Apoptosis, autophagy, cell cycle arrest, cell invasion and BSA-binding studies in vitro of ruthenium(II) polypyridyl complexes

Abstract

Ruthenium(II) polypyridyl complexes show high anticancer activity, and can induce apoptosis. Herein, a new ligand AQTP (AQTP = 12-acenaphtho[1,2-b]quinoxalin-9-yl-4,5,9,14-tetraazabenzo[b]triphenylene) and its four ruthenium(II) polypyridyl complexes [Ru(N-N)₂(AQTP)](ClO₄)₂ (N-N = dmb: 4,4′-dimethyl-2,2′-bipyridine 1; bpy: 2,2′-bipyridine 2; phen: 1,10-phenanthroline 3 and dmp: 2,9-dimethyl-1,10-phenanthroline 4) were synthesized and characterized. The cytotoxic activity in vitro of the complexes against BEL-7402, A549, HeLa, HepG2, MG-63 and normal cell HLF was investigated using the MTT method (MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The ROS, mitochondrial membrane potential and autophagy were studied using a fluorescent microscope. The expression of caspases and Bcl-2 family proteins was investigated by western blot analysis. The IC₅₀ values of complexes 1–4 toward A549 cells are 5.0 ± 0.8, 10.0 ± 0.7, 45.0 ± 1.4 and 3.8 ± 0.1 μM. The complexes can increase the levels of reactive oxygen species (ROS), and induce a decrease in the mitochondrial membrane potential. Complexes 1–4 inhibit cell growth at the G0/G1 phase in A549 cells, and the complexes can induce both autophagy and apoptosis, and the complexes induce apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

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1. Introduction

Carcinogenesis is thought to be prompted by changes to the DNA within cells and also by inhibition of growth suppressors, which, in turn, gives rise to uncontrolled cell proliferation, invasion of surrounding and distant tissues, and ultimately leads to a risk of aggressive metastasis.^1–3 After the success of cisplatin and countless platinum candidates under trial, only two other platinum-based drugs, carboplatin and oxaliplatin, are used in routine clinical therapy.⁴ However, these platinum complexes are ineffective against many common types of cancer; inefficient against platinum-resistant tumors; being non-specific have severe side effects including nephrotoxicity, neurotoxicity, ototoxicity, nerve damage, hair loss, nausea, vomiting and abdominal pain.⁵ This has led to the search for complexes of other transition metals with interesting biological properties, wider ranges of activity, and lower systematic toxicities.⁶ Ruthenium complexes, owing to possessing several favorable properties suited to rational anticancer drug design and biological applications, are regarded as promising alternatives to platinum complexes in cancer therapies. In recent years, the anticancer activity of ruthenium(II) polypyridyl complexes has been made a great progress. An attractive feature of Ru(II) polypyridyl complexes that makes them particularly useful for applications as biological probe and effectors is the diversity of the chemical structures that are readily available through modifications of the coordinated ligands.⁷ A number of these complexes reveal interesting bioactivity.^8–13 The complex [Ru(bpy)(phpy)(dppz)]⁺ (phpy = 2-phenylpyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine) was found to be rapidly taken up by cancer cells, and nearly 90% of the complex accumulated in the nuclei of cancer cells after a 2 h incubation.¹⁴ [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, and it is able to disrupt the mitochondria membrane potential.¹⁵ [Ru(dip)₂(PAIDH)]²⁺ (dip = 4,7-dimethyl-1,10-phenanthroline, PAIDH = 2-pyridyl-1H-anthra[1,2-d]imidazole-6,11-dione) was shown to accumulate preferentially in the mitochondria of HeLa cells and induced apoptosis via the mitochondrial pathway, which involved ROS generation, mitochondrial membrane potential depolarization.¹⁶ The complex [Ru(dmp)₂(pddppn)]²⁺ (dmp = 2,9-dimethyl-1,10-phenanthroline, pddppn = phenantheno[1,2-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine) shows very high inhibitory effect on the cell growth in BEL-7401, MG-63 and A549 cell with an IC₅₀ value of 1.6 ± 0.4 μM, 1.5 ± 0.2 μM and 1.5 ± 0.3 μM, respectively.¹⁷ Previously, our group reported some anticancer activity studies of Ru(II) polypyridyl complexes against various cancer cells.^17–19 To extend this work further and to obtain more information of Ru(II) polypyridyl complexes on anticancer activity, in this report, a new ligand AQTP (AQTP = 12-acenaphtho[1,2-b]quinoxalin-9-yl-4,5,9,14-tetraazabenzo[b]triphenylene) and its four ruthenium(II) polypyridyl complexes [Ru(N-N)₂(AQTP)](ClO₄)₂ 1–4 (N-N = dmb: 4,4′-dimethyl-2,2′-bipyridine 1; bpy: 2,2′-bipyridine 2; phen: 1,10-phenanthroline 3; and dmp: 2,9-dimethyl-1,10-phenanthroline 4, Scheme 1) were synthesized and characterized. The cytotoxic activity in vitro of the complexes against BEL-7402, A549, HeLa, HepG2, MG-63 and normal cell HLF was investigated by MTT method (MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). The IC₅₀ values of complexes 1–4 toward A549 cells are 5.0 ± 0.8, 10.0 ± 0.7, 45.0 ± 1.4 and 3.8 ± 0.1 μM. The morphological feature of the cells was assayed with acridine/orange (AO)/ethidium bromide (EB) and Hoechst 33258 staining methods. The DNA damage, reactive oxygen species (ROS), mitochondrial membrane potential, and autophagy were performed under fluorescent microscope. The percentage in the apoptotic cells and cell cycle arrest were studied by flow cytometry. The expression of caspases and Bcl-2 family proteins was assayed by western blot.

Scheme 1 The synthetic route of ligand and complexes.

