Cytotoxicity in vitro, cell migration and apoptotic mechanism studies induced by ruthenium(II) complexes

Abstract

Four new ruthenium(II) polypyridyl complexes [Ru(dmb)₂(DHBT)](ClO₄)₂ (1) (DHBT = 12,14-dihydroxyl-4,5,9,10,11,13-hexaazabenzo[b]triphenylene, dmb = 4,4′-dimethyl-2,2′-bipyridine), [Ru(bpy)₂(DHBT)](ClO₄)₂ (2) (bpy = 2,2′-bipyridine), [Ru(phen)₂(DHBT)](ClO₄)₂ (3) (phen = 1,10-phenanthroline) and [Ru(dmp)₂(DHBT)](ClO₄)₂ (4) (dmp = 2,9-dimethyl-1,10-phenanthroline) were synthesized and characterized. The cytotoxicity in vitro of these complexes was evaluated against human HepG-2, HeLa, A549, MG-63 and BEL-7402 cancer cell lines. The IC₅₀ values of the complexes toward selected cell lines range from 14.9 ± 1.1 to 30.1 ± 2.7 μM. The cytotoxicity and the levels of reactive oxygen species were found to increase with increasing concentrations of the complexes. The complexes are sensitive to MG-63 cells and can inhibit the MG-63 cell migration. Morphological and comet assay studies show that the complexes can effectively induce apoptosis in MG-63 cells. Complex 2 inhibits the cell growth at the G0/G1 phase, whereas the other complexes exhibit the antiproliferative mechanism at the S phase in the MG-63 cell line. The complexes can downregulate the expression of Bcl-2 protein and upregulate the levels of Bad protein in MG-63 cells. The complexes induce MG-63 cells apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

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1. Introduction

Since the discovery of the antitumor and anti-metastatic properties of cisplatin, intensive studies have been performed on cytotoxic compounds with more acceptable toxicity profiles.¹ As a widely used metal-based anticancer drug, cisplatin possesses inherent drawbacks such as serious side effects, general toxicity, and acquired drug resistance.^2–4 These drawbacks have limited the clinical application of cisplatin and its analogs. These important problems in platinum based anticancer drugs have stimulated more research efforts to investigate drugs based on other transition metals.⁵ Ruthenium complexes are regarded as the most promising alternatives to cisplatin as anticancer drugs. So far, two ruthenium complexes, NAMI-A ([ImH][trans-RuCl₄ (DMSO)(Im)], where Im = imidazole and DMSO = dimethylsulfoxide) and KP1019 ([IndH][trans-RuCl₄(Ind)₂], where Ind = indazole) have entered clinical trials.⁶ NAMI-A is effective against lung metastases and KP1019 is active against colon carcinomas.^7,8 In recent years, some new findings of ruthenium complexes as potential cancer agents have been observed, and a variety of studies have demonstrated these complexes can exhibit significant cytotoxicity.^9–16 Complex [Ru(tpy)(5CNU)₃]²⁺ (tpy = 2,2′:6′,2′′-terpyridine and 5CNU = 5-cyanouracil) binds DNA and simultaneously releases biologically active 5CNU, this complex has the potential to be a dual-action therapeutic agent.¹⁷ [Ru(phpy)(bpy)(dppn)]⁺ (bpy = 2,2′-bipyridine, dppn = benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine) is 6 times more active than the platinum drug against HeLa cells, and it is able to disrupt the mitochondria membrane potential.¹⁸ [Ru(bpy)₂(2,9-dimethyl-dpq)]²⁺ has no cytotoxicity toward A549 cells in the dark with an IC₅₀ value of 250 (±5) μM, but on irradiation with >450 nm light for 3 min, the complex shows a high cytotoxic effect with an IC₅₀ value of 1.2 (±0.1) μM.¹⁹ A series of ruthenium(II) complexes containing a β-carboline alkaloid as ligand trigger release of reactive oxygen species (ROS) and show ROS-dependent apoptosis.²⁰ Schatzschneider reported that [Ru(bpy)₂(dppn)]²⁺ exhibits cytotoxic activity toward HT-29 cells comparable to cisplatin under identical conditions, whereas complex [Ru(bpy)₂(dppz)]²⁺ shows low inhibitory effect on the cell growth in MCF-7 cells with an IC₅₀ value of 90.2 ± 19.6 μM.²¹ In our previous works, we found that the complexes with the structures similar to dppz show high cytotoxicity.^15,22,23 To further investigate the anticancer activity of this kind of ruthenium complexes, in this report, a new ligand DHBT (DHBT = 12,14-dihydroxyl-4,5,9,10,11,13-hexaazabenzo[b]triphenylene) and its four ruthenium(II) polypyridyl complexes: [Ru(N–N)₂(DHBT)](ClO₄)₂ 1–4 (N–N = dmb: 4,4′-dimethyl-2,2′-bipyridine; bpy: 2,2′-bipyridine; phen: 1,10-phenanthroline; and dmp: 2,9-dimethyl-1,10-phenanthroline, Scheme 1) were synthesized and characterized by elemental analysis, ES-MS and ¹H NMR. The cytotoxicity in vitro of the complexes against HepG-2, A549, HeLa, MG-63 and BEL-7402 cells was evaluated by MTT (MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay. The apoptosis of MG-63 cells induced by the complexes was studied with AO/EB staining method and comet assay. The cell cycle arrest of MG-63 cells was analyzed by flow cytometry. The reactive oxygen species and mitochondrial membrane potential induced by the complexes were investigated. The expression of the caspase-3, caspase-7 and Bcl-2 family proteins was assayed by western blot.

