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Introduction

Leukemia is one of the causes of cancer-related death in humans (1,2). According to the Department of Health, Executive Yuan, Taiwan R.O.C. (2010), approximately 4.2 per 100,000 people in Taiwan succumb to leukemia each year (3,4). Currently, treatment of leukemia in clinics includes hematopoietic stem cell transplantation, radiotherapy and chemotherapy agents; however, these outcomes are not fully satisfactory (5–7). Thus, numerous studies have been focused on discovering a novel compound from natural products that blocks the development of cancer, including leukemia. The most effective strategy for killing cancer cells is to induce apoptosis. Evidence has shown that increased consumption of a plant-based diet may reduce the risk of cancer (8,9).

For a number of years, Glycyrrhiza glabra (Licorice) has been used as a traditional Chinese medicine for the treatment of liver disease. It was reported that glycyrrhizic acid (GA) and 18β-glycyrrhetinic acid are the biologically active compounds in Licorice (10). GA, one of the triterpenoid saponin glycoside, was found to protect PC12 cells from 1-methyl-4-phenylpyridinium-induced cytotoxicity (11). GA alters inflammatory processes by modulating NF-κB activities (12) and by blocking the activation of NF-κB in primary neurons (13). Furthermore, it was reported that the neuroprotective effects of GA in PC12 cells is via modulation of the PI3K/Akt pathway (14). GA was found to modulate critical end points of oxidative stress-induced apoptosis and may be beneficial against liver diseases (15). Recently, it was reported that GA exerts an anti-inflammatory effect, at least in part, by inhibiting HMGB1 secretion (16). However, there is no available evidence demonstrating that GA induces apoptosis in leukemia cells. Therefore, the present study investigated the effects of GA on the cytotoxicity of mouse leukemia cells (WEHI-3). Our findings indicated that GA induced apoptosis in WEHI-3 cells through caspase- and mitochondria-dependent pathways.

Materials and methods

Chemicals and reagents

GA, agarose, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), dimethyl sulfoxide (DMSO), propidium iodide (PI), Triton X-100, Tris-HCl and ribonuclease A were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCF-DA), 3,3′-dihexyloxacarbocyanine iodide (DiOC6), RPMI-1640 medium, fetal bovine serum (FBS), L-glutamine, trypsin-EDTA and penicillin/streptomycin were purchased from Invitrogen/Life Technologies (Carlsbad, CA, USA). The caspase-3 activity assay kit and the caspase-3-specific inhibitor (z-Asp-Met-Gln-Asp-fluoromethyl ketone; z-DEVD-fmk) were obtained from R&D Systems (Minneapolis, MN, USA). Primary and secondary antibodies used for western blotting were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cell morphology and viability determined in WEHI-3 cells

WEHI-3 cells were placed in 24-well plates at a density of 2×105 cells/well overnight and were then incubated with 0, 200, 250, 300, 350 and 400 μM of GA at 37°C, in 5% CO2 and 95% air for 24 and 48 h. In order to examine morphological changes, cells from each treatment were examined and images were captured under a phase-contrast microscope at a magnification of ×200. Cells in each well were harvested, stained with PI (5 μg/ml) and then the total number of viable cells for all samples was determined using flow cytometry (Becton-Dickinson, San Jose, CA, USA) as previously described (17,18).

Assay of cell cycle distribution using flow cytometry

WEHI-3 cells (2×105 cells/ml) in 24-well plates were incubated with 200, 250, 300, 350 and 400 μM of GA and were incubated for 0, 24 and 48 h. Cells from each treatment were collected, fixed in 70% ethanol overnight, washed twice with PBS and re-suspended in 500 μl of 192 mM Na2HPO4, 4 mM citric acid and pH 7.8 at 25°C for 30 min. Then all samples were individually stained with 0.5 ml of PBS containing 1 mg/ml RNase and 10 μg/ml PI for 30 min in the dark and were directly analyzed using flow cytometry as previously described (18).

DAPI staining and comet assay for examining the DNA damage in WEHI-3 cells

Approximately 5×104 cells/ml of WEHI-3 cells in 24-well plates were individually treated with 0, 200, 250, 300, 350 and 400 μM of GA for 24 and 48 h. After incubation, all samples from each treatment were harvested. For DAPI staining, cells were stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride), examined and images were captured using a fluorescence microscope as previously described (9,19). For comet assay, cells were harvested, isolated and examined for DNA damage by using the comet assay as previously described (9,17).

