Treatment with dibenzoxanthenes inhibits proliferation and induces apoptosis of HepG2 cells via the intrinsic mitochondrial pathway

Abstract

Dibenzoxanthenes were reported to possess antitumor biological activity. In this study, we synthesized four dibenzoxanthenes analogs (4a–4d) with CN and COOCH₃ groups, and revealed the underlying molecular biological mechanism. MTT assay indicated that compounds showed high cytotoxic activities against HepG2, HeLa, SGC-7901 and A549 cells. These compounds also caused DNA damage and G2/M phase arrest in HepG2 cells. Moreover, compounds induced cell apoptosis, which was associated with ROS generation, and a decrease of the mitochondrial membrane potential. Western blotting showed cytochrome c release, caspase-3 and caspase-9 activation, downregulation of Bcl-2, Bcl-xl and upregulation of Bax, Bid. Molecular location experiments showed that compounds 4a–4d entered the nuclei of cells. A cell scratch test and transwell assays indicated that compounds could suppress HepG2 cell migration and invasion. Taken together, our experiment results suggest that the four dibenzoxanthenes (4a–4d) exert their cytotoxicity on HepG2 cells by inducing ROS-mediated mitochondrial dysfunction apoptosis.

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1. Introduction

Apoptosis is a very important biological program due to its maintenance of cellular homeostasis by regulating cell division and cell death. Cancer may be described as the abnormal growth of cells by uncontrolled proliferation. Therefore, the tumor cell apoptosis which is trigged by cytotoxic drugs is potential tumor treatment.^1–3 At present, apoptotic signals involve two main pathways: the extrinsic or death receptor pathway and intrinsic or mitochondria-regulated pathway.^4–6 The intrinsic pathway is regulated by the anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family members, resulting in translocation of cytochrome c from mitochondria into the cytosol. This activates the formation of apoptosome, a complex composed of cytochrome c, Apaf-1 and procaspase-9. Formation of the apoptosome is followed by the activation of caspase-9, subsequently caspase-9 promotes the activation of caspase-3 and caspase-7, ultimately inducing DNA damage and cell apoptosis.^7–9 Recently, thousands of natural or synthesized organic compounds were identified to have pro-apoptotic merits and were considered as promising candidates for novel cancer therapeutics.^10–12 It is well known that metastatic behavior is highly correlated with prognosis after tumor surgical resection.¹³ Metastasis is responsible for most cancer deaths. Thus, therapeutic strategies that prevent development of metastase may reduce cancer mortality.¹⁴

Benzoxanthenes possess analgesic, anti-inflammatory, antibacterial, and antiviral activities^15–18 and can be used as dyes,¹⁹ pH sensitive fluorescent materials for visualization of biomolecules²⁰ and in laser technologies.²¹ Due to their wide range of interesting biological and therapeutic properties, benzoxanthenes attract the attentions of chemists and pharmaceutical researchers.^22–25 Our previous studies have demonstrated that dibenzoxanthenes are capable of inhibiting cancer cell proliferation and inducing apoptosis of various cancer cell lines.^26–28 It is observed that dibenzoxanthenes promote intracellular reactive oxygen species which mediates multiple cellular responses, including cell cycle progression, apoptotic cell death. Dibenzoxanthenes activate caspase cascade signaling and induce cell apoptosis by alteration in the mitochondria membrane potential, mitochondria function protein (such as Bcl-2, Bcl-xl and Bax) and caspase activity (caspase-3, caspase-7).²⁹

According to analyzing structure–activity relationship of dibenzoxanthenes, we found that substrate binaphthol with substitution group at 6 or/and 6′ position carbon, the formed dibenzoxanthene showed higher cytotoxicity against cancer cells.²⁴ Therefore, in order to discover more potential activity and lower toxicity anti-cancer drugs, we synthesized dibenzoxanthenes, 4a–4d, using the corresponding 6 position monosubstituted or bis-substituted binaphthols, 3a–3d, possessing CN or COOCH₃ group. In this study, we examined the cytotoxicity of compounds in vitro by MTT assay. The apoptosis of cells induced by compounds was studied by fluorescent microscopy and flow cytometry. The reactive oxygen species (ROS), mitochondrial membrane potential and cell cycle arrest were analyzed by flow cytometry. We also investigated the apoptotic pathway by western blot analysis. Moreover, we performed the scratch test and transwell assays to observe the inhibition migration and invasion in tumor cells.