Scheme 1 Synthetic route of ligand and complexes.

2. Results and discussion

2.1. Cytotoxicity in vitro

The cytotoxic activity of 1–4 against HepG-2, HeLa, MG-63, A549 and BEL-7402 cell lines was investigated in comparison with the widely used drug cisplatin using the MTT assay. The above cells were exposed to the different concentrations of the ligand DHBT and Ru(II) complexes for 48 h, the IC₅₀ values of the complexes are listed in Table 1. DHBT is unexpectedly found to display no cytotoxic activity towards the selected cell lines. The complexes show high cytotoxic activity. Complexes 1 and 4 are sensitive to MG-63 cells and complex 3 shows the least cell killing ability against HepG-2 cells. Complex 2 shows moderate cytotoxic activity on the selected cell lines. The cytotoxicity of the complexes are higher than those of [Ru(phen)₂(mitatp)]²⁺ (IC₅₀ = 27.0 μM)²⁴ and [Ru(Hdpa)₂(4HEPIP)]²⁺ (IC₅₀ = 45 ± 0.5 μM)²⁵ against HeLa cells, but their cytotoxic activity is lower than cisplatin under the identical conditions. Comparing the IC₅₀ values of DHBT and its complexes, the cytotoxic activity is enhanced when the ligand bonded to metal to form Ru(II) complexes. Because complexes 1–4 exhibit relative high cytotoxic activity against MG-63 cells, this cell line was used for further investigation on apoptosis, comet assay, cellular uptake, reactive oxygen species, mitochondrial membrane potential, cell cycle arrest and western blot analysis.

