Cytotoxic Compounds from Juglans sinensis Dode Display Anti-Proliferative Activity by Inducing Apoptosis in Human Cancer Cells

Phytochemical investigation of the bark of Juglans sinensis Dode (Juglandaceae) led to the isolation of two active compounds, 8-hydroxy-2-methoxy-1,4-naphthoquinone (1) and 5-hydroxy-2-methoxy-1,4-naphthoquinone (2), together with 15 known compounds 3–17. All compounds were isolated from this plant for the first time. The structures of 1 and 2 were elucidated by spectroscopic data analysis, including 1D and 2D NMR experiments. Compounds 1–17 were tested for their cytotoxicity against the A549 human lung cancer cell line; compounds 1 and 2 exhibited significant cytotoxicity and additionally had potent cytotoxicity against six human cancer cell lines, MCF7 (breast cancer), SNU423 (liver cancer), SH-SY5Y (neuroblastoma), HeLa (cervical cancer), HCT116 (colorectal cancer), and A549 (lung cancer). In particular, breast, colon, and lung cancer cells were more sensitive to the treatment using compound 1. In addition, compounds 1 and 2 showed strong cytotoxic activity towards human breast cancer cells MCF7, HS578T, and T47D, but not towards MCF10A normal-like breast cells. They also inhibited the colony formation of MCF7, A549, and HCT116 cells in a dose-dependent manner. Flow cytometry analysis revealed that the percentage of apoptotic cells significantly increased in MCF7 cells upon the treatment with compounds 1 and 2. The mechanism of cell death caused by compounds 1 and 2 may be attributed to the upregulation of Bax and downregulation of Bcl2. These findings suggest that compounds 1 and 2 may be regarded as potential therapeutic agents against cancer.

The extracts of J. sinensis show antiasthma effects [5] and antioxidant activities on liver damage [6] and acute renal failure [7].
In previous reports on the anticancer effects of Juglans species, the extracts of root barks, fruits, or seeds of J. regia showed anti-proliferative activity against Caco-2 human colon cancer cells, HepG2 human liver cancer cells, and MDA-MB-231 human breast cancer cells [8][9][10]; the extract of seeds of J. sinensis protected UVB-induced human keratinocytes apoptosis [11]. Sesquiterpenes and triterpenes isolated from the leaves and twigs of J. sinensis inhibited the proliferation of immortalized rat hepatic stellate cells through apoptosis [1]; however, the mechanism of action of the anti-proliferation activity of the phenolic compounds of J. sinensis has not been investigated in detail.
Therefore, in continuation of our search for novel natural anticancer agents, we performed a bioactivity-guided fractionation to isolate and identify cytotoxic compound(s) from J. sinensis. Herein, we describe the separation and structure elucidation of such cytotoxic compounds, and furthermore, we evaluated their anti-proliferative and apoptotic activity to study mechanism of the cytotoxicity of these compounds in human cancer cells.

Identification of Cytotoxic Compounds
The cytotoxic activities of the MeOH extract, solvent-partitioned fractions, and the compounds isolated from J. sinensis were examined on the A549 human non-small cell lung cancer cell line at various concentrations for 24 h. Inhibitory concentration (IC50) values were calculated from their cell viability curves. Because the MeOH extract showed cytotoxic activity against A549 cells, this extract was partitioned into hexane, ethyl acetate, butanol, and aqueous soluble fractions. As shown in Table 1, the ethyl acetate fraction was the major fraction responsible for the cytotoxic activity compared to other fractions.
Individual compounds were isolated from the ethyl acetate fraction, and their IC50 values were determined against A549 cells. The cells were treated with 0-50 μM of compounds 1 to 17 for 24 h. Compounds 1 and 2 showed strong cytotoxicity against A549 cells, with the IC50 values of 1.82 and 1.33 μM, respectively, whereas other compounds were inactive (IC50 > 10 μM, Table 1). Based on the cytotoxic potency and selectivity, compounds 1 and 2 were selected as the potential anticancer compounds and for further investigation of their cytotoxicity against different human cancer cell lines.