2. Results and discussion

2.1. Synthesis, characterization and structure

The synthetic rout of the target compounds was shown in Scheme 1. Dibenzoxanthene derivatives 4a–4d were prepared in a facile two-step sequences in high yield. The commercially available 6-cyano-2-naphthol took place a self-couple reaction or a crossing-couple reaction with 2-naphthol to afford 6-cyano-dinaphthol 3a or 6,6′-dicyano-dinaphthol 3b. Then, 3a or 3b was oxidated in the present of copper–amine complex to obtain dibenzoxanthene 4a or 4b. As shown in Scheme 1, compounds 4c and 4d were prepared by following similar sequences for the synthesis of 4a and 4b.

Scheme 1 The synthetic route of compounds 4a–4d.

2.2. In vitro cytotoxicity activity

We started our studies with the cytotoxicity of dibenzoxanthene analogs. The antiproliferative activities of compounds 4a–4d were evaluated against four human cancer cell lines, including HepG2 cells line, HeLa cell line, SGC-7901 cell line and A549 cell line. We can find three results from Table 1, (1) the cytotoxicity of four compounds in vitro was higher than that of the other dibenzoxanthenes reported in previous literatures^24–26 (2) Compounds 4a and 4b displayed significant antitumor activities with IC₅₀ values of lower than 7.1 μM, which indicated that the introduction of CN group into 6- or 6′-position, the strong electron-withdrawing group, can obviously enhance the antitumor activities of dibenzoxanthene analogs; however, compounds 4c and 4d showed, in general, weaker antitumor activities than 4a and 4b, which indicated that the presence of COOCH₃ group at 6- or 6′-position, the strong electron-donating group, would decrease the antitumor activities. Therefore, the presence of strong electron-withdrawing group at 6- or 6′-position is favorable for enhancing antitumor activities of dibenzoxanthene molecules. (3) We also examined the cytotoxic activities of four compounds against human normal live cell line LO2. Interestingly, these compounds almost showed lower inhibitory effect against LO2 cell line with IC₅₀ values of 10.43–27.31 μM than that these IC₅₀ values towards tumor cells. These results indicated the compounds 4a–4d were potential antitumor agents. Comparing the IC₅₀ values, the anticancer activities of four compounds against HepG2 cells were higher than other tumor cells. So we chosed the HepG2 cell line to assess the biological properties of compounds in following experiments.

Table 1 The IC₅₀ values for compounds 4a–4d

Compounds	IC50 (μM)
	HepG2	HeLa	SGC-7901	A549	LO2
4a	1.34 ± 0.16	4.23 ± 0.25	5.40 ± 0.17	7.09 ± 0.32	11.29 ± 1.24
4b	1.05 ± 0.26	6.79 ± 0.36	5.39 ± 0.18	4.75 ± 0.18	10.43 ± 0.97
4c	4.63 ± 0.19	5.97 ± 0.35	7.63 ± 0.17	9.33 ± 0.96	13.23 ± 1.06
4d	11.39 ± 1.52	30.12 ± 3.03	10.39 ± 1.13	19.56 ± 1.76	27.31 ± 1.29

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2.3. DNA damage by comet assay

The interactions of small molecules with DNA can cause DNA damage in cancer cells, blocking the division of cancer cells, and resulting in cell death.^30–32 The comet assay is a very helpful and versatile technique for assessing DNA damage on the single cell level. The commonly used alkaline modification of the comet assay detects DNA strand breaks. The results of DNA damage induced by compounds 4a–4d in HepG2 cells were shown in Fig. 1. The control cells failed to show a comet like appearance. However, treatment of HepG2 cells with 5 μM of compounds 4a–4d showed significant comet tail. It was obvious that the length of the comet tail represented extent of DNA damage.

Fig. 1 Comet assay of EB-stained HepG2 control (a) exposure to 5 μM of compound 4a (b), 4b (c), 4c (d) and 4d (e) for 24 h.

2.4. Analysis of cell cycle arrest by flow cytometry

To investigate the distribution of HepG2 cells cycle progression, flow cytometry was performed on cells which were treated with 5 μM of compounds 4a–4d. Significantly, accumulation of the sub-G1 population indicates characteristics of apoptosis.^33,34 Seen from Fig. 2 control cells displayed 0.28% of the phase, whereas compounds-treated cells displayed increased sub-G1 phases of 0.56% for 4a, 0.64% for 4b, 1.28% for 4c and 0.76% for 4d, respectively. Flow cytometric analysis also showed compounds 4a–4d exposure resulted in progressive and sustained accumulation of cells in G2/M phase. After compounds treatment for 24 h, the percentage of G2/M phase reached 37.11%, 25.38%, 32.02% and 31.52% for compounds 4a–4d, respectively. These percentages were comparatively higher about 32.53–93.78% than the proportion of control cells. Furthermore, the accumulation of cells in G2/M phase was accompanied by a decrease in the population of cells in G1 phase. Therefore, our findings showed that dibenzoxanthenes promoted cell grown inhibition by inducing G2/M phase arrest.