Table 1 The IC₅₀ values of complexes 1, 2, 3 and 4 against HepG-2, A549, HeLa, MG-63 and BEL-7402 cell lines

Complexes	IC50 (μM)
	HepG-2	A549	HeLa	MG-63	BEL-7402
DHBT	>200	>200	>200	>200	>200
1	20.6 ± 1.7	18.8 ± 1.5	20.8 ± 2.2	14.9 ± 1.1	17.9 ± 1.3
2	24.2 ± 2.4	25.6 ± 2.7	19.4 ± 1.6	19.1 ± 1.5	20.2 ± 1.2
3	30.1 ± 2.7	26.1 ± 2.3	18.2 ± 1.4	21.1 ± 1.6	18.6 ± 1.4
4	28.4 ± 2.5	20.7 ± 2.1	17.6 ± 1.7	15.9 ± 1.2	21.6 ± 2.4
Cisplatin	11.5 ± 1.2	7.5 ± 1.3	7.3 ± 1.4	6.8 ± 0.8	11.5 ± 1.3

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2.2. Induction of apoptosis in MG-63 cells by the complexes

The design of chemotherapeutic drugs in order to understand the complexities of apoptosis evolved by cancer cells and the development of strategies to selectively induce apoptosis in cancer cells have turned into a unique target in cancer drug development.²⁶ The morphological changes induced by complexes 1–4 were evaluated using AO/EB-stained MG-63 cancer cells. As shown in Fig. 1a, in the control, living cells have uniformly green fluorescing nuclei with highly organized structure. After the treatment of MG-63 cells with 12.5 μM of 1–4, apoptotic cells with apoptotic features such as nuclear shrinkage and chromatin condensation were found. Necrotic cells, having uniformly orange to red fluorescing nuclei were also observed (Fig. 1b–e). All these morphological changes indicate that the complexes can effectively induce apoptosis in MG-63 cells.

Fig. 1 Apoptosis in MG-63 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.

2.3. Comet assay

DNA fragmentation is a hallmark of apoptosis, mitotic catastrophe or both.²⁷ Apoptosis eventually entails DNA damage providing another experimental endpoint to validate the existence of apoptotic processes in the cells. The single cell gel electrophoresis assay known as comet assay was performed to asses DNA integrity. As shown in Fig. 2a, in the control, MG-63 cells fail to show a comet like appearance. Treatment of MG-63 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 (Fig. 2b–e). These results clearly indicate that the four complexes indeed induce DNA fragmentation, which is further evidence of apoptosis.

Fig. 2 Comet assay of EB-stained control (a) and 25 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) treated MG-63 cancer cells at 24 h incubation.

2.4. Cellular uptake and localization studies

The cellular uptake characteristics of a small molecule are critical to its application as a therapeutic and diagnostic agent.²⁸ The cellular uptake and localization was performed by the treatment of MG-63 cells with complexes 1–4 for 24 h, and the cells were stained with DAPI and observed under fluorescent microscope. As shown in Fig. 3, the DAPI-stained cell nuclei emit blue fluorescence with excitement at 340 nm. Complexes 1–4 emit red luminescence with an excitation wavelength at 455 nm. The results of cell uptake and localization were observed by merging the blue fluorescent picture and red fluorescent points. Seen from the images, we found a plenty of red fluorescent spots in the cells. This observation indicates that the complexes can be successfully uptaken by MG-63 cells.

Fig. 3 Images of MG-63 cells exposure to 12.5 μM of complexes 1–4 and stained with DAPI at 37 °C for 24 h.

2.5. The wound healing assay

Metastatic cancers have several important characteristics, including the migratory and invasive activities of tumor cells.²⁹ To determine the effects of complexes 1–4 on cancer cell migration, MG-63 cells were exposed to the 3.13 μM of the complexes at 0, 6, 12 and 24 h. As shown in Fig. 4, at 6 and 12 h, the complexes show weak inhibitory effects on the MG-63 cells migration. When the treatment of MG-63 cell with the complexes at 24 h, the complexes can strongly suppress MG-63 cell migration to the wound area and the inhibition of cell migration was more in the cells treated with complexes 2 and 3. The results indicate that the complexes can effectively inhibit MG-63 cell migration thereby metastasis.

Fig. 4 Temporal progress of the wound healing in MG-63 cells (a) treated with 3.13 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) at different time intervals.

