Cytotoxicity,Cell Cycle Arrest,Antioxidant Activity and Interaction of Dibenzoxanthenes Derivatives with DNA

Abstract

In this study, we report the DNA interaction and cytotoxicity of four dibenzoxanthene compounds 1–4. The binding behaviors of these compounds to calf thymus DNA were studied by absorption titration, viscosity measurements. The DNA binding constants of compounds 1, 2, 3, and 4 are 5.05×10⁴, 2.13×10³, 5.10×10⁴, and 3.03×10³ M⁻¹, respectively. The lipophilicity of the compounds was determined by the shake flask method. The cytotoxicity of these compounds has been assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These compounds exhibit high activity against BEL-7402, Hela, MG-63, and SKBR-3 cells. The cell cycle arrest was analyzed by flow cytometry. These compounds inhibit S phase of BEL-7402 and SKBR-3 cells. The experiments on antioxidant activity show that these compounds exhibit good antioxidant activity against hydroxyl radical (^•OH).

Introduction

X anthenes and benzoxanthenes with active heterocycles have been extensively studied (Bhattacharya and Rana, 2007; Dabiri et al., 2008; Gong et al., 2009; Kumar et al., 2010). These heterocycles display useful spectroscopic properties and are used as dyes (Bhowmik and Ganguly, 2005) and fluorescent materials for the visualization of biomolecules (Bekaert et al., 1992). Additionally, they also show important biological activity. Some evidence suggests that the xanthene core structure also may be useful in the design and development of pharmacological agents (Chibale et al., 2003; Bhattacharya, et al., 2009). Naya studied the structure–activity relationships of a series of 2,9-disubstituted xanthenes as chemokine receptor (CCR1) antagonists (Naya et al., 2001). Ornstein reported a 9-xanthylmethyl amino acid that was certified to be a selective and potent antagonist of the metabotropic glutamate receptor (Ornstein et al., 1998). David and Ferguson described the substituted xanthenes to inhibit cancer cell growth (Goodell et al., 2006; Giri et al., 2010). These xanthenes showed high cytotoxicity in vitro. To explore the relation between DNA-binding and the biological effect of benzoxanthenes derivatives in this article, we investigate the DNA-binding properties and bioactivities of the four dibenzoxanthenes compounds: mddx (mddx=13c-methoxy-1-oxo-1,13c-dihydro-dibenzo[a,kl]xanthene) 1, dbmddx (dbmddx=5,11-dibromo-13c-methoxy-1-oxo-1,13c-dihydro-dibenzo[a,kl]xanthene) 2 (Tan et al., 2001), dheddx (dheddx=2,8-dihydromethyl-13c-ethoxy-1-oxo-1,13c-dihydro-dibenzo[a,kl]xanthene) (Wang et al., 2006), and hhddx (hhddx=2-hydromethyl-13c-hydroxy-1-oxo-1,13c-dihydro-dibenzo[a,kl]xanthene, Fig. 1) 4. Their DNA-binding behaviors were studied by electronic absorption titration and viscosity measurements. The lipophilicity of the compounds was determined by the shake flask method. The cytotoxicity of these compounds has been evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The cell cycle arrest was analyzed by flow cytometry. The antioxidant activity of these compounds against hydroxyl radical (^•OH) was also explored in vitro.

FIG. 1.

The structures of the compounds 1–4.

Materials and Methods

Calf thymus DNA (CT DNA) was obtained from the Sino-American Biotechnology Company. Dimethyl sulfoxide (DMSO) was purchased from Sigma. Cell lines of BEL-7402 (hepatocellular), Hela (Human epithelial carcinoma), MG-63 (Human osteosarcoma), and SKBR-3 cells (Human breast cancer) were purchased from American Type Culture Collection. 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 (Reichmann et al., 1954). The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M⁻¹ cm⁻¹) at 260 nm (Chaires et al., 1982). UV/Vis spectra were recorded on a Shimadzu UV-3101PC spectrophotometer at room temperature.

Physical measurements

¹H NMR and ¹³C NMR spectra were recorded on a Varian-300 spectrometer. All chemical shifts were given relative to tetramethylsilane. Electrospray mass spectra (ES-MS) was recorded on a LCQ system (Finnigan MAT) using methanol as a mobile phase. Microanalysis was carried out with a Perkin-Elemer 240Q elemental analyzer.