Identification of Cytotoxic Compounds
The cytotoxic activities of the MeOH extract, solvent-partitioned fractions, and the compounds isolated from J. sinensis were examined on the A549 human non-small cell lung cancer cell line at various concentrations for 24 h. Inhibitory concentration (IC50) values were calculated from their cell viability curves. Because the MeOH extract showed cytotoxic activity against A549 cells, this extract was partitioned into hexane, ethyl acetate, butanol, and aqueous soluble fractions. As shown in Table 1, the ethyl acetate fraction was the major fraction responsible for the cytotoxic activity compared to other fractions.
Individual compounds were isolated from the ethyl acetate fraction, and their IC50 values were determined against A549 cells. The cells were treated with 0-50 μM of compounds 1 to 17 for 24 h. Compounds 1 and 2 showed strong cytotoxicity against A549 cells, with the IC50 values of 1.82 and 1.33 μM, respectively, whereas other compounds were inactive (IC50 > 10 μM, Table 1). Based on the cytotoxic potency and selectivity, compounds 1 and 2 were selected as the potential anticancer compounds and for further investigation of their cytotoxicity against different human cancer cell lines.

Identification of Cytotoxic Compounds
The cytotoxic activities of the MeOH extract, solvent-partitioned fractions, and the compounds isolated from J. sinensis were examined on the A549 human non-small cell lung cancer cell line at various concentrations for 24 h. Inhibitory concentration (IC 50 ) values were calculated from their cell viability curves. Because the MeOH extract showed cytotoxic activity against A549 cells, this extract was partitioned into hexane, ethyl acetate, butanol, and aqueous soluble fractions. As shown in Table 1, the ethyl acetate fraction was the major fraction responsible for the cytotoxic activity compared to other fractions.
Individual compounds were isolated from the ethyl acetate fraction, and their IC 50 values were determined against A549 cells. The cells were treated with 0-50 µM of compounds 1 to 17 for 24 h. Compounds 1 and 2 showed strong cytotoxicity against A549 cells, with the IC 50 values of 1.82 and 1.33 µM, respectively, whereas other compounds were inactive (IC 50 > 10 µM, Table 1). Based on the cytotoxic potency and selectivity, compounds 1 and 2 were selected as the potential anticancer compounds and for further investigation of their cytotoxicity against different human cancer cell lines.

Cytotoxic Effects of Compounds 1 and 2 against Various Human Cancer Cells
Different human cancer cell lines were treated with compounds 1 and 2 using serial dilution concentrations (10, 5, 2.5, 1.25, 0.625, and 0 µM). As shown in Figure (Table 2 and Figure 4A). Especially, compound 1 displayed strong activity against MCF7 breast cancer, HCT116 colon cancer, and A549 lung cancer cells. A549 and MCF7 cells were used to further evaluate the cytotoxic effect of compounds 1 and 2. In addition, when compounds 1 or 2 were treated in A549 and MCF7 cells for 24 h, the cell morphology became more round and floated compared to the untreated healthy cells, showing a dissimilar cytoskeleton ( Figure 4B).

Anti-Proliferative Activity of Compounds 1 and 2
To explore the anticancer properties of compounds 1 and 2, colony formation assays were performed. MCF7 cells were incubated with compounds 1 and 2 at various concentrations (0, 0.5, 1, and 2 μM) for two weeks. Compounds 1 and 2 suppressed the colony formation of MCF7 breast cancer cells in a dose-dependent manner, and compound 1 was more sensitive than compound 2 for MCF7 cells ( Figure 6A). To further confirm these results, the inhibitory effect of colony formation was also examined using another two cell lines, HCT116 colon cancer cells and A549 lung cancer cells, at the same concentrations of compounds 1 and 2 ( Figure 6B,C). The results show that compounds 1 and 2 can inhibit the colony formation capacity; especially compound 1 was also more sensitive than compound 2 for both A549 and HCT116 cells, which was consistent with that of MCF7 cells. Taken together, compounds 1 and 2 showed the inhibitory effect of the colony formation in human cancer cells, and compound 1 was more sensitive than compound 2 for human cancer cells.

Anti-Proliferative Activity of Compounds 1 and 2
To explore the anticancer properties of compounds 1 and 2, colony formation assays were performed. MCF7 cells were incubated with compounds 1 and 2 at various concentrations (0, 0.5, 1, and 2 µM) for two weeks. Compounds 1 and 2 suppressed the colony formation of MCF7 breast cancer cells in a dose-dependent manner, and compound 1 was more sensitive than compound 2 for MCF7 cells ( Figure 6A). To further confirm these results, the inhibitory effect of colony formation was also examined using another two cell lines, HCT116 colon cancer cells and A549 lung cancer cells, at the same concentrations of compounds 1 and 2 ( Figure 6B,C). The results show that compounds 1 and 2 can inhibit the colony formation capacity; especially compound 1 was also more sensitive than compound 2 for both A549 and HCT116 cells, which was consistent with that of MCF7 cells. Taken together, compounds 1 and 2 showed the inhibitory effect of the colony formation in human cancer cells, and compound 1 was more sensitive than compound 2 for human cancer cells.
Molecules 2016, 21, 120 6 of 13 Figure 5. Cytotoxic effects in the normal and breast cancer cell lines. Cells were exposed to 2.5 μM of compounds 1 and 2 for 24 h. ** p < 0.01, compared to the MCF10A cells. Data presented are mean ± SD from three independent observations.