Fig. 2 Cell cycle analysis of HepG2 cells treated with compounds. Data represent the arithmetic mean ± SD of three separate experiments. *P < 0.05.

2.5. Apoptosis assay by microscopy and flow cytometry

To observe the morphologic characteristics of apoptotic nuclei, HepG2 cells were stained with AO/EB after exposure to 5 μM and 8 μM concentrations of compounds 4a–4d for 24 h and detected by fluorescence microscopy. Representative images of the cells treated with vehicle and compounds were shown in Fig. 3. Control cells exhibit homogeneous nuclear staining, and after treatment with compounds apoptotic cells increased gradually in a dose-dependent manner and displayed typical apoptotic changes (e.g. staining bright, condensed chromatin and fragmented nuclei) (Fig. 3b–i). To further quantify dibenzoxanthenes-induced apoptosis, we performed Annexin V/PI double-staining experiments to examine apoptosis of cells. Flow cytometry was used to detect positive cells after treatment with compounds 4a–4d for 24 h. Annexin V staining and Annexin V/PI double staining population represented early apoptosis and late apoptosis or necrosis. The results were shown in Fig. 4, with increasing compounds concentrations, a higher proportion of early apoptosis and late apoptosis was detected in HepG2 cell lines. After treatment with 5 μM compounds concentration, the proportion of early apoptosis increased about 88.2–188.2% than control cells (Fig. 4b–e). The proportion of late apoptosis increased dramatically compared with the control cells. At a dose of 8 μM, the proportion of early apoptosis increased about 142.8–259.6% (Fig. 4f–i). And the proportion of late apoptosis was from 7.32% to 13.2%. Together, these results suggested compounds induced apoptosis in HepG2 cells in dose-dependent concentration.

Fig. 3 AO/EB staining HepG2 cells (a) exposure to 5 μM compounds 4a–4d (b–e) and 8 μM compounds 4a–4d (f–i) for 24 h.

Fig. 4 The percentage of living (L), early apoptosis (EA) and late apoptosis (LA) after HepG2 cells (a) exposed to 5 μM of 4a–4d (b–e) and 8 μM of 4a–4d (f–i).

2.6. Dibenzoxanthenes increase intracellular ROS generation

Chemopreventive agents that enhance reactive oxygen species (ROS) reaching a toxic threshold can encourage apoptosis in cancer cells with minimal toxicity to normal counterparts. A mild increase in the level of ROS may promote cell proliferation, whereas a severe increase of ROS in cancer cells can trigger cell death.^35,36 ROS accumulation is also considered to be a pivotal phenomenon, which is related to the apoptotic mechanisms induced by DNA damage.^37,38 The generation of intracellular ROS in HepG2 cells was evaluated using 2′,7′-dichloro-dihydrofluorescein diacetate (DCHF-DA) fluorescent probe which can be deacetylated by intracellular esterase to the non-fluorescent DCFH, then can be oxidized by ROS to the fluorescent compound dichlorofluorescein (DCF). Cells exposed to 5 μM compounds 4a–4d exhibited increased fluorescence intensity in flow cytometry experiment (Fig. 5). The increment rate of fluorescence intensity is 133% for 4a, 246% for 4b, 98% for 4c and 71% for 4d, respectively (Fig. 5B). Compounds 4a and 4b showed higher generation ROS than compounds 4c and 4d. We examined the effect of ROS scavenger antioxidant vitamin C on ROS generation in cells. As shown in Fig. 5B, the fluorescent intensity of ROS reduced about from 19–54% for compounds in the presence of vitamin C. These results indicated that compounds promoted an increase of ROS production.

Fig. 5 (A) HepG2 cells were treated with compounds 4a–4d (and in the presence VC) to examine ROS production by flow cytometry. (B) Relative DCF fluorescence intensity was induced by compounds 4a–4d. *P < 0.05 versus control.