Synthesis of compound 4

To a stirred solution of CuCl₂ (0.350 g, 2 mmol) and ethanolamine (0.120 g, 2 mmol) in 15 mL, MeCN was added with 3-hydroxymethyl-2-binaphthol (0.316 g, 0.1 mmol) at 60°C. The mixture was stirred for 15 h, the reaction was quenched with 5% NH₃·H₂O and the mixture was extracted with EtOAc. The organic extract was washed with water and dried over anhydrous Na₂SO₄. The solvent was evaporated and the crude product was purified by column chromatography on a neutral alumina (EtOAc-petroleum ether, 1:2 v:v). Compound 4 as yellow powder was obtained. Yield: 70%. ¹H NMR (Acetone-d₆) δ: 8.32–8.10 (m, 1H), 7.95 (d, J=8.7 Hz, 1H), 7.86–7.83 (m, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.40–7.33 (m, 3H), 7.21–7.16 (m, 1H), 7.20 (s, 1H), 7.09 (d, J=8.4 Hz, 1H), 6.01 (s, 1H), 4.64∼4.54 (m, 2H), 4.26 (s, 1H). ¹³C NMR (Acetone-d₆) δ: 200.7, 150.3, 149.6, 139.3, 134.2, 133.5, 132.7, 131.6, 131.2, 129.3, 128.4, 128.3, 126.5, 125.6, 124.6, 124.2, 117.8, 117.2, 116.3, 112.7, 59.4. IR (KBr, cm⁻¹) ν: 3336, 2924, 2860, 1704, 1627, 1581, 1513, 1458, 1347, 1271, 1231, 1166, 1049, 815, 752. ESI-MS, m/z: 313 ([M⁺-HOH). Anal. Calcd. For C₂₁H₁₄O₃: C, 80.24; H, 4.49. Found: C, 79.86; H, 4.63%.

DNA-binding studies

The DNA-binding experiments were performed at room temperature. The absorption titrations of the compounds in a buffer were performed using a fixed concentration (5–20 μM) for compounds to which increments of the DNA stock solution were added. Compound–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 aromatic band with increasing concentration of DNA using the following equation (Wolf et al., 1987): \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*}[DNA] / ( \varepsilon_a - \varepsilon_f ) = [ DNA ] / ( \varepsilon_b - \varepsilon_f ) + 1 / K_b ( \varepsilon_b - \varepsilon_f ) \tag{1} \end{align*}\end{document}

where [DNA] is the concentration of DNA in base pairs, ɛ_a , ɛ_f , and ɛ_b correspond to the apparent absorption coefficient Aobsd/[compound], the extinction coefficient for the free compound and the extinction coefficient for a compound in the fully bound form, respectively. In plots of [DNA]/(ɛ_a −ɛ_f ) versus [DNA], K _b is given by the ratio of slope to the intercept.

Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a constant temperature at 25.0 (±0.1)°C in a thermostatic bath. DNA samples ∼200 base pairs in average length were prepared by sonication to minimize complexities arising from DNA flexibility (Chaires et al., 1982). Flow time was measured with a digital stopwatch, and each sample was measured three times, and an average flow time was calculated. Relative viscosities for DNA in the presence and absence of compounds were calculated from the relation η=(t−t₀)/t₀, where t is the observed flow time of the DNA-containing solution and t₀ is the flow time of the buffer alone (Satyanarayana et al., 1992, 1993). Data were presented as (η/η₀)^1/3 versus a binding ratio (Cohen and Eisenberg, 1969), where η is the viscosity of DNA in the presence of compounds and η₀ is the viscosity of DNA alone.

Partition coefficient determination

The lipophilicity of the compounds was determined by the shake flask method using the phosphate buffer (0.129 M NaCl, pH=7.4) and n-octanol as the solvent (Graham, 1995). The octanol and buffer solution were presaturated with each other before use. The compound was dissolved in n-octanol. 5 mL of the above solution and 5 mL of the buffer solution were added in 25-mL round-bottomed flask. The mixture was shaken vigorously for 24 h, and then the organic phase and the buffer solution were separated by centrifugation (2500 rpm, 10 min). Each phase was measured by the UV/Vis spectrophotometer and compared with a standard curve to obtain the concentration of compound in two phases. Experiments were performed in triplicate. The partition coefficients P were calculated from the ratio Coct/C_PBS, where Coct and C_PBS were the concentrations of the compound in the n-octanol and in the phosphate buffer solution, respectively. Equilibration and absorption measurements were made at room temperature (Angeles-Boza et al., 2006).