Anti-Proliferative Activity of Compounds 1 and 2
To explore the anticancer properties of compounds 1 and 2, colony formation assays were performed. MCF7 cells were incubated with compounds 1 and 2 at various concentrations (0, 0.5, 1, and 2 μM) for two weeks. Compounds 1 and 2 suppressed the colony formation of MCF7 breast cancer cells in a dose-dependent manner, and compound 1 was more sensitive than compound 2 for MCF7 cells ( Figure 6A). To further confirm these results, the inhibitory effect of colony formation was also examined using another two cell lines, HCT116 colon cancer cells and A549 lung cancer cells, at the same concentrations of compounds 1 and 2 ( Figure 6B,C). The results show that compounds 1 and 2 can inhibit the colony formation capacity; especially compound 1 was also more sensitive than compound 2 for both A549 and HCT116 cells, which was consistent with that of MCF7 cells. Taken together, compounds 1 and 2 showed the inhibitory effect of the colony formation in human cancer cells, and compound 1 was more sensitive than compound 2 for human cancer cells.

Apoptotic Activity of Compounds 1 and 2
To determine whether compounds 1 and 2 caused apoptosis, flow cytometry was performed using Annexin V-FITC and propidium iodide (PI) double staining assay in MCF7 cells. At 24 h after treatment with 1 and 2, the proportion of apoptotic cells was 61% higher in 2 μM compound 1-treated MCF7 cells than in the control cells (72.19% ± 0.36% vs. 10.79% ± 0.19%, p < 0.01) and 76% higher in 2 μM compound 2-treated MCF7 cells than in the control cells (87.02% ± 0.18% vs. 11.04% ± 0.26%, p < 0.01) ( Figure 7A).  Moreover, compound 1 induced cell necrosis at 2 µM compared to the untreated control group (2.02%˘0.62% vs. 21.02%˘1.12%); however, compound 2 did not induce cell necrosis ( Figure 7A). These findings indicate that compounds 1 and 2 could induce apoptosis and inhibit cancer cell growth. To study the mechanism of compounds 1 and 2-induced apoptosis, the expression levels of apoptosis-related proteins were measured using western blotting ( Figure 7B). The treatment with these compounds increased the expression level of Bax, a pro-apoptotic protein, whereas the expression level of Bcl2, an anti-apoptotic protein, decreased by the treatment with these compounds. When the expression level of Bax was represented as ratios to the level of Bcl2, the treatment with these compounds increased the ratio of Bax/Bcl2, and especially a significant elevation in the ratio of Bax/Bcl2 in compound 1-treated cells was observed. Altogether, these findings demonstrate that compounds 1 and 2 induced apoptosis by regulating pro-and anti-apoptotic genes.

Discussion
Breast cancer is the second most common reason for death in females worldwide [30]. In this study, 17 phenolic compounds 1-17 were isolated from the bark of J. sinensis; their cytotoxic activities were tested against diverse human cancer cells. Among them, compounds 1 and 2 exhibited cytotoxic activities and inhibited human cancer cell growth, which was in agreement with the previous reporters for similar compounds [31][32][33].
2-Methoxy-1,4-naphthoquinone (MNQ) exerts anticancer activity by the induction of apoptosis [34]. Our data demonstrate that compounds 1 and 2 induced apoptotic characteristics such as cytoplasm retraction, bleb formation, and the condensation of nuclear material [35,36], in a dose-dependent manner ( Figure 4B). Identified apoptotic pathways in cells can divide into two pathways mediated by: (i) the death receptor and (ii) mitochondria [37,38]. Recent report showed that MNQ promotes cancer cell death by a reactive oxygen species (ROS)-dependent mechanism [34]. Since compounds 1 and 2 have the same skeleton as MNQ, compounds 1 and 2 should have similar activity as MNQ based on their structures. The mitochondrial-mediated apoptosis is regulated by the Bcl2 protein family [39]. Pro-apoptotic protein Bax transposes to the mitochondrial outer membrane, and anti-apoptotic protein Bcl2 expression decreases followed by cytochrome c release inducing cell apoptosis. Our results show that compounds 1 and 2 induced cell apoptosis by reducing Bcl2 protein and increasing Bax protein, therefore they may induce cell apoptosis by a mitochondrial-mediated pathway.

