Synthesis, characterization, crystal structure, cytotoxicity, apoptosis and cell cycle arrest of ruthenium(II) complex [Ru(bpy)₂(adpa)](PF₆)₂ (bpy = 2,2′-bipyridine, adpa = 4-(4-aminophenyl)diazenyl-N-(pyridin-2-ylmethylene)aniline)

Abstract

A novel ruthenium(II) complex [Ru(bpy)₂(adpa)](PF₆)₂ (bpy = 2,2′-bipyridine, adpa = 4-(4-aminophenyl)diazenyl-N-(pyridin-2-ylmethylene)aniline) (Ru-adpa) was synthesized, characterized and confirmed by X-ray diffraction studies. The cytotoxic potential of the complex was tested on AGS, BGC823 and SGC7901 cancer cells, the viability of the treated cells was evaluated by MTT assay and colony formation assay. The mode of cell death was assessed by different morphological studies of DNA damage and apoptosis assays (Hoechst staining, acridine orange & ethidium bromide (AO & EB) staining and flow cytometry assay). The Ru-adpa induced cell death in a dose- and time-dependent manner, and the mode of cell death were essentially apoptosis though necrosis was also noticed. Cell cycle analysis by flow cytometry indicated that the Ru-adpa caused cell cycle arrest and accumulated cells in the S phase. The DNA binding properties with Ru-adpa were studied by gel electrophoresis.

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1. Introduction

Development of more efficient anticancer drugs with better selectivity but less toxic side effects is currently an area of intense research in bioinorganic chemistry.^1,2 Since the two ruthenium complexes, NAMI-A and KP1019 entered clinical trials, more and more attention has been paid to the ruthenium complexes as potential anticancer drugs.^2–7 Recently, many Ru(II) complexes were reported to exhibit high anticancer activity in vitro and in vivo.^8–17 For example, in the field of antitumoral and antimetastatic arene ruthenium complexes pioneered by Dyson and Sadler, Ru(η⁶-p-cymene)Cl₂(pta) (pta = 1,3,5-triaza-7-phospha-tricyclo-[3.3.1.1]decane) and [(η⁶-C₆H₅Ph)Ru(N,N-en)Cl]⁺ (en = 1,2-ethylenediamine), exhibited high activity to lung metastases in mice.^18,19 The Ru(II) polypyridyl complexes [Ru(dip)₂(1-Py-βC)]²⁺ (dip is 2,2′-bipyridine or 1,10-phenanthroline, 1-Py-βC is 1-(2-pyridyl)-b-carboline) exhibited very high cytotoxicity toward Hela cells.²⁰ [Ru(bpy)₂(dppz)]²⁺ (bpy is 2,2′-bipyridine, dppz is dipyrido[3,2-a;2′,3′-c]phenazine) exhibited moderate anticancer activity against HT-29 cells (IC₅₀ = 26.9 μM).²¹

On the other hand, Schiff bases have regularly been studied as ligands in coordination chemistry as a result of their good metal binding ability. The bidentate and multidentate Schiff bases with delocalized π-orbitals are suitable ligands for the metals of biological importance, and the study of the chemical properties of such metal–Schiff base complexes also represents a good strategy for the design and synthesis of models of biological systems.^22–25 For example, the Ru(II) complex Ru(II)-2,6-bis(2,6-diisopropylphenyliminomethyl)pyridine exhibits very high cytotoxicity toward cancer cells,²⁶ and Ru(II)-bis(arylimino)pyridine complexes with the co-ligands 1,10-phenanthroline (phen), 2,2′-dipyridyl (bpy) can effectively inhibit the proliferation of the cancer cell lines with a low half-maximal inhibitory concentration (IC₅₀).²⁷ These studies stimulate us to combine Ru metal with Schiff base to study it's cytotoxicity toward cancer cells. So in this work, a new Ru-adpa, [Ru(bpy)₂(adpa)](PF₆)₂ (bpy = 2,2′-bipyridine, adpa = 4-(4-aminophenyl)diazenyl-N-(pyridin-2-ylmethylene)aniline) was synthesized and characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), ¹H NMR spectroscopy and X-ray diffraction. Its cytotoxicity in vitro was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The morphological apoptosis and the percentage of necrotic and apoptotic AGS (human gastric carcinoma) cells induced by Ru-adpa were also studied by fluorescence microscopy and flow cytometry. The cell cycle distribution of AGS cells was investigated by flow cytometry. The DNA binding properties with Ru-adpa was studied by gel electrophoresis.