2. Results and discussion

2.1. Synthesis and characterization

The ligand AQTP was prepared by the reaction of TTBD²⁰ (4-(4,5,9,14-tetraazabenzo[b]triphenylen-12-yl)benzene-1,2-diamine) with acenaphthylene-1,2-dione in glacial acetic acid. The chemical structure of the ligand was confirmed by elemental analysis, ESI-MS, IR and ¹H NMR. The corresponding Ru(II) mixed-ligand complexes were synthesized by the direct reaction of AQTP with the appropriate precursor complexes in ethylene glycol. The desired Ru(II) complexes were isolated as the perchlorate and purified by column chromatography. The formation of the complexes was confirmed by elemental analysis, ESI-MS, ¹H NMR and IR. In the IR spectra, 2916.5 cm⁻¹ for AQTP, 2965.9 cm⁻¹ for 1, 3054.5 cm⁻¹ for 2, 3049.8 cm⁻¹ for 3 and 3052.3 cm⁻¹ for 4 are assigned to C–H stretch vibration. In the ESI-MS spectra for the Ru(II) complexes, all of the expected signals [M − 2ClO₄ − H]⁺ and [M − 2ClO₄]²⁺ were observed. The measured molecular weights were consistent with expected values.

The UV-Vis spectra of the complexes in DMF, DMSO, EtOH, CH₃OH and CH₃CN are shown in Fig. S1 (ESI†), the UV-Vis spectra of the complexes consist of three well resolved bands in the range 200–600 nm. The low-energy absorption band at 405–430 nm are assigned to a metal-to-ligand charge-transfer (MLCT) transition, the bands at the range of 300–400 nm are attributed to π–π* transitions, and the bands below 300 nm are attributed to an intraligand (IL) π–π* transition. The complexes show high solubility in DMSO, DMF, CH₃CN, EtOH and MeOH, whereas ligand can not be dissolved in these solvents.

2.2. Cytotoxicity in vitro evaluation

The cytotoxic activity in vitro of the ligand and complexes was evaluated by MTT method. 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 the ligand and complexes 1–4 against BEL-7402, A549, HeLa, HepG2, MG-63 and normal HLF cells are listed in Table 1. As expectation, ligand AQTP shows no cytotoxic effect on the selected cell lines. Complexes 1, 2 and 4 exhibit high cytotoxic activity toward A549 cells with a low IC₅₀ value of 5.0 ± 0.8, 10.0 ± 0.7, 3.8 ± 0.1 μM, respectively. Complexes 2 and 4 display strongly inhibitory effect on the cell growth in BEL-7402 cells. It is unexpected to find that the complexes 1–4 show no or very low cytotoxicity against HeLa, HepG2 and MG-63 cells. The cytotoxic activity of complex 4 toward A549 cells is higher than that of other ruthenium(II) complexes: Λ-[Ru(bpy)₂(o-tFPIP)]²⁺ (IC₅₀ = 15.6 μM),²¹ but lower than those of [Ru(bpy)(phpy)(dppz)]²⁺ (IC₅₀ = 1.4 ± 0.2 μM)¹⁴ and [Ru(dmp)₂(pdppn)]²⁺ (IC₅₀ = 1.5 ± 0.3 μM).¹⁷ Table 1 shows that complex 4 displays higher cytotoxic effect on BEL-7402 and A549 cells than cisplatin under identical conditions. Additionally, complex 4 also shows high inhibition of the cell growth against normal cell HLF (IC₅₀ = 9.8 ± 0.7 μM). Thus, it is very difficult to find a drug which can effectively inhibit the cancer cell growth and has no influence on the normal cells. The difference in cytotoxic activity of the complexes 1–4 toward the same cell line may be caused by different ancillary ligands, ancillary ligand dmp has larger hydrophobicity than dmb and bpy or phen, thus, complex 4 shows relatively higher cytotoxicity against BEL-7402, A549 and HepG2 cells than the other three complexes. Because A549 cell was sensitive to the complexes, this cell was selected for the following experiments.

Table 1 The IC₅₀ values (μM) of ligand and the complexes toward the selected cell lines

Complex	BEL-7402	A549	HeLa	HepG2	MG-63	HLF
AQTP	>200	>200	>200	>200	>200	>200
1	9.4 ± 0.4	5.0 ± 0.8	>100	>100	>100	21.9 ± 2.2
2	>100	10.0 ± 0.7	>100	>100	>100	>100
3	>100	45.0 ± 1.4	>100	>100	65.8 ± 14.8	>100
4	6.4 ± 0.5	3.8 ± 0.1	>100	32.9 ± 3.1	>100	9.8 ± 0.7
Cisplatin	11.1 ± 1.2	6.3 ± 1.1	7.4 ± 1.3	25.4 ± 3.3	6.5 ± 0.4	34.8 ± 3.5

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2.3. Apoptosis assay with AO/EB and Hoechst 33258 staining methods

AO is a crucial dye that stains nuclear DNA across an intact cell membrane and EB only stains cells that have lost membrane integrity. To observe the morphological changes, A549 cells were treated with the complexes for 24 h and the cells were imaged under a fluorescent microscope. As shown in Fig. 1a, in the control, the living cells are stained bright green spots. However, A549 cells were exposed to 3.13 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h, the apoptotic cells appear green and contain apoptotic characteristics such as cell blebbing, nuclear shrinkage and chromatin condensation. In addition, after the treatment of A549 cells with the complexes, the cells were stained with Hoechst 33258 (f–j), the morphological apoptotic features were also observed. These morphological changes suggest that the complexes can induce apoptosis in A549 cells. Similar phenomena were found in our previous reports.^17–19

Fig. 1 Apoptosis in A549 cells (a and f) exposure to 3.13 μM of complexes 1 (b and g), 2 (c and h), 3 (d and i) and 4 (e and j) for 24 h and stained with AO/EB (a–e) and Hoechst 33258 (f–j).