DNA gel electrophoresis for DNA fragmentation of WEHI-3 cells

Approximately 1×106 cells/ml of WEHI-3 cells on 10-cm dishes were individually treated with 0, 200, 250, 300, 350 and 400 μM of GA for 24 and 48 h. At the end of incubation, all samples from each treatment were harvested for DNA isolation (Genomic DNA Purification kit; Genemark Technology Co., Ltd., Tainan, Taiwan) and DNA gel electrophoresis was performed to examine the DNA fragmentation as previously described (20,21).

Flow cytometric detection of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and caspase-3 activity

WEHI-3 cells were placed in 24-well plates at the density of 2×105 cells/ml and were then exposed to 300 μM of GA for indicated intervals of time. ROS and ΔΨm were assessed by cell permeable probes H2DCF-DA (10 μM) and DiOC6 (500 nM) using a flow cytometer, respectively, as previously described (9,22). Cells were pretreated with or without NAC (5 mM) or Z-VAD-FMK (5 μM), were incubated with 300 μM GA for various time periods and were then harvested and lysed in a lysis buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT]. Cell lysates (50 μg protein) were incubated with caspase-3 specific substrates (DEVD-pNA) for 1 h at 37°C and the caspase-3 activity was determined by measuring OD405 of the released pNA as previously described (23,24).

Western blot analysis for examining the associated protein levels in WEHI-3 cells

WEHI-3 cells were placed in 10-cm dishes at the density of 5×105 cells/ml and were exposed to 300 μM of GA for 0, 6, 12, 24 and 48 h. Then cells from each treatment were harvested and were dissolved in the PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Seongnam-si, Gyeonggi-do, Korea). All lysed samples were boiled at 100°C for 10 min with 4X protein loading dye. All samples were individually subjected to SDS-polyacrylamide gel electrophoresis as previously described (9,17). All proteins in the gel were transferred onto an Immobilon-P PVDF membrane (Merck Millipore, Bedford, MA, USA) and incubated with the primary antibodies Fas, FasL, caspase-3, Bid, Bax, apoptosis-inducing factor (AIF), cytochrome c, Endo G, IRE-1α, calpain 1, caspase-12, GRP 78, SOD (Cu/Zn), SOD (Mn) and catalase overnight (1:1,000 dilution), were then washed and then were incubated with horseradish peroxide-linked secondary antibody (1:8,000 dilution) and analyzed using the Immobilon Western Chemiluminescent HRP substrate (Millipore) as previously described (9,25).

Confocal laser microscopy

WEHI-3 cells at the density of 5×104 cells/well were plated on 4-well chamber slides and were then treated without (control) or with 300 μM of GA for 24 h. At the end of incubation, cells were washed with PBS and fixed with 4% formaldehyde in PBS for 15 min, followed by permeabilization for 1 h using 0.3% Triton X-100 in PBS containing 2% BSA for blocking non-specific binding sites. Cell samples were then stained by anti-AIF, anti-Endo G or anti-cytochrome c antibodies (1:100 dilution, respectively) for 24 h, washed twice with PBS and then stained with a secondary antibody (FITC-conjugated goat anti-mouse IgG at 1:100 dilution) for 40 min, followed by PI staining for DNA analysis. Examinations and photomicrographs were captured using a Leica TCS SP2 Confocal Spectral Microscope, as previously described (26,27).

Statistical analyses

The data from individual experiments are presented as the means ± SD. The differences between the GA-treated and -untreated (control) groups were analyzed using the Student's t-test; a probability of P<0.05 indicated a statistically significant difference.

Results

GA induces cell morphological changes and decreases the percentage of viable WEHI-3 mouse leukemia cells

WEHI-3 cells were treated with various concentrations (0, 200, 250, 300, 350 and 400 μM) of GA or DMSO for 24 and 48 h. Cells were examined and images were captured using a contrast-phase microscope at a magnification of ×200. The results showed that GA induced cell morphological changes in a dose-dependent manner (Fig. 1A). The percentage of total viable cells in each treatment was determined by flow cytometric assay. Results demonstrated that GA concentrations of 200–400 μM decreased the cell number (inhibited cell growth) in a dose- and time-dependent manner (Fig. 1B).

Figure 1 GA induces cell morphological changes and reduces the percentage of viable WEHI-3 mouse leukemia cells. (A) Cells (2×105 cells/well) placed in 24-well plates were treated with 0, 200, 250, 300, 350 and 400 μM of GA for 24 and 48 h. Cells were examined and images were captured using a contrast phase microscope. (B) All cells from each treatment were harvested and the percentage of viable cells was determined as described in Materials and methods. Data are presented as the means ± SD in triplicate. P<0.05 indicated a statistically significant difference compared to the GA and DMSO-treated groups.