2.7. Mitochondrial membrane potential assay

Considering that decline in mitochondrial membrane potential is an important proapoptotic index for early apoptosis, we chose a mitochondria-specific and voltage-dependent fluorescent probe, JC-1, to observe whether there was loss in mitochondrial membrane potential in HepG2 cells after treatment with compounds. JC-1 with aggregates emits red fluorescence corresponding to high mitochondrial membrane potential, whereas monomeric JC-1 emits green fluorescence corresponding to low mitochondrial membrane potential.^39–41 As shown in Fig. 6, exposure to 5 μM compounds 4a–4d for 24 h resulted in decrease in the percentage of cells with red fluorescence (Fig. 6B). The ratio of red/green fluorescence intensity reduced about 55.6% for 4a, 51.1% for 4b, 54.2% for 4c and 48.0% for 4d, respectively, which indicated a remarkable decrement of mitochondrial membrane potential in compounds-treated HepG2 cells.

Fig. 6 (A) Assay of HepG2 cells mitochondrial membrane potential. HepG2 cells (a). exposed to 5 μM of 4a (b), 4b (c), 4c (d) and 4d (e). (B) The fluorescent intensity ratio red/green. *P < 005 versus control.

2.8. The expression of caspases and Bcl-2 family members by western blotting

The proteins of Bcl-2 family play a major role in regulation of apoptosis by functioning as promoters (e.g. Bax or Bak) or inhibitors (e.g. Bcl-2 or Bcl-xl) of cell death.^42–44 To explore the involvement of Bcl-2 family members in apoptosis induced by compounds 4a–4d, the expression levels of Bcl-2, Bcl-xl, Bak and Bid were analyzed by western blotting. We found that treatment of HepG2 cells with compounds for 24 h resulted in a reduction in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl compared with the levels of control cells. And an increasement in the expression of proapoptotic protein Bak was obtained (Fig. 7). A key step in the intrinsic apoptotic pathway is release of cytochrome c, which in turn on the caspase cascaded.^45,46 As shown in Fig. 7, treatment with compounds effectively increased the expression of cytochrome c in HepG2 cells. It is well known that the release of cytochrome c promotes activation of caspase-9, followed by the activation of effector caspase-3 in mitochondria pathway apoptosis.⁴⁷ So we also examined whether compounds 4a–4d induced the activation of caspase-9, caspase-3 and procaspase-7 in HepG2 cells. Western blotting analysis revealed that the treatment with compounds leaded to an increase in the expression of caspase-9 and caspase-3. But there was a significant decrease in the expression of procaspase-7. These results suggested that a mitochondria-dependent pathway was involved in dibenzoxanthenes-induced apoptosis in HepG2 cells.

Fig. 7 Western blot analysis of caspase-3 -9, procaspase 7, Cyto C, Bc1-2, Bc1-x and Bax, Bid in HepG2 cells treated with compounds 4a–4d for 24 h, GAPDH was used as internal control.

2.9. Cellular uptake and localization studies

The cellular uptake feature of small molecule is important to its application as a therapeutic or diagnostic agent.^48,49 The cellular uptake of compounds 4a–4d was examined in vitro with fluorescence microscopy. HepG2 cells were incubated with compounds at 5 μM for 24 h at room temperature and the cells were stained with DAPI. As shown in Fig. 8, the green channel showed the luminescence of compounds 4a–4d with an excitation wavelength of 458 nm, the blue channel displayed DAPI stained nuclei with an excitation wavelength of 340 nm, and the overplay represented cellular association of compounds. Green channel could completely overlay with blue channel, which showed that compounds were uptaken by HepG2 cells and the compounds gradually penetrated into the interior of the cell nucleus.

Fig. 8 Images of HepG2 cells exposure to 5 μM of 4a–4d and DAPI stained for 24 h.

2.10. Effects of dibenzoxanthenes on the HepG2 cells migration and invasion

Metastatic cancers have important characteristics, including the migratory and invasive activities of tumor cells. The effect of compounds towards migration ability of HepG2 was assessed by in vitro scratch assay. As shown in Fig. 9A the wound gap of the control group significantly reduced after incubation for 24 h. However, the wound gap of compounds-treated groups had almost no changes for 24 h comparing to the wound gap at 0 hour. To further evaluate the effects of compounds on cell migration activity, we also performed transwell cell invasion assays. The results showed that compounds significantly inhibited the invasion of HepG2 cells (Fig. 9B). Compared to the control group, the HepG2 cell invasion ability was notably weakened in compounds group. The suppression ratio was 77.6% for 4a, 73.5% for 4b, 74.8% for 4c and 60.9% for 4d, respectively (Fig. 9C). These results confirmed that compounds 4a–4d were effective in the suppression of HepG2 cells migration and invasion.