2.4. DNA damage assay

DNA fragmentation is a hallmark of apoptosis, mitotic catastrophe or both.²² Apoptosis eventually entails DNA damage providing another experimental endpoint to validate the existence of apoptotic processes in the cells. Single cell gel electrophoresis (comet assay) in an agarose gel matrix was used to study DNA fragmentation. When a cell with damaged DNA is subjected to electrophoresis and then stained with ethidium bromide (EB), it appears as a comet, and the length of the comet tail represents the extent of DNA damage.¹¹ As shown in Fig. 2a, in the control, A549 cells do not show any comet like appearance. Exposure of A549 cells to 3.13 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h demonstrates statistically significant and well-formed comets, and the length of the comet tail represents the extent of DNA damage. These results clearly suggest that the four complexes indeed induce DNA fragmentation, which is further evidence of apoptosis.

Fig. 2 Comet assay of EB-stained A549 cells (a) exposure to 3.13 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h.

2.5. The detection of percentage in apoptotic cells

The morphological characteristics and comet assay indicate that the complexes can cause apoptosis in A549 cells. To quantitatively compare the apoptotic effect, the percentage in the apoptotic cells was determined by flow cytometry. In the control (Fig. S2, ESI†), the percentages in the early apoptotic cells are 0.11%. A549 cells were exposed to 3.13 and 6.25 μM of complexes 1 (b and c), 2 (d and e), 3 (f and g) and 4 (h and i) for 24 h, the percentages in the early apoptotic cells are 2.56% and 6.09% for 1, 5.76% and 10.94% for 2, 1.92% and 4.15% for 3, 2.97% and 6.98% for 4, respectively. The apoptotic effect follows the order of 2 > 4 > 1 > 3. Moreover, the apoptotic effect shows a concentration-dependent manner. Complex 3 shows the least apoptotic effect. This is consistent with its cell cytotoxicity in vitro.

2.6. The cellular uptake and localization studies

The cellular uptake characteristics of a small molecule are critical to its application as a therapeutic or diagnostic agent.²³ To testify whether or not ruthenium complexes can be transported into the cellular interior, A549 cells were incubated with 3.13 μM of the complexes 1–4 for 24 h and the cells were stained with DAPI. As shown in Fig. S3 (ESI†), the blue channel displays DAPI stained nuclei, the red channel shows the luminescence of complexes 1, 2, 3 or 4, and the merge represents cellular association of complexes 1–4. The overlay of the blue channel with the red channel demonstrates that the complexes can be taken up by A549 cells, and the complexes gradually penetrate into the interior of the nucleus and show diffuse cytoplasmic and nuclear fluorescence.

2.7. Reactive oxygen species levels assay induced by the complexes

Reactive oxygen species (ROS) play an important role in cancer cell death and apoptosis. In the evaluation of the ROS generation, 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA) was used as fluorescence probe, which is cleaved by intracellular esterases into its non-fluorescent form (DCHF). Then DCFH is oxidized by intracellular free radicals to produce a fluorescent product, namely, dichlorofluorescein (DCF).^24,25 In the control (Fig. 3A(a)), no any obvious green fluorescent points were found. After the treatment of A549 cells with Rosup (positive control, (b)) and 3.13 μM of 1 (c), 2 (d), 3 (e) and 4 (f) for 24 h, the bright fluorescent images were observed. The fluorescent intensity of DCF is proportional to the amount of peroxide (ROS) produced by the cells.²⁶ The DCF fluorescent intensity was determined by flow cytometry. As shown in Fig. 3B, in the control, the DCF fluorescent intensity is 229. After A549 cells were exposed to the complexes 1–4 for 24 h, the DCF fluorescent intensity is 501, 537, 315 and 440, respectively. The treatment of A549 cells induces an increase of 2.19 for 1, 2.34 for 2, 1.38 for 3 and 1.92 times for 4 than the original. Obviously, complex 2 induces the largest increase in the ROS levels among the complexes. The results demonstrate that the complexes can enhance the ROS levels.

Fig. 3 (A) Intracellular ROS was detected in A549 cells (a) exposure to Rosup (positive control, (b)) and 3.13 μM of complexes 1 (c), 2 (d), 3 (e) and 4 (f) for 24 h. (B) The DCF fluorescent intensity induced by 1–4 was determined by flow cytometry.

2.8. The detection of the changes in the mitochondrial membrane potential

Mitochondria act as a point of integration for apoptotic signals originating from both extrinsic and intrinsic apoptotic pathways.^27,28 The changes in mitochondrial membrane potential were assayed using JC-1 as fluorescent probe. As shown in Fig. 4A(a), in the control, JC-1 forms aggregates and emits a red fluorescence corresponding to high mitochondrial membrane potential. After A549 cells were exposed to cccp (positive control, (b)) and 3.13 μM of complexes 1 (c), 2 (d), 3 (e) and 4 (f) for 24 h, JC-1 forms monomers, which emit a green fluorescence peak corresponding to low mitochondrial membrane potential. The changes from the red to green fluorescence suggest that the complexes can induce a decrease of mitochondrial membrane potential. To quantitatively determine the ratio of red/green fluorescence, the mitochondrial membrane potential was assayed by flow cytometry. In the control (Fig. 4B(a)), the ratio of red/green fluorescence is 4.94. The treatment of A549 cells with complexes shows the ratio of 3.31 for 1, 2.58 for 2, 3.38 for 3, and 1.34 for 4, respectively. The reduction in the ratio of red/green indicates that the red fluorescent intensity decreases and the green fluorescent intensity increases. The ability of the complexes inducing the changes in the mitochondrial membrane potential follows the order of 4 > 2 > 1 > 3.

Fig. 4 (A) Assay of A549 cells mitochondrial membrane potential with JC-1 as fluorescent probe. A549 cells (a) exposed to cccp (positive control, (b)) and 3.13 μM of complexes 1 (c), 2 (d), 3 (e) and 4 (f) for 24 h. (B) The ratio of red/green fluorescence was determined by flow cytometry. A549 cells (a) exposure to 3.13 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h. Ratio stands for red/green fluorescence.