2. Experimental

2.1 Materials and instrumentation

All reagents and solvents were of commercial origin and were used without further purification unless otherwise noted. Ultrapure Milli-Q water was used in all experiments. Dimethyl sulfoxide (DMSO) and RPMI 1640 were purchased from Sigma. AGS (human gastric carcinoma), BGC823 (human gastric carcinoma), and SGC-7901 (human gastric carcinoma) cell lines were purchased from the American Type Culture Collection. RuCl₃·3H₂O was purchased from the Chang Cheng shiji, 4,4′-diaminoazobenzene²⁸ and Ru(bpy)₂·2H₂O²⁹ were prepared by published methods.

Microanalysis (C, H, and N) was conducted with a Perkin-Elmer 240Q elemental analyzer. Electrospray ionization mass spectra (ESI-MS) were recorded with an LCQ system (Finnigan MAT, USA) using CH₃OH as the mobile phase. 300 MHz ¹H NMR spectroscopic measurements were performed on a Bruker AM-300 NMR spectrometer, using d₆-DMSO as solvent and TMS (SiMe₄) as an internal reference at 25 °C. Infrared spectra were recorded on a Perkin-Elmer Spectrum FT-IR spectrometer using KBr pellets. UV-vis spectra were obtained at room temperature on Shimmadzu 3100 spectrophotometer in DMSO.

2.2 Synthesis

2.2.1 Synthesis of adpa. The ligand adpa was synthesised according to its analogues.³⁰ Yield: 75%. Anal. calcd (%) for C₁₈H₁₅N₅: C, 71.76; H, 4.98; N, 23.25. Found (%): C, 71.80; H, 4.94; N, 23.26. ¹H NMR (300 MHz, CDCl₃, TMS) [ppm]: δ = 6.27 (s, 2H, –NH₂), 7.40 (d, 2H, Ph, J = 9.0 Hz), 7.47 (m, 1H, Py), 7.50 (m, 1H, Py), 7.55 (m, 1H, Ph), 7.84 (m, 2H, Ph), 7.92 (d, 2H, Ph, J = 6.0 Hz), 7.99 (d, 2H, Ph, J = 9.0 Hz), 8.22 (d, 1H, Py), 8.66 (s, 1H, –CH=N), 8.74 (d, 1H, Py). IR (KBr) [cm⁻¹]: 3308 (ν_N–H), 1626, 1586, 1464, 1349 (ν_C=C, ν_C=N, ν_N=N), 856, 740 (δ_C–H).