Fig. 9 (A) Compounds 4a–4d reduced migration of HepG2 cells after 24 h. (B) HepG2 cells invasion was inhibited by compounds 4a–4d. (C) Numbers of invading were measured treated with 3 μM of compounds. *P < 0.05 versus control.

3. Conclusions

In summary, four dibenzoxanthene analogs 4a–4d with CN or COOCH₃ groups were successfully synthesized. In vitro cytotoxicity assay demonstrated that these reagents exhibited effective activities of killing cancer cells. High toxicities were observed in tumor cell lines, such as HepG2, HeLa, SCG-1709 and A549 cell lines. In addition, these compounds showed less toxic to normal live LO2 cells. Further study suggested that compounds induced inhibition HepG2 cell proliferation was associated with DNA damage and G2/M phase arrest. In addition, compounds 4a–4d significantly triggered apoptosis in HepG2 cells, which was characterized by chromatin condensation. The cellular study with or without an ROS quencher showed these compounds function through ROS-dependent mitochondrial mechanism. Compounds promoted ROS production in HepG2 cells, which led to disruption of the out mitochondrial membrane and increased the ratio of Bax/Bcl-2, resulting in the release of cytochrome c. This in turn promoted activation of caspase-9, following by the activation of effector caspase-3, finally induced apoptosis. Compounds could also enter the nuclear of HepG2 cells and inhibit the migration and invasion of cells. Collectively, these data provided utility and selectivity of these compounds which should inspire further and effective application in potential cancer chemotherapies.

4. Experimental

4.1. Materials and methods

NMR spectra were recorded on a Varian spectrometer. All chemical shifts were given relative to tetramethylsilane (TMS). LC mass spectra (LC-MS) were recorded on a LCQ system (Finnigan MAT, USA) using methanol as mobile phase. HRMS were recorded on a Finnigan MAT 95 spectrometer using the DART positive technique. TLC-analysis was performed on glass-backed plates (Merck) coated with 0.2 mm silica 60F₂₅₄. Melting points were recorded using a digital melting point apparatus (IA9000 series, ThermoFischer Scientific, Rochford, U.K.) and are given without correction. Common reagent-grade chemicals are commercially available and are used without further purification. Binaphthols were prepared according to literature procedure.²⁴ 4,6-Diamidino-2-phenylindole (DAPI), an ECL-Plus Kit and Cell Cycle and Apoptosis Analysis Kit were purchased from Beyotime (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Sigma-Aldrich. The fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) and JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) were purchased from Roche Diagnostics (Indianapolis, IN). Polyclonal antibodies against Bax, Bcl-2, Bid and Bcl-xl were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-procaspase-7, caspase-3 and caspase-9 antibodies were purchased from Cell Signaling Technology (Beverly, MA).

4.2. Cell culture

Five cell lines were used in this study, a normal human liver cell line (LO2) and four cancer cell lines: human hepatocellular carcinoma cell line (HepG2), cervical cancer cell line (HeLa), human gastric carcinoma cell line (SGC-7901), and human lung adenocarcinoma epithelial cell line (A549). Cell lines of HepG2, HeLa, SGC-7901 and A549 cells were purchased from American Type Culture Collection. These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; TBD Science), 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin (Ameresco, USA) at 37 °C with 5% CO₂.

4.3. Synthesis procedure of binaphthol compounds 3a–3d

A round-bottomed flask was charged with 6-cyano-2-naphthanol 1 and 2-naphthol 2 (1 mmol), CuCl₂ (1 mmol), TMENDA (2 mmol) in MeOH (20 mL). The mixture was then stirred for 10 h at room temperature. The solution was extracted with ethyl acetate (2 × 20 mL). The combined organic layers were dried over Na₂SO₄ and filtered. The solvent was removed under reduced pressure, and the residue was subjected to column chromatography on silica gel (100–200 mesh) with a mixture of EtOAc–petroleum ether (1 : 2, v/v) as eluent. The pure products 3a–3d were obtained as yellow solid.