2.6. Reactive oxygen species assay

To determine whether reactive oxygen species (ROS) are involved in apoptotic mechanism induced by the complexes 1–4, intracellular generation of ROS was evaluated using dye H₂DCF-DA as fluorescence probe. This dye is stable and diffuses inside the cells. Then it is hydrolyzed by intracellular esterase to yield 2′,7′-dichlorofluorescein (DCFH). Reactive oxygen species produced within the cells oxidize DCFH to the highly fluorescent compound, namely, 2′,7′-dichlorofluorescein (DCF). The fluorescent intensity of DCF is proportional to the amount of peroxide (ROS) produced by the cells.³⁰ In the control (Fig. 5a), no fluorescent point is observed. After MG-63 cells were exposed to Rosup (Fig. 5b, positive control) and 12.5 μM of complexes 1–4 (Fig. 5c–f), bright fluorescent points are found. The fluorescent intensity of DCF was determined with microplate analyzer. As shown in Fig. 6, MG-63 cells were exposed to 12.5 μM of complexes 1–4, the fluorescent intensities are 210.3, 206.0, 209.7 and 210.7, respectively. Compared the complexes 1–4 with the control, the fluorescent intensities of DCF grow 2.44, 2.39, 2.43 and 2.52 times than the original. After the treatment of MG-63 cells with 25.0 μM of complexes 1–4, the fluorescent intensities of DCF increase by 2.94, 3.05, 3.10 and 3.56 times than the original. Obviously, ROS generation induced by complexes 1–4 follows the order of 4 > 3 > 2 > 1 at 25 μM. Moreover, the level of ROS shows a concentration-dependent manner. These observations show that complexes 1–4 can enhance the levels of ROS.

Fig. 5 Intracellular ROS was detected in MG-63 cells (a) exposure to Rosup (b) and 12.5 μM of complexes 1 (c), 2 (d), 3 (e) and 4 (f) for 24 h. Rosup was used as positive control.

Fig. 6 Effects on ROS generation induced by different concentrations of complexes 1 (), 2 (), 3 () and 4 () in MG-63 cells. Data were calculated from three independent experiments.

2.7. Mitochondrial membrane potential detection

Mitochondria act as a point of integration for apoptotic signals originating from both the extrinsic and intrinsic apoptotic pathways.^31,32 The changes of mitochondrial membrane potential in complexes 1–4-treated MG-63 cells were investigated with JC-1 as fluorescent probe. As shown in Fig. 7a, in the control, JC-1 forms aggregates and emits red light corresponding to high mitochondrial membrane potential. Treatment of MG-63 cells with cccp (Fig. 7b, positive control) and 12.5 μM of complexes 1–4 (Fig. 7c–f), JC-1 exists with monomers and emits green with little red light corresponding to low mitochondrial membrane potential. The changes from red to green fluorescence suggest the mitochondrial membrane potential decreases. The ratio of the red/green fluorescent intensity was determined with microplate analyzer. As shown in Fig. 8, in the control, the ratio of red/green is 2.72. MG-63 cells were exposed to 12.5 μM of complexes 1–4, the ratios of the red/green are 1.56, 1.56, 1.72 and 1.36, respectively. After the treatment of MG-63 cells with 25 μM of complexes 1–4, the ratios of red/green are 0.70 for 1, 0.85 for 2, 0.75 for 3 and 0.69 for 4, respectively. Comparing these ratios, complexes 1 and 4 induce larger changes in mitochondrial membrane potential than complexes 2 and 3 under the same conditions. The decrease of the ratios of red/green indicates that the intensity of red light decreases and the intensity of green fluorescence increases. Moreover, Fig. 8 also shows that the changes in mitochondrial membrane potential are a concentration-dependent manner. These results indicate that the complexes can induce the decrease of mitochondrial membrane potential.