Materials and Chemicals
The barks of J. sinensis were collected at the Medicinal Plant Garden, College of Pharmacy, Ewha Womans University, in August 2010, and identified by Prof. Je-Hyun Lee (Dongguk University, Geongju 780-714, Korea). A voucher specimen (No. EA310) was deposited at the Natural Product Chemistry Laboratory, College of Pharmacy, Ewha Womans University. Cell culture reagents were purchased from WelGENE (Daegu, Korea). The EZ-CyTox Cell Viability assay kit was purchased from Daeil Lab Service Co. (Seoul, Korea). Formaldehyde, crystal violet and DMSO were obtained from Sigma (St. Louis, MO, USA). The EzWay Annexin V-FITC apoptosis detection kit was purchased from KOMA Biotech (Seoul, Korea). Xpert protease inhibitor cocktail was purchased from GenDEPOT (Barker, TX, USA). Primary antibodies against Bcl2, Bax and β-actin were purchased by Santa Cruz Biotechnology (Santa Cruz, CA, USA), and horseradish peroxidase (HRP)-conjugated secondary antibody was obtained from Jackson Immuno Research laboratories, Inc. (West Grove, PA, USA). The enhanced chemiluminescence (ECL) kit was obtained from Advansta Inc. (Advansta, CA, USA).

Cell Viability Assay
Cells were cultured on 96-well plates at a density of 5ˆ10 4 cells/mL and treated with compounds at the indicated concentrations. After 24 h of incubation, cell viability was analyzed according to the manufacturer's instructions using the EZ-CyTox Cell Viability assay kit. Briefly, 10 µL of kit solution was added to each well for additional 4 h incubation. Absorbance was detected at 450 nm using a VERSA max microplate reader (Molecular Devices, Sunnyvale, CA, USA) and used to calculate the percentage of viable cells compared to the untreated cells. Results were expressed as cell viability (%) = (mean absorbency in test wells/mean absorbency in control wells)ˆ100. Cytotoxicity was expressed as the concentration of inhibiting cell growth by 50% (IC 50 value).

Colony Formation Assay
Cells were treated with compounds 1 and 2 at indicated concentrations for two weeks. The medium was changed every three days by the treatment with compounds 1 and 2. After that, the supernatant was thrown away, and the cells were washed three times with phosphate-buffered saline (PBS). The cells were fixed with 4% formaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. Colonies were photographed, and the number of colonies was counted using Image J (National Institutes of Health, Bethesda, MD, USA) from three independent experiments.

Flow Cytometry Analysis
Annexin V positive MCF7 cells were detected using an EzWay Annexin V-FITC apoptosis detection kit according to the manufacturer's protocol. Briefly, MCF7 cells were seeded and incubated with the indicated concentration of compounds 1 and 2 for 24 h. The cells were harvested, washed three times with PBS, and incubated with 1ˆBinding Buffer. Then, the cells were incubated with 1.25 µL of Annexin V-FITC and 10 µL of propidium iodide (PI) at room temperature for 15 min in the dark.
The samples were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). The apoptosis percentage was calculated as the number of PI positive and Annexin-V positive cells divided by the total number of cells. The experiments were repeated three times independently.

Western Blotting
Cells were washed with PBS and lysed in lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 50 mM EDTA, 1% Triton X-100) containing a protease inhibitor cocktail at 4˝C for 20 min. After centrifugation at 12,000 rpm for 15 min, the supernatants were collected. Protein concentration was determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins were subjected to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were then blocked with 5% skim milk for 1 h and incubated with primary antibody. After washing, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Proteins bands were visualized using an enhanced chemiluminescence (ECL) system.

Statistical Analysis
All the data are presented as the mean˘SD and are representative of at least three independent experiments. Comparisons between the two groups were analyzed by Student's t-test. A p value of less than 0.05 was considered to be statistically significant.

Conclusions
The leaves and (or) twigs of J. sinensis have usually been subjected to phytochemical and biological studies previously; however, this is the first report of the phytochemical study on the bark of this plant. In this study, bioassay-guided fractionation of an ethyl acetate-soluble fraction of the bark of J. sinensis using the A549 cell line, led to the isolation of 17 phenolic compounds 1-17, which were found in this plant for the first time. Moreover, 14 and 16 have never been isolated from the family Juglandaceae. This study suggests that compounds 1 and 2 are the main compounds responsible for the biological activity for the bark extract of J. sinensis. The most active compound 1 is a potential candidate for antitumor drug based on its effective cytotoxic and apoptotic activities.