6-Cyano-1,1′-binaphthalene-2,2′-diol (3a). Yield, 75%, ¹H-NMR (300 MHz, acetone-d₆) δ: 8.42 (s, 1H), 8.09 (d, J = 9.0 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.45 (dd, J = 9.0 Hz 1.8 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.33–7.21 (m, 2H), 7.19 (d, J = 8.7 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H). ¹³C NMR (75 MHz, acetone-d₆) δ: 157.34, 154.67, 137.28, 135.27, 135.05, 131.33, 131.04, 129.96, 129.05, 128.89, 127.59, 127.39, 126.79, 125.01, 123.84, 121.39, 120.13, 119.51, 116.09, 113.86, 106.71. LC-MS, m/z: 311 [M]⁺

6,6′-Dicyano-1,1′-binaphthalene-2,2′-diol (3b). Yield, 85%, ¹H-NMR (300 MHz, DMSO-d₆) δ: 10.08 (s, 2H), 8.51 (s, 2H), 8.06 (d, J = 9.0 Hz, 2H), 7.50–7.45 (m, 4H), 7.02 (d, J = 9.0 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ: 156.13 (2C), 135.59 (2C), 134.45 (2C), 130.19 (2C), 126.98 (2C), 126.68 (2C), 125.25 (2C), 120.19 (2C), 119.60 (2C), 114.78 (2C), 104.47 (2C). LC-MS, m/z: 335 [M − H]⁻.

6,6′-Methoxyacyl-1,1′-binaphthalene-2,2′-diol (3c). Yield, 90%, ¹H-NMR (300 MHz, DMSO-d₆) δ: 9.82 (s, 2H), 8.60 (s, 2H), 8.01 (d, J = 9.0 Hz, 2H), 7.70 (dd, J = 9.0 Hz 1.8, 2H), 7.43 (d, J = 9.0 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 2.50 (s, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ: 166.49 (2C), 155.55 (2C), 136.42 (2C), 130.95 (2C), 130.79 (2C), 127.03 (2C), 125.05 (2C), 124.53 (2C), 123.37 (2C), 119.49 (2C), 115.02 (2C), 51.92 (2C). LC-MS, m/z: 401 [M − H]⁻.

6-Methoxyacyl-1,1′-binaphthalene-2,2′-diol (3d). Yield, 80%, ¹H-NMR (300 MHz, DMSO-d₆) δ: 9.54–9.52 (m, 2H), 8.61 (s, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.90–7.85 (m, 2H), 7.71 (dd, J = 9.0 Hz 1.8, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 9.0 Hz, 1H), 7.25–7.18 (m, 2H), 7.06 (d, J = 9.0 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 3.87 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ: 166.54, 155.54, 153.03, 136.61, 133.92, 130.91, 130.52, 128.92, 128.10, 127.92, 127.03, 126.01, 124.87, 124.79, 124.12, 123.29, 122.34, 119.50, 118.53, 115.74, 114.68, 51.91. LC-MS, m/z: 343 [M − H]⁻.

4.4. Synthesis of compounds 4a–4d

To a stirred solution of CuCl₂ (0.350 g, 2 mmol) and ethanolamine (0.120 g, 2 mmol) in 15 mL MeOH was added binaphthol 3a–3d (1 mmol) at 50 °C. The mixture was stirred for 4 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 crude product was purified by column chromatography on silica gel (100–200 mesh) with a mixture of EtOAc–petroleum ether (1 : 2, v/v) as eluent. Yellow powder were obtained.

1-Oxo-11-cyano-13c-methoxy-1,13c-dihydroxyl-dibenzo[a,kl]xanthene (4a). Yield, 80%, mp 260.3–261.5 °C, ¹H-NMR (300 MHz, DMSO-d₆) δ: 8.59 (s, 1H), 8.21 (d, J = 9.0 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.73 (dd, J = 9.0 Hz 1.8 Hz, 1H), 7.62 (d, J = 9.0 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.46 (d, J = 10.2 Hz, 1H), 7.35–7.27 (m, 2H), 6.33 (d, J = 9.9 Hz, 1H), 3.34 (s, 3H). ¹³C-NMR (75 MHz, DMSO-d₆) δ: 197.42, 153.05, 151.32, 139.85, 134.56, 134.39, 132.93, 132.63, 131.87, 129.74, 128.64, 126.26, 125.58, 125.19, 119.22, 118.94, 116.48, 114.84, 107.56, 107.02, 74.78, 51.27. HRMS (m/z): calcd for C₂₂H₁₃NO₃, 340.0968; found 340.0971.