Fig. 7 Assay of MG-63 cells mitochondrial membrane potential with JC-1 as fluorescence probe staining method. MG-63 cells (a) exposed to cccp (b) and 12.5 μM of complexes 1 (c), 2 (d), 3 (e) and 4 (f) for 24 h. cccp was used as positive control.

Fig. 8 Assay of MG-63 cells mitochondrial membrane potential with JC-1 as fluorescence probe staining method. MG-63 cells exposed to 12.5 and 25 μM of complexes 1 (), 2 (), 3 () and 4 () for 24 h. *p < 0.05 represents significant differences compared with control.

2.8. Cell cycle distribution studies

To investigate further the inhibitory effect of complexes 1–4 on cell growth, the cell cycle distribution of MG-63 cells was examined by flow cytometry. As shown in Fig. 9, the percentage of 9.19% in MG-63 cells at the G0/G1 phase was increased by treatment with complex 2 at 25 μM. However, treatment of the cells with 25 μM of complexes 1, 3 and 4, the increase of 2.42% for 1, 6.37% for 3 and 4.53% for 4 in cells at the S phase was observed. These data suggest that the anti-proliferative mechanism induced by 2 was a G0/G1 phase, by 1, 3 and 4 was S phase arrest on MG-63 cells.

Fig. 9 Cell cycle distribution of MG-63 cells exposure to 25 μM of complexes 1, 2, 3 and 4 for 24 h.

2.9. The expression of caspase, antiapoptotic proteins and proapoptotic proteins

Caspases are known to mediate the apoptotic pathways.^33,34 To assess the apoptotic pathway activated by complexes 1–4, we performed western blot analysis to test the levels of caspase-3, caspase-7, antiapoptotic protein Bcl-2 and proapoptotic protein Bad after MG-63 cells incubated with 12.5 μM of complexes 1–4 for 24 h. As shown in Fig. 10, incubation of MG-63 cells with the complexes caused a decrease in the levels of caspase-3 and -7. Treatment of Bcl-2 with complexes 1–4, downregulation in the expression levels of Bcl-2 was observed. Incubation of proapoptotic protein Bad with the complexes, up-regulation in the expression levels of Bad was found. These results indicate that the complexes can regulate the expression of Bcl-2 and Bad proteins.

Fig. 10 Western blot analysis of caspase-3, -7, Bcl-2 and Bad in MG-63 cells treated with 12.5 μM of complexes 1–4. β-Actin was used as internal control.

3. Conclusions

Four new Ru(II) complexes were synthesized and characterized. The cytotoxicity in vitro assay suggests that complexes 1–4 can effectively inhibit tumor cells proliferation. Ligand DHBT shows no cytotoxicity towards the selected cell lines. However, when DBHT bonded metal ruthenium to form complexes, the antiproliferative activities of complexes 1–4 are much higher than that of the ligand DBHT. The morphological changes and comet assay show that complexes 1–4 can effectively induce apoptosis of MG-63 cells. The wound healing assay demonstrates that the complexes can inhibit MG-63 cell migration. Additionally, complexes 1–4 induce a decrease of the mitochondrial membrane potential and an increase in ROS levels. The cell cycle arrest indicates that complex 2 inhibit the proliferation in the G0/G1 phase and complexes 1, 3 and 4 induce an arrest in the S-phase on MG-63 cells. The complexes can downregulate the levels of Bcl-2 and upregulate the expression of Bad protein. In summary, the complexes induce apoptosis of MG-63 cells through the mitochondrial signal transduction pathway and regulate the expression levels of Bcl-2 and Bad.

4. Experimental sections

4.1. Materials and methods

All reagents and solvents were purchased commercially and used without further purification unless otherwise noted. Ultrapure MilliQ water was used in all experiments. DMSO, 2,2′-bipyridine, 4,4′-dimethyl-2,2′-bipyridine, 2,9-dimethyl-1,10-phenanthroline, and RPMI 1640 were purchased from Sigma. 1,10-Phenanthroline was obtained from the Guangzhou Chemical Reagent Factory. Cell lines of HepG-2 (Human hepatocellular carcinoma), A549 (Human lung carcinoma), BEL-7402 (Hepatocellular), MG-63 (Human osteosarcoma) and HeLa (Human cervical cancer) were purchased from the American Type Culture Collection. RuCl₃·3H₂O was purchased from the Kunming Institution of Precious Metals.

Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental analyzer. Electrospray ionization mass spectra (ES-MS) were recorded on a LCQ system (Finnigan MAT, USA) using acetonitrile 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.

4.2. The synthesis of ligand and complexes

4.2.1. 12,14-Dihydroxyl-4,5,9,10,11,13-hexaazabenzo[b]triphenylene (DHBT). 5,6-Diamino-2,4-dihydroxypyrimidine sulfate dihydrate (0.573 g, 1.5 mmol) and K₂CO₃ (0.270 g, 1.5 mmol) were dissolved in 15 mL of water. A mixture of phen-5,6-dione (0.63 g, 3 mmol)³⁵ and ethanol (120 mL) was slowly added into the above solution and refluxed at 90 °C for 13 h. After cooling, the solution was filtered and the precipitate was washed with acetic acid (10 mL) and water (30 mL) and dried under in vacuo, a yellow powder was obtained. Yield: 80%. Anal. calcd for C₁₆H₈N₆O₂: C, 60.76; H, 2.55; N, 26.57. Found: C, 60.58; H, 2.67; N, 26.46%. FAB-MS: m/z = 317 [M + 1]. ¹H NMR (DMSO-d₆): 9.25 (dd, 2H, J = 4.5, J = 4.5 Hz), 9.16 (dd, 2H, J = 4.5, J = 4.5 Hz), 7.98–7.92 (m, 2H), 3.35 (s, 2H).

4.2.2. Synthesis of [Ru(dmb)₂(DHBT)](ClO₄)₂ (1). A mixture of cis-[Ru(dmb)₂Cl₂]·2H₂O³⁶ (0.288 g, 0.50 mmol) and DHBT (0.158 g, 0.50 mmol) in ethylene glycol (30 mL) was refluxed under argon for 8 h to give a clear red solution. Upon cooling, a red precipitate was obtained by dropwise addition of saturated aqueous NaClO₄ solution. The crude product was purified by column chromatography on neutral alumina with a mixture of CH₃CN–toluene (3 : 1, v/v) as eluent. The red band was collected. The solvent was removed under reduced pressure and a red powder was obtained. Yield: 71%. Anal. calc. for C₄₀H₃₂N₁₀Cl₂O₁₀Ru: C, 48.79; H, 3.28; N, 14.22%. Found: C, 48.93; H, 3.15; N, 14.30%. ¹H NMR (DMSO-d₆): δ 9.22 (d, 1H, J = 8.0 Hz), 9.14 (d, 1H, J = 8.0 Hz), 8.72 (d, 4H, J = 8.5 Hz), 8.21 (d, 1H, J = 5.0 Hz), 8.08 (d, 1H, J = 5.0 Hz), 7.93 (t, 1H, J = 5.5 Hz), 7.86 (t, 1H, J = 5.5 Hz), 7.63 (dd, 2H, J = 6.0, J = 5.5 Hz), 7.57 (d, 1H, J = 6.0 Hz), 7.52 (d, 1H, J = 6.0 Hz), 7.41 (d, 2H, J = 6.0 Hz), 7.19 (d, 2H, J = 7.5 Hz), 3.42 (s, 2H), 2.55 (s, 6H), 2.46 (s, 6H). ES-MS (CH₃CN): m/z 784.5 ([M − 2ClO₄ − H]⁺), 392.6 ([M − 2ClO₄]²⁺).