2.2.2 Synthesis of the [Ru(bpy)₂(adpa)](PF₆)₂ (Ru-adpa). An ethanol solution of AgNO₃ (0.17 g, 1.0 mmol) was added to an ethanol solution of [Ru(bpy)₂Cl₂]·2H₂O (0.26 g, 0.5 mmol). After refluxing for 30 min and filtering to remove the deposited AgCl, the ligand adpa (0.15 g, 0.5 mmol) was added, the solution was refluxed for 12 h under N₂, and then evaporated to 5 mL, on cooling, a red precipitate was obtained by dropwise addition of saturated aqueous KPF₆, then filtered, the filtrate was placed and evaporated, two weeks later, [Ru(bpy)₂(adpa)](PF₆)₂ was obtained as a red crystal. Yield: 70%. Anal. calcd (%) for C₃₈H₃₁N₉P₂F₁₂Ru: C, 45.42; H, 3.09; N, 12.55. Found (%): C, 45.43; H, 3.09; N, 12.59. ¹H NMR (300 MHz, d₆-DMSO, TMS) [ppm]: δ = 6.29 (s, 2H, –NH₂), 6.66 (d, 2H, Ph, J = 7.5 Hz), 7.41 (d, 4H, bpy, J = 9.0 Hz), 7.51 (m, 1H, Py), 7.62 (m, 1H, Py), 7.76 (m, 4H, bpy), 7.84 (m, 2H, Ph), 7.90 (d, 2H, Ph, J = 6.0 Hz), 7.99 (d, 2H, Ph, J = 9.0 Hz), 8.21 (d, 4H, bpy), 8.22 (d, 1H, Py), 8.62 (s, 1H, –CH=N), 8.75 (d, 1H, Py), 8.85 (d, 4H, bpy). IR (KBr) [cm⁻¹]: 3388 (ν_N–H), 1591, 1515, 1381, 1129 (ν_C=C, ν_C=N, ν_N=N), 835, 759 (δ_C–H). ESI-MS: m/z 860.0 for [Ru(bpy)₂(adpa)·PF₆]⁺, 357.6 for [Ru(bpy)₂(adpa)]²⁺. UV-vis (DMSO, λ_max/ε) [nm/dm³ mol⁻¹ cm⁻¹]: 292 (6.90 × 10⁴), 460 (2.02 × 10⁴).

2.3 Methods

2.3.1 Single crystal X-ray structure determinations. The X-ray diffraction experiments were carried out with a Bruker ApexIICCD area detector diffractometer, using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å) at 100(2) K. Absorption corrections were applied semi-empirically using the APEX2 program.³¹ The structures were solved by direct methods and refined by the full-matrix least-squares against F² in an anisotropic (for non-hydrogen atoms) approximation. All calculations were performed using the SHELXTL software.³²

2.3.2 Cytotoxicity assay in vitro. Standard MTT assay procedures were used.³³ Cells were placed in 96-well microassay culture plates (8 × 10³ cells per well) and grown overnight at 37 °C in a 5% CO₂ incubator. The complex tested was then added to the wells to achieve final concentrations ranging from 10⁻⁷ to 10⁻⁴ M. Control wells were prepared by addition of culture medium (200 μL). The plates were incubated at 37 °C in a 5% CO₂ incubator for 48 h. On completion of the incubation, stock MTT dye solution (20 μL, 5 mg mL⁻¹) was added to each well. After 4 h, 150 mL dimethylsulfoxide (DMSO) was added to solubilize the MTT formazan. The optical density of each well was then measured with a microplate spectrophotometer at a wavelength of 490 nm. The IC₅₀ values were determined by plotting the percentage viability versus the concentration and reading off the concentration at which 50% of the cells remained viable relative to the control. Each experiment was repeated at least three times to obtain the mean values. Three different tumor cell lines were the subjects of this study: AGS, BGC823 and SGC-7901. These cells were purchased from the American Type Culture Collection (Rockville, MD, USA).

2.3.3 Colony formation assay. The cells were plated in six-well plates at 2000 cells per well. 24 h later, the complex was added to the plates. After treatment, fresh medium was applied to the plates. The cells were allowed to grow for ten additional days before staining with Crystal Violet (Sigma). All experiments were repeated at least three times, and similar results were obtained in each trial.

2.3.4 Apoptosis assay by acridine orange (AO) and ethidium bromide (EB) staining³⁴. AGS cells were seeded in 6-well plates and allowed to reach 70% confluence. The cells were then treated with different concentrations of the complex for 24 or 48 h. The cells were trypsinized and pelleted, and then suspended in PBS. A drop of cell suspension was placed on a glass slide and stained with AO & EB and a cover slip was laid over to reduce light diffraction. At random 300 cells were observed in a fluorescent microscope (Nikon, Yokohama, Japan) fitted with a 377–355 nm filter, and observed at ×400 magnification.

