level of expression of procaspase 3 and antiapoptotic protein Bcl-x, and upregualtes the levels of expression of caspase 7 and proapoptotic proteins of Bad, Bax and Bid. In summary, these results suggest that complex 2 induces apoptosis of BEL-7402 cells through activation of caspase 7, upregulation of proapoptotic protein and ROS-mediated mitochondrial dysfunction pathways. Acknowledgements This work was supported by the National Nature Science Foundation of China (No. 31070858). References [1] M. Demeunynck, C. Bailly, W.D. Wilson (Eds), DNA and RNA Binders: From Small Molecules to Drugs, 2003, Wiley-VCH, Weinheim. [2] M. Gielen, E.R.T. Tiekink (Eds), Metallotherapeutic Drugs and Metal-Based Diadnostic Agents: The Use of Metal in Medicine, 2005, John Wiley & Sons. [3] A. Sigel, H. Sigel (Eds), Metal Ions in Biological Systems, 1996, vol. 33, Marcel Dekker, New York. [4] H.L. Huang, Z.Z. Li, Z.H. Liang, Y.J. Liu, Cell cycle arrest, cytotoxicity, apoptosis, DNA-binding, photocleavage and antioxidant activity of octahedral ruthenium(II) complexes, Eur. J. Inorg. Chem. 36 (2011) 5538–5547. [5] C.S. Devi, D.A. Kumar, S.S. Singh, N. Gabra, N. Deepika, Y.P. Kumar, S. Satyanarayana, Synthesis, interaction with DNA, cytotoxicity, cell cycle arrest and apoptotic inducing properties of ruthenium(II) molecular ‘‘light switch’’ complexes, Eur. J. Med. Chem. 64 (2013) 410421. [6] T.S. Kamatchi, N. Chitrapriya, S.K. Kim, F.R. Fronczek, K. Natarajan, Influence of carboxylic acid functionalities in ruthenium (II) polypyridyl complexes on DNA binding, cytotoxicity and antioxidant activity: Synthesis, structure and in vitro anticancer activity, Eur. J. Med. Chem. 59 (2013) 253–264. [7] V. Rajendiran, M. Palaniandavar, V.S. Periasamy, M.A. Akbarsha, New [Ru(5,6dmp/3,4,7,8-tmp)2(diimine)]2+ complexes: non-covalent DNA and protein binding, anticancer activity and fluorescent probes for nuclear and protein components, J. Inorg. Biochem. 116 (2012) 151–162. [8] N. Deepika, Y.P. Kumar, C.S. Devi, P.V. Reddy, A. Srishailam, S. Satyanarayana, Synthesis, characterization, and DNA binding, photocleavage, cytotoxicity, cellular uptake, apoptosis, and on–off light switching studies of Ru(II) mixedligand complexes containing 7-fluorodipyrido[3,2-a:20 ,30 -c]phenazine, J. Biol. Inorg. Chem. 18 (2013) 751–766. [9] F. Gao, H. Chao, J.Q. Wang, Y.X. Yuan, B. Sun, Y.F. Wei, B. Peng, L.N. Ji, Targeting topoisomerase II with the chiral DNA-intercalating ruthenium(II) polypyridyl complexes, J. Biol. Inorg. Chem. 12 (2007) 1015–1027. [10] S. Shi, H.L. Huang, X. Gao, J.L. Yao, C.Y. Lv, J. Zhao, W.L. Sun, T.M. Yao, L.N. Ji, A comparative study of the interaction of two structurally analogous ruthenium complexes with human telomeric G-quadruplex DNA, J. Inorg. Biochem. 121 (2013) 19–27. [11] J.J. Liu, L.J. Xie, X.N. Sun, L.F. Tan, Binding properties of Ru(II) polypyridyl complexes with poly(U)poly(A)⁄poly(U) triplex: the ancillary ligand effect on third-strand stabilization, J. Biol. Inorg. Chem. 18 (2013) 71–80. [12] N. Deepika, Y.P. Kumar, C.S. Devi, P.V. Reddy, A. Srishailam, S. Satyanarayana, Synthesis, characterization, and DNA binding, photocleavage, cytotoxicity, cellular uptake, apoptosis, and on–off light switching studies of Ru(II) mixedligand complexes containing 7-fluorodipyrido[3,2-a:20,30-c]phenazine, J. Biol. Inorg. Chem. 18 (2013) 751–766. [13] Q. Wu, C.D. Fan, T.F. Chen, C.R. Liu, W.J. Mei, S.D. Chen, B.G. Wang, Y.Y. Chen, W.J. Zheng, Microwave-assisted synthesis of arene ruthenium(II) complexes that induce S-phase arrest in cancer cells by DNA damage-mediated p53 phosphorylation, Eur. J. Med. Chem. 63 (2013) 57–63. [14] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, A molecular light switch for DNA: Ru(bpy)2(dppz)2+, J. Am. Chem. Soc. 112 (1990) 4960– 4962. [15] C. Hiort, P. Lincoln, B. Norden, DNA binding of .DELTA.- and .LAMBDA.[Ru(phen)2DPPZ]2+, J. Am. Chem. Soc. 115 (1993) 3448–3454. [16] I. Haq, P. Lincoln, D. Suh, B. Nordén, B.Z. Chowdry, J.B. Chaires, Interaction of .DELTA.- and .LAMBDA.-[Ru(phen)2DPPZ]2+ with DNA: A calorimetric and equilibrium binding study, J. Am. Chem. Soc. 117 (1995) 4788–4796. [17] H.J. Yu, Y. Chen, L. Yu, L.H. Zhou, Synthesis, visible light photocleavage, antiproliferative and cellular uptake properties of ruthenium complex [Ru(phen)2(mitatp)]2+, Eur. J. Med. Chem. 55 (2012) 146–154.

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