1-Oxo-5,11-dicyano-13c-methoxy-1,13c-dihydroxyl-dibenzo[a,kl]xanthene (4b). Yield, 87%, mp 340.1–341.6 °C, ¹H-NMR (300 MHz, DMSO-d₆) δ: 8.70 (s, 1H), 8.31 (d, J = 9.0 Hz, 1H), 8.10 (d, J = 9.0 Hz, 1H), 7.99 (s, 1H), 7.87 (s, 1H), 7.80 (dd, J = 9.0 Hz 1.8 Hz, 1H), 7.69 (d, J = 9.0 Hz, 1H), 7.55 (d, J = 10.2 Hz, 1H), 6.53 (d, J = 9.9 Hz, 1H), 3.39 (s, 3H). ¹³C-NMR (75 MHz, DMSO-d₆) δ: 196.21, 152.77, 150.96, 138.32, 134.48, 134.28, 133.08, 131.65, 129.91, 128.62, 128.52, 127.31, 126.81, 120.73, 119.96, 119.03, 118.84, 117.32, 114.10, 107.37, 107.09, 74.39, 51.76. HRMS (m/z): calcd for C₂₃H₁₂N₂O₃, 365.0921; found 365.0895.

1-Oxo-5,11-dimethoxyacyl-13c-methoxy-1,13c-dihydroxyl-dibenzo[a,kl]xanthene (4c). Yield, 88%, mp 304.5–342.4 °C, ¹H-NMR (300 MHz, DMSO-d₆) δ: 8.66 (s, 1H), 8.33 (d, J = 9.0 Hz, 1H), 8.03 (d, J = 9.0 Hz, 1H), 7.91 (dd, J = 8.1 Hz 1.8 Hz, 2H), 7.75 (s, 1H), 7.58–7.55 (m, 2H), 6.43 (d, J = 10.2 Hz, 1H), 3.92 (s, 6H), 2.71 (s, 3H). ¹³C-NMR (75 MHz, DMSO-d₆) δ: 196.76, 166.06, 164.79, 152.66, 151.19, 139.12, 134.95, 133.77, 131.49, 130.75, 129.99, 128.62, 127.81, 126.31, 125.77, 124.79, 124.53, 119.61, 118.29, 116.98, 106.96, 74.74, 52.69, 52.21, 51.49. HRMS (m/z): calcd for C₂₅H₁₈O₇, 431.1125; found 431.1130.

1-Oxo-5,11-dimethoxyacyl-13c-methoxy-1,13c-dihydroxyl-dibenzo[a,kl]xanthene (4d). Yield, 85%, mp 230.5–231.8 °C, ¹H-NMR (300 MHz, DMSO-d₆) δ: 8.65 (s, 1H), 8.31 (d, J = 9.0 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.90 (dd, J = 9.0 Hz 1.8 Hz, 1H), 7.58–7.53 (m, 2H), 7.45 (d, J = 9.9 Hz, 1H), 7.33 (d, J = 6.9 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H H), 6.34 (d, J = 10.2 Hz, 1H), 3.92 (s, 3H), 2.69 (s, 3H). ¹³C-NMR (75 MHz, DMSO-d₆) δ: 197.51, 166.10, 152.70, 151.38, 139.68, 135.11, 133.48, 132.99, 131.77, 130.71, 129.87, 127.88, 125.69, 125.57, 125.03, 124.63, 118.36, 116.42, 114.98, 107.31, 74.89, 52.19, 51.18. HRMS (m/z): calcd for C₂₃H₁₆O₅, 373.1070; found 373.1071.

4.5. In vitro cytotoxicity assay

Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide (MTT) assay procedures were used. 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 prior to use. Control cells were prepared by addition of culture medium containing the same amount of DMSO 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 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 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.

4.6. Comet assay

DNA damage was investigated by means of comet assay. HepG2 cells in culture medium were incubated with 5 μM of compounds 4a–4d for 2 h at 37 °C. Then the cells were incubated 24 h. 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). 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.

4.7. Flow cytometric analysis of cell cycle distribution

HepG2 cells were seeded into six-well plates at a density of 2 × 10⁵ cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C and 5% CO₂. The medium was removed and replaced with medium (final DMSO concentration, 0.05%, v/v) containing compounds (5 μM). After incubation for 24 h, the cell layer was trypsinized and washed with cold phosphate buffered saline (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 incubated at 37 °C for 30 min. Then the samples were analyzed by a FACS Calibur flow cytometer (Becton Dickinson & Co., Franklin Lakes, NJ). The number of cells analyzed for each sample was 10 000.