GA induces G0/G1 phase arrest in WEHI-3 cells

Flow cytometry was used for evaluating the cell cycle distribution of WEHI-3 cells with or without GA treatment for 24 and 48 h. As shown in Fig. 2A and B, exposure to 200–400 μM GA caused an increase in the G0/G1 phase fraction from 39.2 to 72.3% and from 41.1 to 80.3%, as compared to the control samples following 24 and 48 h of treatment, respectively. These effects of GA on G0/G1 phase arrest were exhibited in a dose- and time-dependent manner. These data suggest that GA-induced G0/G1 phase arrest accounts for the decrease in the percentage of viable WEHI-3 cells by GA.

Figure 2 GA induces G0/G1 phase arrest in WEHI-3 cells. Cells at a density of 2×105 cells/well were placed in 24-well plates and were then treated with 0, 200, 250, 300, 350 and 400 μM of GA for 24 and 48 h. Cells were harvested for measuring the cell cycle distribution by flow cytometric assay as described in Materials and methods. The representative of profiles of DNA content and the percentage of cells in G0/G1, S and G2/M phase in WEHI-3 cells at 24 h (A) and 48 h (B) following GA treatment. Data are representative of 3 experiments with similar results.

GA induces DNA damage and apoptosis in WEHI-3 cells

DAPI staining and comet assay were used for investigating GA-induced DNA damage and chromatin condensation (apoptosis) of WEHI-3 cells after exposure to various concentrations of GA. GA induced DNA condensation as shown by an increased fluorescent intensity (Fig. 3A) indicating that GA induced apoptosis. GA induced DNA damage as noted by a longer comet tail (Fig. 3B). The higher the GA concentration, the longer the comet tail and the lower the number of cells (Fig. 3B).

Figure 3 GA induces apoptosis and DNA damage in WEHI-3 cells. Cells (2×105 cells/well) were placed in 24-well plates and grown for 24 h. The cells were incubated with 0, 200, 250, 300, 350 and 400 μM of GA for 24 h and and 48 h were then isolated for the examination of apoptosis using DAPI staining (A); or for the examination of DNA damage using the comet assay (B). Images were captured under fluorescence microscopy as in Materials and methods. *P<0.05 and ***P<0.001 show a significant difference.

GA induces apoptotic death of WEHI-3 cells

After treatment with GA for 24 and 48 h, WEHI-3 cells were harvested for DNA isolation followed by DNA gel electrophoresis (Fig. 4). GA induced DNA fragmentation (DNA ladder) in WEHI-3 cells at all examined concentrations.

Figure 4 GA induces DNA fragmentations of WEHI-3 cells. Cells (1×106 cells/dish) were placed in 10-cm dishes and were treated with 0, 200, 250, 300, 350 and 400 μM of GA for 24 h (Left) and 48 h (Right) to examine apoptosis. Cells were harvested for DNA isolation and then DNA gel electrophoresis was performed as described in Materials and methods.

GA affects reactive oxygen species (ROS) production, decreases the level of ΔΨm and promotes caspase-3 activity in WEHI-3 cells

WEHI-3 cells were treated with 300 μM GA for various time periods and were then harvested for the measurements of ROS production, the levels of ΔΨm and caspase-3 activity. GA promoted the production of ROS (Fig. 5A) and caspase-3 activities (Fig. 5C) but decreased the levels of ΔΨm (Fig. 5B) in WEHI-3 cells.

Figure 5 GA affects reactive oxygen species (ROS) production, decreases the level of mitochondrial membrane potential (ΔΨm) and promotes caspase-3 activity in WEHI-3 cells. Cells (2×105 cells/well) in 24-well plates were treated with or without 300 μM of GA for various time periods and were harvested and re-suspended in 500 μl of H2DCF-DA for determination of ROS (A); DiOC6 for assessment of ΔΨm (B); and caspase-3 substrate solution for determination of caspase-3 activity (C). Cells were then incubated at 37°C for 30 min and were analyzed by flow cytometry as described in Materials and methods. Columns, mean (n=3); bars, SD. *P<0.05 and ***P<0.001 indicate a significant difference compared with the control.

GA affects levels of apoptosis-associated proteins in WEHI-3 cells

Cells treated with GA were then harvested for determination of apoptotic-associated protein levels by using western blotting. Results indicated that GA promoted the protein expression of Fas, FasL and caspase-3 (Fig. 6A); Bid, Bax, AIF, cytochrome c and Endo G (Fig. 6B); IRE-1-α, calpain-1, caspase-12 and GRP 78 (Fig. 6C); catalase, SOD (Cu/Zn) and SOD (Mn) (Fig. 6D). This indicated that GA induced apoptosis in WEHI-3 cells through caspase-dependent, ER stress and mitochondria-dependent pathways.