2.9. Cell cycle arrest studies

Inhibition of cancer cell proliferation by cytotoxic drugs could be the result of induction of apoptosis or cell cycle arrest or a combination of these. The status of cell cycle for cells treated with complexes 1–4 for was analyzed. As shown in Fig. 5, in the control, the percentage in the cell at G0/G1 phase is 69.48%. After the A549 cells were incubated with 6.25 μM of complexes 1, 2, 3 or 4 for 24 h, the percentage in the cell at G0/G1 is 73.15%, 77.59%, 73.31% and 77.22%, respectively. The increase of 3.67% for 1, 8.11% for 2, 3.83% for 3, and 7.74% for 4 at G0/G1 phase was found, which was accompanied by the corresponding reduction in the S phase. The data indicate that the complexes induce the cell cycle arrest at G0/G1 phase in A549 cells. Obviously, complex 2 shows greater effect on the cell cycle arrest than complexes 1 or 3 or 4 under identical condition. The effect on the cell cycle arrest is not consistent with the cytotoxic activity of the complexes.

Fig. 5 Cell cycle arrest of A549 cells exposure to 6.25 μM of complexes 1, 2, 3 and 4 for 24 h. G0/G1 (blue), S (red) and G2/M (green). *P < 0.05 represents significant differences compared with control.

2.10. Autophagy induced by the complexes

Autophagy, or type II programmed cell death, has been proposed as a third mode of cell death besides apoptosis and necrosis.^29,30 Most cancer chemotherapy drugs act through induction of apoptosis.³¹ The relationship between autophagy and apoptosis is complex and depends on particular cell type, stimulus, and environment. The induction of autophagy may be used as a potential therapy for some apoptosis resistant cancers (i.e., breast and pancreatic cancers).³² The autophagy induced by the complexes was investigated using monodansylcadaverine (MDC) as fluorescent probe. It is well known that MDC fluorescent dye is a specific, in vivo marker for autophagic vacuoles,³³ and MDC incorporation is an indicator of autophagic activity. As shown in Fig. 6A, in the control, MDC emits weak green fluorescence. A549 cells were treated with 3.13 μM of the complexes for 24 h, the interspersed and punctate MDC labeling green fluorescent points in the cytoplasm were observed, which suggests that the autophagic vacuoles were formed. LC3 is a hallmark of autophagy, and the conversion of LC3-I to LC3-II shows autophagy induction.³⁴ The amount of LC3-II is closely correlated with number of autophagosomes.³⁵ As shown in Fig. 6B, the protein level of LC3-II was up-regulated in A549 cells after treatment with 3.13 μM of complexes 1–4, and the ratios of the amount of LC3-II/LC3-I were increased. It is well known that Beclin-1 is necessary to form autophagosomes in autophagy. The expression levels of Beclin-1 protein induced by the complexes were also examined. As seen in Fig. 6B, there were significant increases in the protein expression of Beclin-1 in A549 cells treated with 3.13 μM of the complexes. Taken the above results together, the results indicate that the complexes can induce autophagy in A549 cells.

Fig. 6 (A) Autophagy in A549 cells induced by 3.13 μM of complexes 1–4 for 24 h. Autophagy was detected using MDC staining. (B) The assay of LC-3 protein by western blot.

2.11. Cell invasion assay

Since metastasis poses a foremost threat for cancer deaths, inhibiting metastasis is an urgent therapeutic need.³⁶ It is interesting to investigate whether complexes 1–4 can inhibit invasion of A549 cells. The transwell invasion assay in A549 cells was treated by Matrigel invasion assay. As shown in Fig. 7A, A549 cells (a) were exposed to 6.25 μM of complexes 1 (b), 2 (c), 3 (d) and 4 (e) for 24 h, the number of the invasion cells decrease gradually, and the percentages of the complexes inhibiting cell invasion are shown in Fig. 7B. Complex 1 displays the highest inhibiting effect on the cell invasion of A549 cells among the complexes, and at 6.25 μM of the complexes, the inhibiting effect follows the order of 1 > 3 > 4 > 2. The inhibiting cell invasion is not consistent with that of cytotoxicity of complexes 1–4 toward A549 cells, and the complexes show a concentration-dependent manner in the inhibiting cell invasion. These results demonstrate that the complexes can effectively inhibit A549 cell invasion.

Fig. 7 (A) Microscope images of invading A549 cells that have migrated through the Matrigel: the extent of inhibition of cell invasion by complexes 1–4 against A549 cells can be seen from the decrease in the numbers of invading cells. (B) The percentage of inhibition cell invasion induced by different concentration of complexes 1 (black), 2 (red), 3 (yellow) and 4 (green) for 24 h.

2.12. The expression of caspases and Bcl-2 family protein

Caspases are known to mediate the apoptotic pathway.^37,38 Caspase-3 and -7 are executioners of apoptosis as processing of their substrates lead to morphological changes associated with apoptosis, including DNA degradation, chromatin condensation, and membrane blebbing.³⁹ The activation of caspase 3 and caspase 7 was assayed by the western blot analysis. After A549 cells were incubated with 6.25 μM of complexes 1–4 for 24 h, the expression of caspase 3 was down-regulated. The expression of caspase 7 induced by complexes 2–4 was up-regulated, whereas complex 1 caused the decrease in the expression of caspase 7 (Fig. 8). Bcl-2 family proteins consist of antiapoptotic such as Bcl-2, Bcl-x and proapoptotic proteins such as Bax and Bid, etc. The treatment of A549 cells with the complexes led to the down-regulation in the expression levels of Bcl-2 and Bcl-x proteins, and the expression levels increased in Bid. In addition, complexes 1 and 2 increased the expression of Bax, complexes 3 and 4 downregulated the expression levels of Bax. This may be caused by the different ancillary ligands of the complexes. These results demonstrate that the complexes induce cell death through activation of caspase 3, caspase 7 and regulation of Bcl-2 family proteins.