In vitro cytotoxicity assay

Cytotoxicity of compounds 1–4 toward tumor cells was evaluated by the standard MTT method. Cells were placed in 96-well microassay culture plates (1×10⁴ cells per well) and grown overnight at 37°C in a 5% CO₂ incubator. Compounds tested were dissolved in DMSO and diluted with RPMI 1640 to the required concentrations before use. Control wells were prepared by addition of a culture medium (100 μL). Wells containing the 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, a stock MTT dye solution (20 μL, 5 mg mL⁻¹) was added to each well. After 4 h incubation, a 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 cell viability versus concentration on a logarithmic graph and reading off the concentration at which 50% of cells remaining 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, MG-63, and SKBR-3 cells.

Flow cytometric analysis

BEL-7402 and SKBR-3 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% fetal bovine serum and incubated at 37°C and 5% CO₂. The medium was removed and replaced with a medium (final DMSO concentration, 1% v/v) containing compounds (25 μM). After incubation for 24 h, the cell layer was trypsinized and washed with cold phosphate-buffered saline (PBS) and fixed with 70% ethanol. About 20 mL of RNAse (0.2 mg/mL) and 20 mL of propidium iodide (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 (Becton Dickinson & Co.). The number of cells was 10,000 (Lo et al., 2008).

Scavenger measurements of ^•OH

The ^•OH in aqueous media was generated by the Fenton system (Li et al., 2008). The solution of the tested compounds was prepared in DMF. The 4 mL of assay mixture contained following reagents: safranin (28.5 μM), EDTA–Fe(II) (100 μM), H₂O₂ (44.0 μM), the tested compounds (0.5–3.5 μM) and a phosphate buffer (67 mM, pH=7.4). The assay mixtures were incubated at 37°C for 30 min in a water bath. After which, the absorbance was measured at 520 nm. All the tests were run in triplicate and expressed as the mean. A_i was the absorbance in the presence of the tested compound; A₀ was the absorbance in the absence of tested compounds; A_c was the absorbance in the absence of tested compound, EDTA–Fe(II), H₂O₂. The suppression ratio (η_a) was calculated on the basis of (A_i−A₀)/(A_c−A₀)×100%.

Results

Electronic absorption spectra studies

Due to the intercalative mode involving a stacking interaction between the aromatic chromophore and the DNA base pairs, the large change in absorbance can be observed, while a compound binds to DNA through intercalation. Figure 2 shows the absorption spectra of compound 4 in the presence of increasing concentration of DNA.

FIG. 2.

Absorption spectra of the compound 4 in the Tris-HCI buffer upon addition of calf thymus DNA (CT DNA) [comp 4]=15 μM. Arrows show the absorbance change upon increase of DNA concentration. Inset: Plots of [DNA]/(ɛ_a−ɛ_f) versus [DNA] for the titration of DNA with the compound.

Viscosity measurements

The relative change in viscosity was measured using CT DNA with increasing concentrations of the compounds 1–4. The effects of compounds on the relative viscosity of rod-like DNA are shown in Figure 3. On increasing the concentration of compounds 1–4, the relative viscosity of the DNA increased steadily.

FIG. 3.

Relative viscosity changes of solutions containing 200 μM CT DNA in the presence of different concentration of compounds 1–4.

Partition coefficient determination

The partition coefficient, P, is defined as the ratio of the concentration of the compound in octanol and water phases. The logarithm of the partition coefficient, log P, is most commonly used. The shake flask method used during the course of these studies has been shown to work well for molecules with log P values that range from −2 to +4. Octanol is the preferred partitioning solvent for most studies since, with its hydrophobic tail and polar head group, this liquid alcohol serves as a simplified, but well-established, membrane model. The log P of compounds 1–4 was measured by the shake flask method. The values obtained for the members of this series are show in Table 1.

Table 1.

The Log P and IC ₅₀ Values for Compounds 1–4

		IC₅₀ (μM)
Compounds	Log P ^a	BEL-7402	Hela	MG-63	SKBR-3
1	1.338	17.6	18.5	20.1	15.5
2	1.622	10.1	9.4	25.2	6.2
3	0.924	42.3	28.1	39.2	14.3
4	0.936	45.2	68.8	54.8	26.2
Cisplatin	—	19.8^b	16.9^b	—	6.8^c

Partition coefficient P=Coct/C_PBS (Coct and C_PBS are the compound concentrations in n-octanol and phosphate buffer solution, respectively).