2.3.5 Apoptosis assay by Hoechst 33258 staining. AGS 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% 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 the complex for 24 or 48 h. The medium was removed, and the cells were washed with ice-cold phosphate-buffered saline (PBS), and fixed with formalin (4%, w/v). Cell nuclei were counterstained with Hoechst 33258 (10 mg mL⁻¹ in PBS) for 10 min. Then the cells were observed and imaged by a fluorescence microscope (Nikon, Yokohama, Japan) with excitation at 350 nm and emission at 460 nm.

2.3.6 Apoptosis assay by flow cytometry. After chemical treatment, 2 × 10⁵ cells were harvested, washed with PBS, then fixed with 70% ethanol, and finally, maintained at 4 °C for at least 12 h. Then the pellets were stained with the fluorescent probe solution containing 50 μg mL⁻¹ propidium iodide and 1 mg mL⁻¹ annexin in PBS on ice in the dark for 15 min. The fluorescence was then measured at 530 and 575 nm using 488 nm excitation by a FACSCalibur flow cytometry system. A minimum of 10 000 cells were analyzed per sample.

2.3.7 Cell cycle arrest investigated by flow cytometry. AGS cells were seeded into 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 FBS (10%) and were incubated at 37 °C and 5% CO₂. The medium was removed and replaced with medium (final DMSO concentration, 0.05% v/v) containing complex. After incubation for 24 h, the cell layer was trypsinized and washed with cold PBS and fixed with 70% ethanol. 20 μL of RNase (0.2 mg mL⁻¹) and 20 μL of propidium iodide (0.02 mg mL⁻¹) were added to the cell suspensions and they were incubated at 37 °C for 30 min. Then the samples were analyzed with a FACSCalibur flow cytometry system. The number of cells analyzed for each sample was 10 000.

2.3.8 Gel electrophoresis. The lpBR322 (linear pBR322) was obtained by the cleavage of pBR322 with Bam HI (New England Biolabs, USA). The mixing ratios of complex/EB/H33258 and pBR322 base pairs were 0, 1 : 1, 2 : 1 and 4 : 1. Electrophoresis was conducted at 120 V for 30–40 min in a Tris/borate/EDTA buffer solution using 1% agarose gels to analyze DNA. The gel was stained with EB and photographed using UV illumination.

3. Results and discussion

3.1 Synthesis and characterization

Schiff base ligand (adpa) was synthesized by 4,4′-diaminoazobenzene and 2-pyridine formaldehyde in ethanol. Ru-adpa was prepared by direct reaction of adpa with appropriate precursor complex relatively high yield (Scheme 1). The desired Ru-adpa was isolated as the hexafluorophosphate and purified by recrystallization. ESI-MS spectra of the Ru-adpa exhibited two strong peaks at 357.6 and 860.0, which were assigned to [Ru(bpy)₂(adpa)]²⁺ and [Ru(bpy)₂(adpa)·PF₆]⁺, respectively (the IR, ¹H NMR and ESI-MS spectra of the Ru-adpa in ESI Fig. S1†). The observed peaks revealed that the synthetic complex was Ru(II) center and stable in the solution.

Scheme 1 Synthetic route of the adpa and Ru-adpa.

The molecular structure of Ru-adpa was further confirmed by single crystal X-ray diffraction analysis. The single crystal of Ru-adpa was obtained from ethanol solvent by evaporating. The crystal data and refinement details for Ru-adpa are in Table 1. The ORTEP drawings of Ru-adpa with atomic numbering scheme are depicted in Fig. 1. It can be seen that the coordination sphere around the ruthenium centre constitutes a distorted octahedron with coordination of the bidentate (N–N) ligand adpa and two 2,2′-bipyridine molecules. The Ru–N bond distance for the two bpy ligands are between 2.056 and 2.070 Å, which are shorter than those with the adpa moiety (Ru1–N1 2.072(6) and Ru1–N2 2.072(5) Å, respectively) because of the greater π back-bonding into the bpy moiety to stabilize the additional electron density on the metal center donated by bpy.