Figure 6 GA affects apoptosis-associated proteins in WEHI-3 cells. Cells (1×106 cells/dish) seeded in 10-cm dishes were then treated with 300 μM of GA and were incubated for 0, 6, 12, 24, 36 and 48 h. Cells were harvested for western blotting to examine the protein levels of Fas, Fas L and caspase-3 (A); Bid, Bax, AIF, cytochrome c and Endo G (B); IRE-1-α, calpain-1 (full and cleaved), caspase-12 and GRP 78 (C); catalase, SOD (Cu/Zn) and SOD (Mn) (D) as described in Materials and methods.

GA affects the AIF, cytochrome c and Endo G expression in WEHI-3 cells

Cells were treated with GA for 24 h and the expression levels of AIF, cytochrome c and Endo G were examined (Fig. 7). GA promoted the release of AIF, cytochrome c and Endo G from mitochondria. These results indicated that GA induced apoptosis in WEHI-3 cells via a mitochondria-dependent pathway.

Figure 7 GA affects the AIF, cytochrome c and Endo G expression in WEHI-3 cells. Cells (5×104 cells/well) placed on 4-well chamber slides were treated with 300 μM of GA for 24 h, fixed and stained using anti-AIF, anti-cytochrome c and anti-Endo G (1:100) overnight and then stained with a secondary antibody (FITC-conjugated goat anti-mouse IgG at 1:100 dilution) (green fluorescence) followed by mitochondrial and nuclear counterstaining individually performed with MitoTracker® Red CMXRos (Molecular Probes) and PI (red fluorescence). Photomicrographs were obtained using a Leica TCS SP2 confocal spectral microscope as described in Materials and methods.

Discussion

Although numerous studies have shown that GA induces cytotoxic effects in many type of cancer cells (10,15,28,29), there is no report demonstrating that GA induces apoptosis in mouse leukemia cells. In the present study, we investigated the effects of GA on mouse leukemia WEHI-3 cells and the results indicated that GA decreased the percentage of viable cells and induced apoptosis in WEHI-3 cells. Furthermore, we also demonstrated that GA induced ROS production (Fig. 5A) and decreased the levels of ΔΨm (Fig. 5B) which was assayed by flow cytometry.

It is well documented that apoptosis may be divided into caspase-dependent and -independent pathways (30,31). The results in the present study indicate that GA decreased the percentage of viable cells through the induction of apoptosis based on the observations of DAPI staining (Fig. 3A) and flow cytometric assay (Fig. 2). Additionally, ROS is known to be involved in the induction of apoptosis after cells are exposed to various compounds (32,33). Our results (Fig. 5A) demonstrated that GA promoted ROS production in WEHI-3 cells. These results indicated that GA induced apoptosis via ROS production. The ER stress pathway is also another possible signaling pathway involved in agent-induced apoptosis in cancer cells (34,35). The hallmarkers of ER stress, such as the expression of GRP 78 and GADD153 are able to activate caspase-12 and IRE-1α (34–36). In the present study, we also found that GA promoted the expression of GRP78 (Fig. 6C) and GADD153 (data not shown) which was measured by western blotting. We suggested that GA induced apoptosis in part through ER stress.

It is well documented that agent-induced cancer cell apoptosis occurs through a mitochondria-dependent pathway (31). We also observed that GA decreased the levels of ΔΨm in WEHI-3 cells (Fig. 5B) in a time-dependent manner. Furthermore, results from western blotting also showed that GA promoted the expression of cytochrome c, AIF and Endo G which are released from mitochondria (Fig. 6). Thus, we suggest that GA induces apoptosis, in part, through the caspase-independent and mitochondria-dependent pathways. These findings also were confirmed using a confocal laser systems microscope which demonstrated that GA promoted the release of AIF, cytochrome c and Endo G (Fig. 6B).

Overall, our study showed that the natural compound GA acts as an apoptosis-inducing agent against mouse leukemia cells in vitro. In our WEHI-3 mouse leukemia cell study, GA not only induced cytotoxic effects, but also suppressed cell growth and induced apoptosis (Fig. 8). This highly correlates with the inhibition of numerous biomarkers linked to apoptosis via caspase-dependent and -independent, ER stress and mitochondria-dependent pathways.

Figure 8 The possible signaling pathways for GA-induced apoptosis through ROS, ER stress, caspase-3- and mitochondria-dependent pathways in mouse leukemia WEHI-3 cells.

Acknowledgements

This study was supported by the grant CMU100-ASIA-4 from the China Medical University and by the Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence (DOH101-TD-C-111-005).

References