Cytotoxicity,Apoptosis,Cellular Uptake,Cell Cycle Distribution,and DNA-Binding Investigation of Ruthenium Complexes

Synthesis of BHIP (2-(3-bromo-4-hydroxyphenyl)[4,5]imidazo[1,10]phenanthroline)

A mixture of 1,10-phenanthroline-5,6-dione (0.315 g, 1.5 mmol) (Yamada et al., 1992), 3-bromo-4-hydroxyphenylaldehyde (0.302 g, 1.5 mmol), ammonium acetate (2.31 g, 30 mmol), and glacial acetic acid (30 mL) was refluxed with stirring for 2 h. The cooled solution was diluted with water and neutralized with concentrated aqueous ammonia. The precipitate was collected and purified by column chromatography on silica gel (60–100 mesh) with ethanol as eluent to give the compound as yellow powder. Yield: 80%. Anal. Calcd. for C₁₉H₁₁N₄BrO: C, 58.33; H, 2.83; N, 14.32; Found: C, 58.18; H, 2.95; N, 14.47%. Fast atom bombardment mass spectra (FAB-MS): m/z=392.3 [M+1]⁺. ¹H NMR (500 MHz, dimethyl sulfoxide [DMSO]–d₆ ): 9.92 (d, 2H, J=6.0 Hz), 8.89 (d, 2H, H_i, J=8.0 Hz), 8.41 (d, 1H, J=2.5 Hz), 7.22 (d, 1H, J=6.5 Hz), 7.82 (dd, 2H, J=4.5, J=4.5 Hz), 7.15 (d, 1H, J=8.0 Hz), 3.40 (s, 1H, H_O-H).

Synthesis of [Ru(bpy)₂(BHIP)](ClO₄)₂ (1)

A mixture of cis-[Ru(bpy)₂Cl₂]·2H₂O (0.260 g, 0.5 mmol) (Sullivan et al., 1978) and BHIP (0.196 g, 0.5 mmol) in ethanol (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 a neutral alumina oxide with a mixture of CH₃CN-toluene (3:1, v/v) as eluent. The red band was collected. The solvent was removed under reduced pressure and a red powder was obtained. Yield: 70%. Anal. Calcd. for C₃₉H₂₇BrCl₂N₈O₉Ru: C, 46.68; H, 2.71; N, 11.17; Found: C, 46.52; H, 2.55; N, 11.05%. ESI-MS [CH₃CN, m/z]: 803.5 ([M–2ClO₄–H]⁺), 402.3 ([M–2ClO₄]²⁺). ¹H NMR (500 MHz, DMSO-d₆ ): δ 9.10 (d, 2H, J=8.1 Hz), 8.85 (dd, 4H, J=8.2, J=8.2 Hz), 8.50(d, 1H, J=5.5 Hz), 8.17–8.23 (m, 4H), 8.08 (t, 2H, J=6.64 Hz), 7.91 (d, 2H, J=4.6 Hz), 7.80–7.85 (m, 4H), 7.58 (d, 4H, J=5.6 Hz), 7.35 (t, 1H, J=6.0 Hz), 7.10 (d, 1H, J=8.5 Hz), 3.38 (s, 1H, H_O-H).

Synthesis of [Ru(phen)₂(BHIP)](ClO₄)₂ (2)

This complex was synthesized in a manner identical to that described for complex 1, with cis-[Ru(phen)₂Cl₂]·2H₂O (0.280 g, 0.5 mmol) (Sullivan et al., 1978) in place of cis-[Ru(bpy)₂Cl₂]·2H₂O. Yield: 71%. Anal. Calcd. for C₄₃H₂₇BrCl₂N₈O₉Ru: C, 49.11; H, 2.59; N, 10.66; Found: C, 49.01; H, 2.46; N, 10.45%. ESI-MS [CH₃CN, m/z]: 851.4 ([M–2ClO₄–H]⁺), 426.5 ([M–2ClO₄]²⁺). ¹H NMR (500 MHz, DMSO-d₆): δ 9.09 (d, 2H, J=7.7 Hz), 8.76 (dd, 4H, J=7.3, J=7.4 Hz), 8.50 (d, 1H, J=6.0 Hz), 8.37 (s, 4H), 8.23 (d, 1H, J= 6.9 Hz), 8.07–8.11 (m, 4H), 7.87 (d, 2H, J=4.8 Hz), 7.73–7.77 (m, 4H), 7.68–7.71 (m, 2H), 7.10 (d, 1H, J=8.5 Hz), 3.37 (s, 1H, H_O-H).