Table 1 Crystal data for Ru-adpa

	Ru-adpa
Molecular formula	C38H31F12N9P2Ru
Mr	1004.860
Crystal system	Triclinic
Space group	P1̄
a (Å)	10.430(2)
b (Å)	11.600(2)
c (Å)	19.635(4)
α (°)	89.474(3)
β (°)	84.908(4)
γ (°)	83.509(3)
V (Å^3)	2351.1(8)
Z	2
T (K)	298(2)
Dc (g cm^−3)	1.54932
M (mm^−1)	0.496
No. refs measured	8166
No. unique refs	764
R1	0.0729
wR2	0.1773
Goofs	1.034

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Fig. 1 ORTEP view of the molecule of Ru-adpa with atom-labeling scheme and thermal ellipsoids drawn at 50% probability level. Selected bond distances (Å) and angles (°): Ru1–N1 2.072(6), Ru1–N2 2.072(5), Ru1–N6 2.063(6), Ru1–N7 2.056(5), Ru1–N8 2.064(5), Ru1–N9 2.070(5), N7–Ru1–N6 78.6(2), N7–Ru1–N8 95.1(2), N6–Ru1–N8, 87.5(2), N7–Ru1–N9 173.3(2), N6–Ru1–N9, 97.5(2), N8–Ru1–N9, 79.2(2), N7–Ru1–N1 90.8(2), N6–Ru1–N1 95.8(2), N8–Ru1–N1 173.7(2), N9–Ru1–N1 95.0(2), N7–Ru1–N2 99.5(2), N6–Ru1–N2 173.9(2), N8–Ru1–N2 98.5(2), N9–Ru1–N2 85.0(2), N1–Ru1–N2 78.4(2).

3.2 Cytotoxicity assay in vitro

The cytotoxicity of the Ru-adpa was studied in AGS, BGC823 and SGC7901 cell lines by means of the MTT cell survival assay. AGS, BGC823 and SGC7901 cells were treated with different concentrations of Ru-adpa for 24, 48, 72, 96 and 120 h. For 48 h (Fig. 2a), the complex showed the highest activity in the AGS cell lines with IC₅₀ values of 27.92 μM, and was moderate with IC₅₀ values of 91.84 for BGC823 and 87.78 for SGC7901, respectively. For 72 h (Fig. 2b–d), the IC₅₀ of the complex was 11.09 μM for AGS, 23.32 μM for BGC823 and 38.75 μM for SGC-7901. The IC₅₀ of Ru-adpa to AGS, BGC823 and SGC7901 in different time was displayed in Table 2. Obviously, the cell viability was found to be concentration-dependent and duration-dependent, which indicated that the Ru-adpa entered the cells slowly and killed the cells gradually. The colony formation assay was performed to examine the effect of Ru-adpa on AGS growth. As shown in Fig. 3, AGS cells generated fewer colonies with increasing of Ru-adpa concentration, suggesting that colony was concentration-dependent. Treated with 5 μM of complex, the colony number was about three times as much as 10 μM, treated with 20 μM of complex, the colony number was nearly zero.³⁵

Fig. 2 (a) Cell viability of Ru-adpa toward proliferation of AGS, BGC823 and SGC7901 in 48 h, (b–d) time gradient of Ru-adpa to AGS, BGC823 and SGC-7901.

Table 2 The IC₅₀ of Ru-adpa to AGS, BGC823 and SGC-7901 in different time

Ru-adpa	IC50 (μM)
	AGS	BGC823	SGC-7901
24	>100	>100	>100
48	27.92	91.84	87.78
72	9.82	25.01	37.37
96	9.53	19.43	36.87
120	8.33	14.74	30.83

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Fig. 3 Antineoplastic activity of Ru-adpa by colony formation assay.