Data from Huang et al.(2011).

Data from Cheng et al. (2009).

In vitro cytotoxicity

The cytotoxicity in vitro for compounds was assessed using the method of MTT reduction. After treatment of BEL-7402, Hela, MG-63, and SKBR-3 cell lines with different concentrations of compounds 1–4 (3.125→200 μM) for 48 h, the inhibitory percentage against growth of cancer cells was determined. The cell viability (%) with continuous exposure for 48 h is depicted in Figure 4. The IC₅₀ were calculated and listed in Table 1.

FIG. 4.

Cell viability of compounds 1–4 on tumor BEL-7402 (a), Hela (b), MG-63 (c), and SKBR-3 (d) cell proliferation in vitro. Each data point is the mean±standard error obtained from at least three independent experiments.

Flow cytometric analysis

The effect of compounds on cell cycle of BEL-7402 and SKBR-3 cells was investigated by flow cytometry in PI (propidiumiodide)-stained cells after compounds 1–4 treatment for 24 h. Figure 5 showed the representative DNA distribution histograms of BEL-7402 and SKBR-3 cells in the absence (A for BEL-7402 and C for SKBR-3) and presence of compounds 1–4 (B for compound 1+BEL-7402, D for compound 2+SKBR-3, E for compound 3+SKBR-3, and F for compound 4+SKBR-3).

FIG. 5.

Cell cycle status of the BEL-7402 cell after treatment with compound 1 (50 μM) and the SKBR-3 cell after treatment with compounds 2–4 (50 μM) for 24 h. (A) untreatment of BEL-7402, (B) compound 1+BEL-7402, (C) untreatment of SKBR-3, (D) compound 2+SKBR-3, (E) compound 3+SKBR-3, and (F) compound 4+SKBR-3.

Antioxidant activity against ^•OH

Oxidative damage to DNA has been suggested to contribute to aging and various diseases, including cancer and chronic inflammation. The antioxidant activity of compounds 1–4 was investigated. The inhibitory effect was depicted in Figure 6 . And the suppression ratio is listed in Table 2. As seen in Table 2, the average suppression ratio valued from 3.69% to 71.01% for compound 1, 11.73% to 79.73% for compound 2, 11.19% to 75.91% for compound 3, and 17.96% to 76.06% for compound 4.

FIG. 6.

Scavenging effect of the compounds 1–4 on hydroxyl radicals. Experiments were performed in triplicate.

Table 2.

Scavenging Ratio (%) of Compounds 1–4 Against ^• OH

	Average inhibition (%) for ^•OH
Compounds	0.5	1.0	1.5	2.0	2.5	3.0	3.5 (μM)
1	3.69	7.85	28.75	30.72	40.79	49.63	71.01
2	11.73	33.07	51.73	60.27	67.20	73.87	79.73
3	11.19	23.60	54.26	63.50	68.86	69.83	75.91
4	17.96	37.91	53.86	62.84	68.83	73.32	76.06

•

OH, hydroxyl radical.

Discussion

The band at 256 nm for compound 4 exhibit hypochromism of about 45.5% (71.54% for 1, 22.56% for 2, and 17.58% for 3) and bathochromism 2 nm for 1, 3 nm for 2, 3 nm for 3, and 2 nm for 4. The intrinsic constants K _b were determined by monitoring the changes in absorbance at the band (250 nm for 1, 253 nm for 2, 252 nm for 3, and 256 nm for 4) with increasing the concentration of DNA. The values of K _b are derived to be 5.05×10⁴, 2.13×10³, 5.10×10⁴, and 3.03×10³ M⁻¹ for compounds 1, 2, 3, and 4, respectively.

The viscosity of a DNA solution was sensitive to the addition of organic drugs bound by intercalation. And relative viscosity measurements have proved to be a reliable method for the assignment of the mode of binding compounds to DNA. In general, intercalation of a ligand into DNA is known to cause a significant increase in the viscosity of a DNA solution due to an increase in the separation of the base pairs at the intercalation site and, hence, an increase in the overall DNA molecular length; A partial and/or nonclassical intercalation of ligand could bend (or kink) the DNA helix, reduces its effective length and, concomitantly, its viscosity (Waring, 1965; Satyanarayana et al., 1993). With increasing concentrations of compounds 1–4, the DNA solution viscosity increases gradually and follows the order of 1, 4>2, 3.