4.2.3. Synthesis of [Ru(bpy)₂(DHBT)](ClO₄)₂ (2). This complex was synthesized in a manner identical to that described for 1, with [Ru(bpy)₂Cl₂]·2H₂O³⁶ in place of [Ru(dmb)₂Cl₂]·2H₂O. Yield: 70%. Anal. calc. for C₃₆H₂₄N₁₀Cl₂O₁₀Ru: C, 45.65; H, 2.60; N, 15.08%. Found: C, 45.73; H, 2.72; N, 15.01%. ¹H NMR (DMSO-d₆): δ 8.94 (d, 2H, J = 8.0 Hz), 8.89 (d, 4H, J = 7.0 Hz), 8.23 (d, 4H, J = 8.0 Hz), 8.16 (d, 2H, J = 7.0 Hz), 7.83 (d, 4H, J = 7.0 Hz), 7.59 (t, 4H, J = 6.5 Hz), 7.42 (t, 2H, J = 6.5 Hz), 3.38 (s, 2H). ES-MS (CH₃CN): m/z 728.3 ([M − 2ClO₄ − H]⁺), 364.7 ([M − 2ClO₄]²⁺).

4.2.4. Synthesis of [Ru(phen)₂(DHBT)](ClO₄)₂ (3). This complex was synthesized in a manner identical to that described for 1, with [Ru(phen)₂Cl₂]·2H₂O³⁶ in place of [Ru(dmb)₂Cl₂]·2H₂O. Yield: 70%. Anal. calc. for C₄₀H₂₄N₁₀Cl₂O₁₀Ru: C, 49.19; H, 2.48; N, 14.34%. Found: C, 49.31; H, 2.34; N, 14.47%. ¹H NMR (DMSO-d₆): δ 8.80 (d, 2H, J = 8.5 Hz), 8.77 (d, 4H, J = 8.5 Hz), 8.40 (s, 4H), 8.22 (dd, 4H, J = 6.0, J = 6.0 Hz), 8.05 (d, 2H, J = 7.0 Hz), 7.81–7.75 (m, 6H), 3.38 (s, 2H). ES-MS (CH₃CN): m/z 776.5 ([M − 2ClO₄ − H]⁺), 388.7 ([M − 2ClO₄]²⁺).

4.2.5. Synthesis of [Ru(dmp)₂(DHBT)](ClO₄)₂ (4). This complex was synthesized in a manner identical to that described for 1, with [Ru(dmp)₂Cl₂]·2H₂O³⁷ in place of [Ru(dmb)₂Cl₂]·2H₂O. Yield: 72%. Anal. calc. for C₄₄H₃₂N₁₀Cl₂O₁₀Ru: C, 51.17; H, 3.12; N, 13.56%. Found: C, 51.33; H, 3.44; N, 13.43%. ¹H NMR (DMSO-d₆): δ 9.18 (d, 1H, J = 8.0 Hz), 9.10 (d, 1H, J = 8.5 Hz), 8.94 (d, 2H, J = 8.5 Hz), 8.46 (dd, 4H, J = 8.5, J = 8.5 Hz), 8.27 (d, 2H, J = 8.5 Hz), 8.00 (d, 2H, J = 8.5 Hz), 7.65 (d, 1H, J = 5.5 Hz), 7.58 (t, 2H, J = 4.5 Hz), 7.47 (d, 1H, J = 5.5 Hz), 7.42 (d, 2H, J = 8.5 Hz), 3.39 (s, 2H), 1.95 (s, 6H), 1.78 (s, 6H). ES-MS (CH₃CN): m/z 832.6 ([M − 2ClO₄ − H]⁺), 416.4 ([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.

4.3. Cytotoxic activity in vitro

3-(4,5-Dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay procedures were used.³⁸ 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. Complexes tested 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 dimethylformamide (50%) and sodium dodecyl sulfate (20%) was added to solubilize the MTT formazan. The optical density of each well was measured with a microplate spectrophotometer at a wavelength of 490 nm. The IC₅₀ values were determined by plotting the percentage of cell viability versus concentration on a logarithmic graph and reading off the concentration at which 50% of cells remain viable relative to the control. Each experiment was repeated at least three times to obtain the mean values.