Fig. 8 A549 cells were treated with 6.25 μM of the complexes for 24 h and the expression levels of the apoptosis-related proteins were examined by western blot.

2.13. Electronic absorption spectra of BSA in the presence of the Ru(II) complexes

Absorption spectrum is a useful technique to explore the structural changes of protein and to investigate the protein–ligand complex formation.⁴⁰ In a dynamic quenching, absorption spectrum exhibits insignificant changes due to existence of the transient excited state of fluorophore and quencher, whereas in static quenching formation of the fluorophore–quencher complex causes perturbations.^41–45 As shown in Fig. S4 (ESI†), upon addition of complexes 1–4 to a fixed concentration of BSA led to a gradual increase in BSA absorption. The results imply that the interaction of the tested complexes and BSA is mainly ascribed to be static quenching.

2.14. Fluorescence spectroscopic studies of BSA

Fluorescence quenching of BSA is usually induced by a variety of molecular interactions such as excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collision quenching etc.^46–49 Synchronous fluorescence spectroscopy is a very useful method to study the microenvironment of amino acid residues by measuring the emission wavelength shift and have several advantages such as sensitivity, spectral simplification, spectral bandwidth reduction and avoiding different perturbing effects.⁵⁰ To explore the binding ability of the complexes with BSA, the effect of the complexes on the fluorescence emission intensity of BSA was investigated. As shown in Fig. 9A, the band of BSA at 342 nm was quenched to an extent of about 65.4%, 60.3%, 60.4% and 70.4% from its initial intensity upon the addition of complexes 1–4, respectively. This suggests that complexes 1–4 bind to BSA and binding site is close to tryptophan and tyrosine residues. Obviously, BSA fluorescence emission is quenched. To evaluate the quenching effect of the complexes on BSA solution, the fluorescence quenching constants are calculated according to Stern–Volmer equation:⁵¹

I₀/I = 1 + K_SV[complex] (1)

I₀ and I represent the fluorescence intensities of BSA in the absence and presence of quencher, respectively. K_SV is the linear Stern–Volmer quenching constant and [complex] the molar concentration of the quencher. As shown in Fig. 9B, a linear plot between I₀/I against [complex] was obtained. The K_SV values for complexes 1–4 from the slope were determined to be 1.34 (±0.18) × 10⁵, 1.16 (±0.13) × 10⁵, 1.23 (±0.16) × 10⁵ and 1.83 (±0.11) × 10⁵ M⁻¹, respectively. The quenching effect follow the order of 4 > 1 > 3 > 2. Obviously, the BSA-binding affinities are not completely consistent with the cytotoxic activity of the complexes. The calculated K_SV values for the tested complexes exhibit their strong protein-binding ability.

Fig. 9 (A) Fluorescence quenching of BSA induced by different concentrations of complexes 1, 2, 3 and 4. (B) Linear Stern–Volmer quenching constants for 1–4.

3. Conclusions

Four new ruthenium(II) polypyridyl complexes were synthesized and characterized in detail. The cell morphological features, comet assay and the determination of the percentage in the apoptotic cells suggest that all the complexes can induce apoptosis in A549 cells. The ROS and mitochondrial membrane potential assays show that the complexes increase the ROS levels and cause a decrease in the mitochondrial membrane potential. The cell cycle arrest indicates that complexes 1–4 inhibit the cell growth at G0/G1 phase in A549 cells, and the complexes can effectively inhibit the cell invasion. Additionally, the complexes induce autophagy and regulate the expression of Bcl-2 family proteins. In summary, the effect of the complexes on ROS, mitochondrial membrane potential, cell cycle arrest and BSA-binding constants are not consistent with the cytotoxic activity, namely, the complex with high cytotoxic activity don't shows high effect on the above biological activity. The studies demonstrate that the complexes can induce both autophagy and apoptosis in A549 cells, and the complexes induce A549 apoptosis through a ROS-mediated mitochondrial dysfunction pathway, which was accompanied by the regulation of the expression of caspases and Bcl-2 family protein. These studies are helpful for the design and synthesis of new ruthenium(II) complexes as potent anticancer agents.

4. Experimental sections

4.1. Materials and method

All reagents and solvents were purchased commercially and used without further purification unless otherwise noted. Ultrapure MilliQ water was used in all experiments. DMSO and RPMI 1640 were purchased from Sigma. BEL-7402 (hepatocellular), HeLa (human cervical cancer cell line), A549 (human lung adenocarcinoma cell line), HepG2 (human liver cancer cell), MG-63 (human osteosarcoma) and normal cell HLF (human lung fibroblasts line) were purchased from the American Type Culture Collection. RuCl₃·3H₂O 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 (ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA) using methanol as mobile phase. The spray voltage, tube lens offset, capillary voltage and capillary temperature were set at 4.50 kV, 30.00 V, 23.00 V and 200 °C, respectively, and the quoted m/z values are for the major peaks in the isotope distribution. ¹H NMR spectra were recorded on a Varian-500 spectrometer with DMSO-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-Acenaphtho[1,2-b]quinoxalin-9-yl-4,5,9,14-tetraazabenzo[b]triphenylene (AQTP). TTBD (0.582 g, 1.5 mmol),²⁰ acenaphthylene-1,2-dione (0.273 g, 1.5 mmol) and glacial acetic acid (30 mL) were refluxed with stirring for 5 h. After cooling, the yellow precipitate was washed with water (30 mL) and yellow powder was obtained. The precipitate was collected and purified by column chromatography on silica gel (60–100 mesh) with ethanol as eluent to give the compound as a yellow powder. Yield: 80%. Anal. calcd for C₃₆H₁₈N₆: C, 80.87; H, 3.40; N, 15.73. Found: C, 80.75; H, 3.47; N, 17.65%. IR (KBr, cm⁻¹): 2916.5, 1590.7, 1489.2, 1408.7, 1348.8, 1299.1, 1208.4, 1102.8, 1071.4, 821.2, 774.8, 740.6. ESI-MS: m/z = 535 [M + 1].