The logarithm of the octanol–water partition coefficient, log P, is a key physicochemical property for bioactive molecules. It provides valuable information for the overall understanding of the uptake, distribution, biotrasformation, and degradation of drugs (Sangster, 1997). Most bioactive molecules are nowadays designed to have log P values in the range of 1–4. Partition coefficients P were used to evaluate the abilities of compound passing through the biological membrane (Graham, 1995). Comparing the partition coefficients of these compounds (Table 1), the value of log P for compound 2 is largest among the four compounds.

The cytotoxicity of compounds was found to be concentration-dependent. The cell viability decreased with increasing the concentrations of compounds 1–4 (Fig. 4). Comparing the IC₅₀ values of compounds 1–4, compound 2 appeared to be more active against BEL-7402, Hela, and SKBR-3 cell lines than other compounds. The presence of two bromine atoms can enhance the cytoxicity activity of compound 2. Compared 2 with cisplatin (Huang et al., 2011; Cheng et al., 2009), compound 2 showed high cytotoxicity against BEL-7402, Hela, and SKBR-3 cells. The differences in cytotoxicity of compounds 1–4 may be caused by a different log P value. These results showed that the cytotoxic activity of compounds 1–4 is not consistent with their DNA-binding affinities.

After treatment of the BEL-7402 cell with the compound 1, an obvious increase of 5.19% in the percentage of cells at S phase, accompanied by a corresponding reduction in the percentage of cells in the G₀/G₁ phase was observed, indicating the induction of S phase arrest by compound 1. Treatment of the SKBR-3 cell with compounds 2–4, a large increase of 17.12% for 3, 4.71% for 2, and 9.08% for 4 in the percentage of cells at S phase and a corresponding reduction at the G₀/G₁ phase was also observed, which suggested the antiproliferative mechanism on SKBR-3 for compounds 2–4 were also S phase arrest (Fig. 5).

Among all reactive oxygen species, the ^•OH is by far the most potent, and therefore the most dangerous oxygen metabolite; elimination of this radical is one of the major aims of antioxidant administration (Udilova et al., 2003). The antioxidant activity against ^•OH of compounds 1–4 is comparable under the same experimental condition. Compounds 2–4 with substitute groups have higher antioxidant activity than compound 1. The inhibitory effects of compounds on ^•OH were concentration-dependent, and the suppression ratio increased with the increasing sample concentration in the range of 0.5–3.5 μM. The suppression ratio against ^•OH varied from 3.69% to 70.00% for 1, 11.73% to 79.73% for 2, 11.19% to 75.91% for 3, and 17.95% to 76.06% for 4. Compared with 7-methoxychromone-3-carbaldehyde-isonicotinoyl hydrazone (Wang et al., 2009), compounds 1–4 showed high antioxidant activity. The results showed that compounds 1–4 at 3.5 μM can effectively eliminate the ^•OH.

Conclusion

Four dibenzoxanthenes were synthesized and studied on interaction with DNA and cytotoxicity in vitro. The DNA-binding behaviors show that compounds interact with CT-DNA through an intercalative mode. The cytotoxicity assay indicates that compounds 1–4 can inhibit the BEL-7402, Hela, MG-63, and SKBR-3 cells proliferation. The cytotoxicity of compounds 1–4 is not consistent with their DNA-binding affinities. The flow cytometric analysis shows the treatment of BEL-7402 and SKBR-3 cells with compounds 1–4 indicates the induction of S phase arrest. Antioxidant activity shows that the four compounds may be potential drugs to eliminate the hydroxyl radical.

Footnotes

Acknowledgments

This work was supported by the National Nature Science Foundation of China (No 30800227, 31070858) and the Guangdong Pharmaceutical University for financial supports.

Disclosure Statement

No competing financial interests exist.

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Cytotoxicity,Cell Cycle Arrest,Antioxidant Activity and Interaction of Dibenzoxanthenes Derivatives with DNA

Abstract

Introduction

Materials and Methods

Physical measurements

Synthesis of compound 4

DNA-binding studies

Partition coefficient determination

In vitro cytotoxicity assay

Flow cytometric analysis

Scavenger measurements of •OH

Results

Electronic absorption spectra studies

Viscosity measurements

Partition coefficient determination

In vitro cytotoxicity

Flow cytometric analysis

Antioxidant activity against •OH

Discussion

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

References

Scavenger measurements of ^•OH

Antioxidant activity against ^•OH