4.4. Apoptosis assay by AO/EB staining method

MG-63 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 in 5% CO₂. The medium was removed and replaced with medium (final DMSO concentration, 0.05% v/v) containing the complexes (12.5 μM) for 24 h. The medium was removed again, and the cells were washed with ice-cold phosphate buffer saline (PBS), and fixed with formalin (4%, w/v). Cell nuclei were counterstained with acridine orange (AO) and ethidium bromide (EB) (AO: 100 μg mL⁻¹, EB: 100 μg mL⁻¹) for 10 min. The cells were observed and imaged with a fluorescence microscope (Nikon, Yokohama, Japan) with excitation at 350 nm and emission at 460 nm.

4.5. Comet assay

DNA damage was investigated by means of comet assay. MG-63 cells in culture medium were incubated with 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 per 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 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). Cells were stained with 20 μL of EB (20 μg mL⁻¹) 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 fluorescent microscopy.

4.6. Cellular uptake and localisation studies

MG-63 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. The test compounds (12.5 μM) 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 PBS. After removing the culture medium, the cells were stained with DAPI and imaged by fluorescence microscopy.

4.7. Scratch assay

MG-63 cells (4 × 10⁵ cells per mL) were plated in six well tissue culture plates and grown to 90–95% confluence. After aspirating the medium, the centers of the cell monolayers were scraped with a pipette tip to create a denuded zone (gap) of constant width. Then cellular debris was washed with PBS twice and the RMPI 1640 (containing 1% FBS) was added, and MG-63 cells were exposed to 3.13 μM of complexes 1–4. The wound closure was monitored and photographed at different time intervals with an Olympus inverted microscope and camera.

4.8. Reactive oxygen species (ROS) detection

MG-63 cells were seeded into six-well plates (Costar, Corning Corp, New York) at a density of 2 × 10⁵ cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS and incubated at 37 °C in 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 (DCHF-DA, 10 μM) was added to the medium 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, the cell pellets were suspended in PBS–EDTA and imaged with fluorescent microscope. The fluorescent intensity was determined by microplate analyzer (Infinite M200, TECAN, Switzerland) with excitation at 488 nm and emission at 525 nm. The fluorescent intensity was calculated by the determined fluorescent intensity minus the fluorescent intensity of the complexes in the corresponding concentration.

4.9. Mitochondrial membrane potential assay

MG-63 cells were treated for 24 h with complex (12.5 μM) in 12-well plates and were then washed three times with cold PBS. The cells were detached with trypsin–EDTA solution. Collected cells were incubated for 20 min with 1 μg mL⁻¹ of 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 imaged by fluorescence microscopy. The fluorescence intensity was determined by microplate analyzer (Infinite M200, TECAN, Switzerland) with excitation set at 488 nm and emission at 525 nm. The fluorescent intensity was calculated by the determined fluorescent intensity minus the fluorescent intensity of the complexes in the corresponding concentration.

4.10. Cell cycle arrest by flow cytometry

MG-63 cells were seeded into six-well plates (Costar, Corning Corp, New York) at a density of 2 × 10⁵ cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS and incubated at 37 °C in 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⁻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. The number of cells analyzed for each sample was 10 000.³⁹

4.11. Western blot analysis

MG-63 cells were seeded in 3.5 cm dishes for 24 h and incubated with 12.5 μM of the complexes in the presence of 10% FBS. The 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 : 5000 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 secondary antibodies conjugated with horseradish peroxidase at 1 : 5000 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 β-actin.

4.12. Statistical analysis

All of the data were expressed as the mean ± SD. Differences between two groups were analyzed by a two-tailed Student's t test. Differences with *P < 0.05 were considered statistically significant.

Acknowledgements

This work was supported by the 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).

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