3.3 Apoptosis studies by acridine orange and ethidium bromide (AO & EB) staining

AO & EB staining followed by fluorescent microscopy revealed apoptosis from the perspective of fluorescence emission. The cells were classified into four types according to the fluorescence emission and the morphological features of chromatin condensation in the nuclei: (1) the viable cells had uniformly green fluorescing nuclei with a highly organized structure. (2) Early apoptotic cells (which possessed intact membrane but DNA fragmentation was initiated) had green fluorescing nuclei, but peri-nuclear chromatin condensation was visible as bright green patches or fragments. (3) Late apoptotic cells had orange to red fluorescing nuclei with condensed or fragmented chromatin. (4) Necrotic cells had uniformly orange to red fluorescing nuclei with no indication of chromatin fragmentation, and the cells were swollen.³⁶ Representative images of the cells treated with Ru-adpa are shown in Fig. 4. The AGS cells treated with Ru-adpa exhibited higher incidence of apoptosis than necrosis. In the 48 h treatment group the percentage of apoptotic cells was higher than 24 h, which means that the incidence of apoptosis increased in duration-dependent manner.

Fig. 4 Photographs showing the feature of AO/EB stained AGS cancer cells.

3.4 Apoptosis studies by Hoechst 33258 staining and flow cytometry

Hoechst 33258, which stains the cell nucleus, is a membrane permeable dye with blue fluorescence. Live cells with uniformly light blue nuclei were observed under fluorescence microscope after treatment with Hoechst 33258, while apoptotic cells had bright blue nuclei on account of karyopyknosis and chromatin condensation, whereas, the nuclei of dead cells could not be stained.³⁷ AGS cells dealt with Ru-adpa at 10, 20 and 40 μM from 24 to 48 h were stained with Hoechst 33258. AGS cells without dealing with the Ru-adpa was used as control. The results were given in Fig. 5. In the control, cells show homogeneous nuclear staining. After treatment of AGS cells with Ru-adpa, the number of apoptotic cells increases in a dose-dependent and time-dependent manner and they exhibit typical apoptotic features, such as staining brightly, condensed chromatin, and fragmented nuclei. These results show the Ru-adpa can effectively induce the apoptosis against AGS cells.

Fig. 5 Hoechst 33258 staining of AGS cells.

The morphological apoptosis studies showed Ru-adpa can induce apoptosis of AGS cells. To determine the percentages of apoptotic and necrotic cells, AGS cells without dealing with the Ru-adpa was used as control, apoptosis was investigated by flow cytometry, as shown in Fig. 6. In the control, the proportions of living cells and apoptotic cells were 93.1 and 2.2%, respectively. After AGS cells have been exposed to Ru-adpa (5, 10, 20 and 40 μM) for 12 h, the proportions of apoptotic cells were 7.8, 14.4, 14.5 and 36.1%, respectively. Comparing with the control, the proportion of living cells decreased and apoptotic cells increased. For treatment of AGS cells with Ru-adpa (5, 10, 20 and 40 μM) for 24 h, the proportions of apoptotic cells were 12.6, 18.8, 22.4 and 45.3%, respectively. For treatment of AGS cells with Ru-adpa (5, 10, 20 and 40 μM) for 48 h, the proportions of apoptotic cells were 16.9, 38.8, 41.7 and 9.9%, respectively. These data demonstrate that the apoptotic effect on AGS cells for Ru-adpa is concentration-dependent, with increasing concentrations of Ru-adpa, the number of apoptotic cells increases. In addition, the apoptotic effect on AGS cells for Ru-adpa is duration-dependent, as time go on, the number of apoptotic cells increases. However, after AGS cells have been exposed to 40 μM Ru-adpa for 48 h, the proportions of necrotic cells increased.

Fig. 6 The percentage of living (B3), necrotic (B2), and apoptotic (B4) AGS cells.