4.2.2. Synthesis of [Ru(dmb)₂(AQTP)](ClO₄)₂ (1). A mixture of cis-[Ru(dmb)₂Cl₂]·2H₂O⁵² (0.288 g, 0.50 mmol) and AQTP (0.267 g, 0.50 mmol) in ethylene glycol (20 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–ethanol (20 : 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 C₆₀H₄₂Cl₂O₈N₁₀Ru: C, 59.89; H, 3.52; N, 11.65%. Found: C, 59.78; H, 3.45; N, 11.87%. ¹H NMR (DMSO-d₆): δ 9.48 (dd, 2H, J = 8.0, J = 8.5 Hz), 9.31 (d, 1H, J = 6.0 Hz), 8.80 (d, 4H, J = 8.5 Hz), 8.67 (d, 1H, J = 1.5 Hz), 8.64 (d, 1H, J = 2.0 Hz), 8.58 (d, 1H, J = 1.5 Hz), 8.40 (d, 1H, J = 7.0 Hz), 8.33 (dd, 2H, J = 8.5, J = 6.5 Hz), 8.27 (d, 2H, J = 5.0 Hz), 8.20 (d, 1H, J = 6.5 Hz), 8.06 (d, 1H, J = 5.5 Hz), 8.02 (d, 1H, J = 8.5 Hz), 7.94 (d, 2H, J = 5.5 Hz), 7.82 (d, 1H, J = 7.0 Hz), 7.68 (d, 4H, J = 5.5 Hz), 7.60 (d, 1H, J = 8.0 Hz), 7.46 (d, 2H, J = 5.5 Hz), 7.30 (d, 2H, J = 6.0 Hz), 2.56 (s, 6H), 2.50 (s, 6H). ¹³C NMR (DNSO-d₆, 125 MHz): δ 156.31, 156.18, 153.46, 153.35, 151.02, 150.45, 150.22, 150.09, 149.74, 141.90, 140.67, 140.16, 139.57, 135.48, 132.66, 130.63, 130.06, 129.78, 129.34, 128.96, 128.59, 128.37, 127.77, 127.65, 127.53, 126.19, 125.08, 121.95, 20.78, 20.70. IR (KBr, cm⁻¹): 2965.9, 1618.8, 1481.2, 1447.1, 1418.6, 1356.2, 1308.8, 1242.5, 1107.9, 1049.6, 880.0, 827.9, 779.4, 725.9, 624.6. ESI-MS (CH₃CN): m/z 1002.4 ([M − 2ClO₄ − H]⁺), 501.6 ([M − 2ClO₄]²⁺).

4.2.3. Synthesis of [Ru(bpy)₂(AQTP)](ClO₄)₂ (2). This complex was synthesized in a manner identical to that described for 1, with [Ru(bpy)₂Cl₂]·2H₂O⁵² in place of [Ru(dmb)₂Cl₂]·2H₂O. Yield: 71%. Anal. calc. for C₅₆H₃₄Cl₂O₈N₁₀Ru: C, 58.63; H, 2.99; N, 12.22%. Found: C, 58.55; H, 3.08; N, 12.41%. ¹H NMR (DMSO-d₆): δ 9.49 (d, 2H, J = 6.5 Hz), 9.28 (d, 1H, J = 8.0 Hz), 8.95 (d, 4H, J = 5.5 Hz), 8.88 (d, 1H, J = 8.0 Hz), 8.63 (d, 2H, J = 1.5 Hz), 8.54 (d, 1H, J = 2.0 Hz), 8.39 (d, 1H, J = 2.0 Hz), 8.30 (d, 1H, J = 4.0 Hz), 8.27 (d, 4H, J = 5.5 Hz), 8.25 (d, 1H, J = 5.5 Hz), 8.23 (d, 1H, J = 5.0 Hz), 8.19 (d, 2H, J = 7.5 Hz), 8.15 (d, 1H, J = 7.5 Hz), 8.08 (dd, 2H, J = 8.0, J = 8.5 Hz), 7.95 (d, 1H, J = 6.5 Hz), 7.88 (d, 2H, J = 5.0 Hz), 7.83 (d, 2H, J = 7.0 Hz), 7.64 (d, 2H, J = 5.5 Hz). 7.52 (d, 1H, J = 7.5 Hz), 7.48 (d, 2H, J = 6.5 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ 156.78, 156.58, 153.75, 153.53, 153.42, 151.90, 151.43, 150.09, 149.96, 141.95, 141.59, 141.38, 140.77, 140.69, 140.19, 139.60, 138.11, 135.51, 133.14, 131.50, 130.65, 130.12, 129.92, 129.77, 129.36, 128.99, 128.75, 128.51, 127.96, 127.76, 127.64, 126.26, 124.56, 122.00. IR (KBr, cm⁻¹): 3054.5, 1601.2, 1538.1, 1490.8, 1463.9, 1444.4, 1420.9, 1354.8, 1301.1, 1243.2, 1217.1, 1172.9, 892.8, 827.6, 764.4, 727.5, 622.4. ESI-MS (CH₃CN): m/z 946.3 ([M − 2ClO₄ − H]⁺), 473.5 ([M − 2ClO₄]²⁺).