4.8. Apoptosis assessment by AO/EB staining and flow cytometry

A monolayer of HepG2 cells was incubated in the absence and presence of the compounds at a concentration of 5 μM or 8 μM at 37 °C. After incubation for 24 h, then each cell culture was stained with AO/EB solution (100 μg mL⁻¹ AO, 100 μg mL⁻¹ EB) and observed by fluorescence microscopy. It is well known that AO can pass through cell membranes, but EB cannot. Under the fluorescence microscope, living cells appear green. Necrotic cells stain red but have a nuclear morphology resembling that of viable cells. Apoptotic cells appear green, and morphological changes such as cell blebbing and formation of apoptotic bodies will be observed.

Cells were collected from each group and 100 μL binding buffer added to suspend the cells. The cells were then collected by centrifugation and washed twice with PBS. Then 5 μL fluorescein isothiocyanate (FITC) and 5 μL propidium iodide (PI) were added to the mixture and incubated in the dark for 10–15 min at room temperature. Apoptosis of the cells was detected by flow cytometry within 1 h after the addition of 500 μL binding buffer.

4.9. Reactive oxygen species (ROS) detection

HepG2 cells were seeded into six-well plates (Costar, Corning, Corning, NY, USA) 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 and 5% CO₂. The medium was removed and replaced with medium (final DMSO concentration, 0.05% v/v) containing 4a–4d (5 μM). After incubation for 24 h, the medium was then removed. The fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA) was added to the medium at a final concentration of 10 μM to cover the cells. After 30 min the treated cells were then washed with cold PBS–EDTA twice. Cells were harvested by centrifugation at 1500 rpm for 5 min and were resuspended in 500 μL of DCHF-DA solution (10 μM), incubated at for 30 min, washed twice and analyzed by flow cytometry.

4.10. Mitochondrial membrane potential detection

Mitochondrial membrane potential was also measured by flow cytometry. After 5 × 10⁵ cells per well was seeded to a 6 well plate for 24 h, the cells were treated with compounds for 24 h. Then, cells were harvested, centrifuged at 1500 rpm for 5 min, and washed with ice-cold PBS once. Consequently, cells were incubated with JC-1 at 37 °C for 20 min in the dark. Then, the cells were washed twice, resuspended in 1 mL PBS, and analyzed by a BD FASCanto flow cytometer with an excitation wavelength of 484 nm and an emission wavelength of 501 nm. At least 10 000 cells were determined for each sample. The data obtained from flow cytometry were analyzed by CellQuest software.

4.11. Western blot analysis

HepG2 cells were seeded in 3.5 cm dishes for 24 h and incubated with 4a–4d (5 μM) in the presence of 10% FBS. Then the cells were harvested in lysis buffer. After sonication, the samples were centrifuged for 20 min at 13 000 g. 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. Cellular uptake and localization studies

HepG2 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. Complexes 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 mediums in the wells. The cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and visualized by fluorescence microscope.

4.13. HepG2 cells migration assay

HepG2 cell suspensions were diluted to 250 000 cells per mL in complete growth medium containing 10% FBS and seeded onto 24 well plates (Costar; Lowell, U.S.A.) to a final well volume of 1 mL. Cells were allowed to reach confluency after 48 h, at which time a single scratch was made using the tip of a plastic disposable 10 μL pipet tip. All wells were washed twice with 1 mL of DPBS to remove cellular debris. Then 500 μL of starvation medium consisting of DMEM, 2% FBS, and 1% pen-strep was used to prepare solutions with compounds concentration of 5 μM. Cell migration across the scratch was measured under a phase contrast inverted microscope (VWR; Mississauga, Canada) at 0 h and 24 h.

4.14. Transwell assay

Cell invasion activity was evaluated in 8.0 μm pore size transwell 24-insert plate chambers (BD Biosciences, Bedford, MA, USA). Chambers were coated with extracellular Matrigel (BD Biosciences). Cells were cultured at 37 °C in a humidified incubator with 5% CO₂. HepG2 cells (4 × 10⁴ per well) were plated in the upper chambers with serum-free medium. The lower chambers were filled with 10% FBS DMEM medium. After 24 h incubation, the non-migrating cells were wiped off the upper side of the upper chamber with a cotton swab. The lower side of the upper chamber was fixed with methanol and stained with 0.1% crystal violet. Six fields were randomly selected and cells counted.

4.15. 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 combination of production and research projects Guangdong province (2012B091000151) and the Priority Academic Program Development of Guangdong Higher Education Institutions (2013LYM0047).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13901a

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