Synthesis of [Ru(dip)₂(BHIP)](ClO₄)₂ (3)

This complex was synthesized in a manner identical to that described for complex 1, with cis-[Ru(dip)₂Cl₂]·2H₂O (0.418 g, 0.5 mmol) (Caspar et al., 2006) in place of cis-[Ru(bpy)₂Cl₂]·2H₂O. Yield: 71%. Anal. Calcd. for C₆₇H₄₃BrCl₂N₈O₉Ru: C, 59.35; H, 3.20; N, 8.26; Found: C, 59.24; H, 3.09; N, 8.41%. ESI-MS [CH₃CN, m/z]: 1156.3 ([M–2ClO₄–H]⁺), 578.5 ([M–2ClO₄]²⁺). ¹H NMR (500 MHz, DMSO-d₆): δ 8.64 (d, 2H, J=7.6 Hz), 8.38 (d, 1H, J=8.8 Hz), 8.34 (d, 4H, J=5.5 Hz), 8.25 (s, 4H), 8.22 (d, 1H, J=5.7 Hz), 8.13 (d, 2H, J=5.0 Hz), 7.85–7.89 (m, 2H), 7.80 (d, 2H, J=5.5 Hz), 7.75 (d, 2H, J=5.6 Hz), 7.56–7.70 (m, 20H), 3.35 (s, 1H, H_O-H).

Physical measurements

CT-DNA was obtained from the Sino-American Biotechnology Company. pBR322 DNA was obtained from Shanghai Sangon Biological Engineering & Services Co., Ltd. DMSO and RPMI 1640 were purchased from Sigma. Cell lines of hepatocellular origin (BEL-7402), Human epithelial carcinoma (Hela), breast cancer (MCF-7), and Human osteosarcoma (MG-63) were purchased from American Type Culture Collection, agarose and ethidium bromide (EB) were obtained from Aldrich. RuCl₃·χH₂O was purchased from Kunming Institution of Precious Metals. 1,10-phenanthroline was obtained from Guangzhou Chemical Reagent Factory. Double distilled water was used to prepare buffers (5 mM Tris(hydroxymethylaminomethane)-HCl, 50 mM NaCl, pH=7.2). A solution of CT-DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.8–1.9:1, indicating that the DNA was sufficiently free of protein (Marmur, 1961). The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M⁻¹ cm⁻¹) at 260 nm (Reichmann et al., 1954).

Microanalysis (C, H, and N) was carried out with a Perkin-Elmer 240Q elemental analyzer. FAB-MS were recorded on a VG ZAB-HS (fast atom bombardment) spectrometer in a 3-nitrobenzyl alcohol matrix. ESI-MS were recorded on a LCQ system (Finnigan MAT) 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. All chemical shifts were given relative to tetramethylsilane. UV/Vis spectra were recorded on a Shimadzu UV-3101PC spectrophotometer and emission spectra were recorded on a Shimadzu RF-4500 luminescence spectrometer at room temperature.

DNA binding and photoactivated cleavage

The DNA-binding and photoactivated cleavage experiments were performed at room temperature. Buffer A (5 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride, 50 mM NaCl, pH 7.0) was used for absorption titration, luminescence titration, and viscosity measurements. Buffer B (50 mM Tris-HCl, 18 mM NaCl, pH 7.2) was used for DNA photocleavage experiments.

The absorption titrations of the complex in buffer were performed using a fixed concentration (20 μM) for complex to which increments of the DNA stock solution were added. Ru-DNA solutions were allowed to incubate for 5 min before the absorption spectra were recorded. The intrinsic binding constants K, based on the absorption titration, were measured by monitoring the changes in absorption at the metal-to-ligand charge transfer (MLCT) band with increasing concentration of DNA using the following equation (Carter et al., 1989). \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}(\varepsilon_a - \varepsilon_f) / (\varepsilon_b - \varepsilon_f)= ({\rm b} - ({\rm b}^2 - 2{\rm K}^2{\rm C}_t [{\rm DNA}] / {\rm s})) ^{1/2} / 2{\rm KC}_{t} \tag{1a}\end{align*}\end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}( b = 1 + KC_t + K [ DNA ] / 2s ) \tag{1{\rm b}} \end{align*}\end{document}

where [DNA] is the concentration of CT-DNA in base pairs, the apparent absorption coefficients ɛ _a, ɛ _f, and ɛ _b correspond to A _obsed/[Ru], the absorbance for the free ruthenium complex, and the absorbance for the ruthenium complex in fully bound form, respectively. K is the equilibrium binding constant, C _t is the total metal complex concentration and s is the binding site size.

For the gel electrophoresis experiment, supercoiled pBR322 DNA (0.1 μg) was treated with the Ru(II) complexes in buffer B, and the solution was then irradiated at room temperature with a UV lamp (365 nm, 10 W) for 45 min. The samples were analyzed by electrophoresis for 1.5 h at 80 V on a 0.8% agarose gel in TBE (89 mM Tris-borate acid, 2 mM EDTA, pH=8.3). The gel was stained with 1 μg/mL EB and photographed on an Alpha Innotech IS–5500 fluorescence chemiluminescence and visible imaging system.