4.2.4. Synthesis of [Ru(phen)₂(AQTP)](ClO₄)₂ (3). This complex was synthesized in a manner identical to that described for 1, with [Ru(phen)₂Cl₂]·2H₂O⁵² in place of [Ru(dmb)₂Cl₂]·2H₂O. Yield: 70%. Anal. calc. for C₆₀H₃₄Cl₂O₈N₁₀Ru: C, 60.30; H, 2.87; N, 11.73%. Found: C, 60.15; H, 2.75; N, 11.87%. ¹H NMR (DMSO-d₆): δ 9.63 (d, 2H, J = 8.0 Hz), 9.56 (d, 1H, J = 7.5 Hz), 9.40 (d, 1H, J = 8.0 Hz), 9.16 (d, 1H, J = 6.5 Hz), 8.94 (d, 1H, J = 7.5 Hz), 8.82 (d, 4H, J = 6.0 Hz), 8.80 (d, 2H, J = 5.0 Hz), 8.77 (d, 1H, J = 6.0 Hz), 8.74 (d, 1H, J = 5.0 Hz), 8.39 (s, 4H), 8.27 (d, 2H, J = 7.5 Hz), 8.24 (d, 4H, J = 5.0 Hz), 8.20 (d, 1H, J = 5.0 Hz), 8.15 (d, 1H, J = 8.5 Hz), 8.08 (d, 4H, J = 5.5 Hz), 7.98 (d, 1H, J = 6.5 Hz), 7.87 (d, 1H, J = 5.0 Hz), 7.72 (d, 2H, J = 5.0 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ 155.36, 155.21, 154.90, 154.63, 154.47, 154.01, 151.66, 151.53, 148.53, 148.44, 143.12, 142.53, 141.94, 141.88, 141.36, 140.73, 139.30, 138.40, 138.31, 136.67, 134.36, 134.18, 132.54, 131.84, 131.28, 131.06, 130.99, 130.53, 130.21, 129.85, 129.64, 129.42, 128.90, 128.68, 127.71, 127.58, 127.37, 123.31, 123.12. IR (KBr, cm⁻¹): 3048.9, 1599.4, 1491.3, 1424.7, 1355.0, 1300.6, 1206.9, 828.4, 777.0, 721.0, 622.9. ESI-MS (CH₃CN): m/z 994.3 ([M − 2ClO₄ − H]⁺), 497.5 ([M − 2ClO₄]²⁺).

4.2.5. Synthesis of [Ru(dmp)₂(AQTP)](ClO₄)₂ (4). This complex was synthesized in a manner identical to that described for 1, with [Ru(dmp)₂Cl₂]·2H₂O⁵³ in place of [Ru(dmb)₂Cl₂]·2H₂O. Yield: 72%. Anal. calc. for C₆₄H₄₂Cl₂O₈N₁₀Ru: C, 61.43; H, 3.39; N, 11.20%. Found: C, 61.56; H, 3.18; N, 11.37%. ¹H NMR (DMSO-d₆): δ 9.37 (d, 2H, J = 8.0 Hz), 9.31 (d, 1H, J = 7.0 Hz), 8.95 (d, 2H, J = 8.5 Hz), 8.73 (d, 1H, J = 2.0 Hz), 8.67 (d, 1H, J = 6.0 Hz), 8.62 (d, 1H, J = 2.0 Hz), 8.51 (dd, 2H, J = 8.0, J = 8.5 Hz), 8.46 (d, 2H, J = 8.5 Hz), 8.42 (d, 1H, J = 6.0 Hz), 8.36 (d, 2H, J = 7.0 Hz), 8.30 (d, 4H, J = 6.5 Hz), 8.07 (d, 1H, J = 8.0 Hz), 8.01 (dd, 2H, J = 8.5, J = 8.0 Hz), 7.84 (d, 1H, J = 8.0 Hz), 7.72 (d, 1H, J = 7.5 Hz), 7.67 (d, 2H, J = 5.5 Hz), 7.57 (d, 2H, J = 5.0 Hz), 7.48 (t, 2H, J = 8.0 Hz), 1.97 (s, 6H), 1.91 (s, 6H). ¹³C NMR (DMSO-d₆, 125 MHz): δ 168.05, 166.81, 153.92, 153.72, 151.10, 150.99, 148.81, 147.61, 142.00, 141.42, 140.77, 140.51, 138.27, 136.73, 135.70, 133.52, 130.83, 130.78, 130.24, 129.93, 129.57, 129.08, 127.95, 127.58, 127.46, 127.16, 126.88, 126.56, 122.21, 26.09, 24.52. IR (KBr, cm⁻¹): 3052.3, 1623.0, 1589.7, 1508.9, 1495.9, 1421.5, 1357.5, 1301.1, 1215.8, 830.0, 812.4, 779.8, 729.3, 622.8. ESI-MS (CH₃CN): m/z 1050.2 ([M − 2ClO₄ − H]⁺), 525.6 ([M − 2ClO₄]²⁺).

4.3. In vitro cytotoxicity assays

MTT assay procedures were used.⁵⁴ Cells were placed in 96-well microassay culture plates (8 × 10⁴ cells per well) and grown overnight at 37 °C in a 5% CO₂ incubator. The tested complexes 1–4 were then added to the wells to achieve final concentrations ranging from 10⁻⁶ to 10⁻⁴ M. Control wells were prepared by addition of culture medium (100 μL). The plates were incubated at 37 °C in a 5% CO₂ incubator for 48 h. Upon completion of the incubation, stock MTT dye solution (20 μL, 5 mg mL⁻¹) was added to each well. After 4 h, buffer (100 μL) containing N,N-dimethylformamide (50%) and sodium dodecyl sulfate (20%) was added to solubilize the MTT formazan. The optical density of each well was then measured with a microplate spectrophotometer at a wavelength of 490 nm. The IC₅₀ values were calculated by plotting the percentage viability versus concentration on a logarithmic graph and reading off the concentration at which 50% of cells remained viable relative to the control. Each experiment was repeated at least three times to obtain the mean values.