3.5 Cell cycle arrest

The distribution of AGS cells in various compartments during the cell cycle was analyzed by flow cytometry in cells stained with propidium iodide. As shown in Fig. 7, treatment of AGS cells with 5, 10, 20 and 40 μM Ru-adpa for 12 h cause significant enhancement of 3.1, 4.9, 10.8 and 19.7% in S phase compared with the control. On exposure of AGS cells to 5, 10, 20 and 40 μM Ru-adpa for 24 h, cause pronounced increases of 7.1, 11.2, 23.3 and 27.9% in S phase compared with the control. These data show that Ru-adpa induce S-phase arrest in AGS cells (see ESI Fig. S2†).

Fig. 7 Cell cycle distribution on exposure of AGS cells to 5, 10, 20 and 40 μM Ru-adpa.

3.6 Gel electrophoresis

Gel electrophoresis is a general method to evaluate the binding mode of small molecules and DNA base pairs.³⁸ In order to discuss how to interact the Ru-adpa with DNA, a gel electrophoresis study was performed on a 2000 bp length DNA segment from lpBR322 (linear pBR322). We examined the effect of different concentration ratios of the Ru-adpa, as the hexafluorophosphate, on DNA by monitoring the changes in the electrophoretic mobility of DNA in an agarose gel. The electrophoretogram of the DNA sample treated with different concentration ratios of the Ru-adpa is illustrated in Fig. 8. Lane 4 represents the gel moving pattern of pure DNA, as a control. Lanes 1–3 represent the DNA treated with the Ru-adpa in various concentration ratios. This electrophoretogram shows that the DNA samples in lanes 1–3 have moved slowly compared to the free DNA (lane 4) and that the migration of the DNA bands has been gradually retarded with the increase of the Ru-adpa. This observation clearly confirms that there is strong interaction between the Ru-adpa and DNA. Furthermore, the decrease in electrophoretic mobility of the bands demonstrates that the interaction of the Ru-adpa with DNA may occur via an intercalative mechanism, which could be facilitated further as a result of an electrostatic interaction between the Ru-adpa and the negative charges of the DNA. This observation could be explained on the basis that hydrophobic interaction of an intercalating ligand with DNA base pairs causes the double helix to become extended and locally uncoiled. Local unwinding of the double helix by an intercalating species results in an overall increase in the size of the DNA molecule.^39,40 This retardation behavior could also be attributed to the decrease of the overall charge of DNA due to binding with the Ru-adpa. Moreover, it was thought that intercalative interactions result in the band lag of the small molecule–DNA complex, while other binding modes do not affect the speed of band migration. We also ran the gel experiments for the effects of EB and H33258, EB is a classical intercalator of DNA and H33258 is a representative DNA-groove binder.⁴¹ Obviously, EB made the band lag but H33258 did not affect the rate of the band. The results indicated that Ru-adpa, like EB, had obvious intercalative interactions with DNA.

Fig. 8 Gel electrophoresis of DNA in the presence of an increasing amount of Ru-adpa (lane 1–4) and EB and H33258 acted as controls for classic intercalative interaction and groove interaction (lane 5–6). M is the marker.

4. Conclusions

A novel ruthenium(II) complex [Ru(bpy)₂(adpa)](PF₆)₂ was synthesized and characterized. In vitro, the Ru-adpa exhibits higher cytotoxicity toward AGS than BGC823 and SGC7901 cell lines under identical conditions. Hoechst 33258 staining and AO & EB staining demonstrated that Ru-adpa can effectively induce apoptosis of AGS cells. Apoptosis assay by flow cytometry showed the number of apoptotic cells increased with increasing concentration and time. The cell cycle arrest studies demonstrated that the antiproliferative effect induced by Ru-adpa on AGS cells occurs in S phase. In addition, the interactions between Ru-adpa and DNA were demonstrated to be intercalative binding.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 21101121), the Natural Science Fund (no. 2010CDB01301) of Hubei Province. the National Natural Science Foundation of China (no. 31170328 and 30900122), the Fundamental Research Funds for the Central Universities (no. 2014301020203).

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1029790. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra12715c

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