Cytotoxicity assay

Standard MTT assay procedures were used (Mosmann, 1983). 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 complexes tested were dissolved in DMSO and diluted with RPMI 1640 and 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). Wells containing culture medium without cells were used as blanks. 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 incubation, 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 on a microplate spectrophotometer at a wavelength of 490 nm. The IC₅₀ values were determined by plotting the percentage of 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 get the mean values. Four different tumor cell lines were the subjects of this study: BEL-7402, Hela, MCF-7, and MG-63 (purchased from American Type Culture Collection).

Apoptosis assessment by morphology with acridine orange/EB staining

Apoptosis studies were performed with a staining method utilizing acridine orange (AO) and EB (Spector et al., 1998). According to the difference in membrane integrity between necrotic and apoptosis, AO can pass through cell membrane, but EB cannot. Under fluorescence microscope, live cells appear green. Necrotic cells stain red but have a nuclear morphology resembling that of viable cells. Apoptosis cells appear green, and morphological changes such as cell blebbing and formation of apoptotic bodies will be observed. A monolayer of BEL-7402 cells was incubated in the absence and presence of complex 2 at concentration of 25 μM at 37°C and 5% CO₂ for 24 h. Then, each cell culture was stained with AO/EB solution (100 μg mL⁻¹ AO, 100 μg mL⁻¹ EB). Samples were observed under a fluorescence microscope.

Apoptotic assay by flow cytometry

After chemical treatment, 1×10⁶ cells were harvested, washed with phosphate-buffered saline (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 propidium iodide (PI) and 1 mg/mL annexin in PBS on ice in dark for 15 min. Then the fluorescence emission was measured at 530 nm and 575 nm (or equivalent) using 488 nm excitation by a FACS Calibur flow cytometer (Beckman Dickinson & Co.). A minimum of 10,000 cells were analyzed per sample.

Cellular uptake study

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. Compounds tested were then added to the wells. The plates were incubated at 37°C in a 5% CO₂ incubator for 24 h. Upon completion of the incubation, the wells were washed three times with phosphate buffered saline (PBS), after removing the culture media cells were visualized by fluorescent microscopy.

Cell cycle arrest

BEL-7402 cells were seeded into six-well plates (Costar; Corning Corp.) at a density of 2×10⁵ cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of fetal bovine serum (FBS) and incubated at 37°C and 5% CO₂. The medium was removed and replaced with medium (final DMSO concentration, 1% v/v) containing the three complexes (25 μM). After incubation for 24 h, the cell layer was trypsinized, washed with cold PBS, and fixed with 70% ethanol. Twenty milliliters of RNAse (0.2 mg/mL) and 20 mL of PI (0.02 mg/mL) were added to the cell suspensions and incubated at 37°C for 30 min. Then the samples were analyzed by a FACS Calibur flow cytometer (Beckman Dickinson & Co.). The number of cells analyzed for each sample was more than 10⁴ (Lo et al., 2008).

Electronic absorption titration

The electronic absorption spectra of complexes 1, 2, and 3 consist of two or three resolved bands. The band centered at 455–470 nm was assigned to a MLCT transition, the bands at the range of 300–400 nm are assigned to π–π* transitions, the bands below 300 nm are attributed intraligand (IL) π–π* transitions by comparison with the spectra of the other ruthenium polypyridyl complexes (Liu et al., 2009; Tan et al., 2009). Figure 2 shows absorption spectra obtained at increasing amounts of CT-DNA to complexes 1, 2, and 3. With increasing amounts of added DNA hypochromicity observed at the MLCT band of complex 1 (at 460 nm), 2 (at 458 nm), and 3 (at 466 nm) was 33.1%, 28.7%, and 24.5%, and bathochromism of 3, 7, and 3 nm, respectively.

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FIG. 2.

Absorption spectra of complexes in Tris-HCl buffer upon addition of calf thymus DNA (CT–DNA) in the presence of complexes (a) 1, (b) 2, and (c) 3. [Ru]=20 μM. Arrow shows the absorbance change upon the increase of DNA concentration. Plots of (ɛ _a−ɛ _f)/(ɛ _b−ɛ _f) versus [DNA] for the titration of DNA with Ru(II) complexes.

Luminescence of 1, 2, and 3 in the presence of DNA

Complexes 1, 2, and 3 can luminesce in Tris buffer at room temperature with a maximum appearing at 597, 588, and 600 nm, respectively. The luminescence spectra of these complexes in the presence of increasing amounts of DNA in buffer solution are shown in Figure 3. The emission intensities of complexes 1, 2, and 3 grow by a factor of 4.68, 2.25, and 1.25 times larger than original, respectively.

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FIG. 3.