4.4. Apoptosis assay by AO/EB and Hoechst 33258 staining methods

A549 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 FBS and incubated at 37 °C in a 5% CO₂. The medium was removed and replaced with medium (final DMSO concentration 0.05% v/v) containing complexes 1–4 (3.13 μM) 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 (100 μg mL⁻¹ AO, 100 μg mL⁻¹ EB) or Hoechst 33258 (10 μg mL⁻¹ in PBS) for 10 min. Then the cells were imaged under 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. A549 cells in culture medium were incubated with 3.13 μM of complexes 1–4 at 37 °C for 24 h. 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 has been fixed. 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 has been fixed. A third coating of 50 μL of 0.5% low melting agarose was placed on the gel and allowed to place 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. After the removal of the lysis solution, the slides 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 the buffer solution, and the DNA was allowed to unwind for 30 min in the electrophoresis solution. Then the electrophoresis was carried out at 25 V and 300 mA for 20 min. After electrophoresis, the slides were removed, and washed thrice in a neutralization buffer (400 mM Tris, HCl, pH 7.5). Nuclear DNA was stained with 20 μL of EtBr (20 μg mL⁻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 fluorescence microscopy.

4.6. Cellular uptake

A549 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. Different concentrations of complexes 1–4 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 discarding the culture medium, the cells were visualized under fluorescent microscope.

4.7. Reactive oxygen species (ROS) levels studies

A549 cells were seeded into six-well plates (Costar, Corning Corp, New York) at a density of 1 × 10⁶ cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 medium supplemented with 10% of 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 3.13 μM of complexes 1–4 for 24 h. The medium was removed again. The fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) was added 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 was imaged under fluorescent microscope at an excitation wavelength of 488 nm and emission at 525 nm. The fluorescent intensity was determined with flow cytometry.

4.8. Mitochondrial membrane potentials assay

A549 cells were treated with complexes 1–4 for 24 h in 12-well plates and were then washed three times with cold PBS. The cells were then detached with trypsin–EDTA solution. Collected cells were incubated for 20 min with 1 μg mL⁻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 then imaged under fluorescence microscope. The ratio of red/green fluorescent intensity was determined with flow cytometry.

4.9. Apoptosis assay by flow cytometry

After chemical treatment, 1 × 10⁶ cells were harvested, washed with PBS, fixed with 70% ethanol, and finally maintained at 4 °C for at least 12 h. The pellets were stained with a fluorescent probe solution containing 50 μg mL⁻¹ propidium iodide and 1 mg mL⁻¹ annexin V in PBS on ice in the dark for 15 min. The fluorescence emission was measured at 530 nm using 488 nm excitation with a FACSCalibur flow cytometry (Beckman Dickinson & Co., Franklin Lakes, NJ). A minimum of 10 000 cells were analyzed per sample.

4.10. Cell cycle arrest by flow cytometry

A549 cells were seeded into six-well plates (Costar, Corning Corp, New York) at a density of 1 × 10⁶ cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS and incubated at 37 °C and 5% CO₂. The medium was removed and replaced with medium (final DMSO concentration 0.05% v/v) containing complexes 1–4 (3.13 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 the mixtures were incubated at 37 °C for 30 min. The samples were then analyzed with a FACSCalibur flow cytometry. The number of cells analyzed for each sample was 10 000.⁵⁵

4.11. Autophagy induced by the complexes

A549 cells were seeded onto chamber slides in 12-well plates 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 (3.13 μM) for 24 h. The medium was removed again, and the cells were washed with ice-cold PBS twice. Then the cells were stained with MDC solution (50 μM) for 10 min and washed with PBS twice. The cells were observed and imaged under fluorescence microscope. The effect of the complexes on the expression of LC3 protein was assayed by western blot.

4.12. Matrigel invasion assay

The BD Matrigel invasion chamber was used to investigate the cell invasion according to the manufacturer's instructions. SGC-7901 cells (4 × 10⁴) in serum free media and different concentration of the complex were seeded in the top chamber of the two chamber Matrigel system. RPMI-1640 (20% FBS) was added as chemo-attractant into the lower chamber. Cells were allowed to invade for 24 h. After incubation, non-invading cells were removed from the upper surface and cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% of crystal violet. Membranes were photographed and the invading cells were counted under a light microscope. Mean values from three independent assays were calculated.

4.13. Western blot analysis

A549 cells were seeded in 3.5 cm dishes for 24 h and incubated with different concentrations of complex 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 (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) buffer for 1 h. The membranes were incubated with primary antibodies at 1 : 5000 dilutions in 5% non-fat milk overnight at 4 °C, and after washed for four times with TBST for a total of 30 min, then the secondary antibodies conjugated with horseradish peroxidase at 1 : 5000 dilution for 1 h at room temperature and washed for four times with TBST. The blots were visualized with the Amersham ECL Plus western blotting detection reagents according to the manufacturer's instructions. To assess the presence of comparable amount of proteins in each lane, the membranes were stripped finally to detect the GAPDH.

4.14. BSA binding experiments

All BSA solutions were prepared in the 5 mM Tris–HCl/10 mM NaCl buffer to keep pH value constant (pH = 7.4). The BSA stock solution was stored at 4 °C in the dark and used within 2 h. The interaction studies of BSA with the Ru(II) complex were performed using absorption titration experiment at room temperature. A 3.0 mL of BSA solution (1 × 10⁻⁵ M) was titrated by successive additions of stock solutions of the Ru(II) complex and changes in the BSA absorption were recorded after each addition.

In the fluorescence quenching experiment, quenching of BSA was performed by taking a fixed concentration of the BSA solution (1 × 10⁻⁶ M) with increasing amounts of the Ru(II) complex. The fluorescence spectra were recorded at an excitation wavelength of 290 nm and emission was observed between 308 and 460 nm after each addition of the quencher and results were analyzed.

5. Data analysis

All data was expressed as means ± SD. Statistical significance was evaluated by a t-test. Differences were considered to be significant when a *P value was less than 0.05.

Acknowledgements

This work was supported by the Natural Science Foundation of Guangdong Province (No. 2016A030313728) and the High-Level Personnel Project of Guangdong Province in 2013.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11391e

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