Emission spectra of complexes 1 (a), 2 (b), and 3 (c) in Tris-HCl buffer in the absence and presence of CT-DNA. Arrow shows the intensity change upon increasing DNA concentration.

Photoinduced cleavage of pBR322 DNA

The cleavage reaction on plasmid DNA induced by complexes 1, 2, and 3 can be monitored by agarose-gel electrophoresis. When circular plasmid DNA is subjected to electrophoresis, relatively fast migration will be observed for the intact supercoiled form (Form I). If scission occurs on one strand (nicking), the supercoil will relax to generate a slower-moving open circular form (Form II). If both strands are cleaved, a linear form (Form III) that migrates between Form I and Form II will be generated (Barton and Raphael, 1984). Figure 4 shows gel electrophoresis separation of pBR322 DNA after incubation with the Ru(II) complexes and irradiation at 365 nm for 45 min. No obvious DNA cleavage was observed for complex-free plasmids (control) or incubation of the plasmid with the Ru(II) complexes in darkness. With increasing concentration of complexes 1, 2, and 3, the amount of Form I of pBR322 DNA diminished, and the amount of Form II gradually increased.

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FIG. 4.

Photoactivated cleavage of pBR322 DNA in the presence of different complexes upon irradiation at 365 nm for 45 min.

Cytotoxicity in vitro assay

Cytotoxic effects of complexes 1, 2, and 3 were observed after incubating BEL-7402, Hela, MCF-7, and MG-63 cells for 48 h with complexes 1, 2, and 3 in the dose range of 6.25–400 μM. These complexes induced cell death in a dose-dependent manner. The IC₅₀ values for 1, 2, and 3 are listed in Table 1. The cell viabilities are depicted in Figure 5.

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FIG. 5.

Cell viability of complexes 1, 2, and 3 on BEL-7402 (a), Hela (b), MCF-7 (c), and MG-63 (d) cells proliferation in vitro. Each point is obtained from three independent experiments with the mean±standard error.

Complex-induced apoptotic cell death in BEL-7402 cells

To determine the percentage of the apoptosis and necrosis of BEL-7402 cells, the cells were treated with complexes 1, 2, and 3 for 24 h followed by cell apoptosis analyses using flow cytometry. The percentage of living, apoptotic and necrotic cells is shown in Figure 6.

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FIG. 6.

The percentage of living (L), necrotic (N), and apoptotic (A) ruthenium complex-treated BEL-7402 cells as analyzed by fluorescence-activated cell sorting (FACS) calibur flow cytometry. Control (a), 24 h exposed to 25 μM complex 1 (b), 2 (c), and 3 (d).

To confirm whether treatment with complex 2 resulted in apoptotic cell death, the morphologic changes of the cells were examined by fluorescence microscopy ultilizing AO and EB staining method. AO can pass through the cell membrane, but EB cannot. Under fluorescence microscopy, living and apoptotic cells appear green. For apoptotic cells, the morphological changes such as cell blebbing and formation of apoptotic bodies were observed (Fig. 7).

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FIG. 7.

BEL-7402 cells incubated at 37°C and 5% CO₂ and observed under fluorescence microscopy: Control (A), 24 h exposed to 25 μM complex 2 (B). Cells in a, b, and c are living, apoptotic and necrotic cells, respectively.

Cellular uptake studies

Complexes 1 (25 μM), 2 (25 μM), and 3 (25 μM) were added to the wells (5×10⁻⁴ cells per well). The plates were incubated at 37°C in a 5% CO₂ incubator for 24 h. After completion of the incubation, the culture medium was discarded, and the wells were washed three times with PBS buffer to remove the residual culture medium. Then the cells were observed under fluorescence microscopy. The results are shown in Figure 8.

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FIG. 8.

An image observed by fluoresecnce microscopy after BEL-7402 cells incubated with complexes 1 (25 μM, a), 2 (25 μM, b), and 3 (25 μM, c) for 48 h. Note that the cytoplasm is stained with the Ru(II) complexes.

Cell cycle distribution

Complex 2 was chosen for investigating on the cell cycle arrest as it showed higher cytotoxicity than complexes 1 and 3 toward all cell lines. Induction of apoptosis of cell cycle arrest or a combination of these can inhibit the cancer cell proliferation. After treatment of BEL-7402 cells with complex 2 for 24 h, the cell cycle of BEL-7402 cells was investigated by flow cytometry in PI-stained cells method. Incubation of fixed and permeabilized cells with PI resulted in quantitative PI binding with total cellular DNA, and the fluorescence intensity of PI-labeled cells was proportional to the DNA content (Fig. 9).

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FIG. 9.

Cell cycle distribution of BEL-7402 cells as analyzed by FACS calibur flow cytometry. Control (a), exposed to complexes 1 (25 μM, b), 2 (25 μM, c), and 3 (25 μM, d) for 24 h.