2.2. Licochalcone A Induces Apoptosis and Cell Arrest

To determine whether programmed cell death was involved in the anti-proliferative effect of Licochalcone A, we analyzed the rate of apoptosis cells in Licochalcone A-treated HOS cells and MG-63 cells by Annexin V and PI staining observed by flow cytometry. The data showed that the rate of Annexin V positive cells was significantly increased after exposure to Licochalcone A (30 μM or 40 μM) for 24 h in both lines of osteosarcoma cells (Figure 2A), indicating Licochalcone A has the potential to induce apoptosis in osteosarcoma cell lines. To determine whether caspase activation was involved in Licochalcone A–induced apoptosis, we measured the protein levels of the activated forms of caspase-3, -8, and -9 and PARP by Western blot analysis in treated HOS cells and MG-63 cells. The data showed that treatment with Licochalcone A (20–40 μM) for 24 h resulted in up-regulated activated forms of caspase 8, caspase 3, and PARP, but decreased activated forms of caspase 9 and Bax (Figure 2B). Besides, we also observed that treatment with Licochalcone A both resulted in down-regulation of pro-survival protein Bcl-2 and inhibitors of the apoptosis protein (IAP) family such as XIAP and survivin (Figure 2B). These findings suggest that Licochalcone A induces apoptosis by caspase 8 and caspase 3 signaling pathway.

Open in a separate window

Figure 2

Licochalcone A induces apoptosis in osteosarcoma cells. Osteosarcoma HOS cells or MG-63 cells were treated with Licochalcone A (30 μM) for 24 h. To detect apoptosis, the HOS cells or MG-63 cells were stained with Annexin V and propidium iodide (PI), and analyzed using flow cytometry. Quantitative results of Annexin V positive cells are shown (A). Expression of apoptosis-related proteins was measured by Western blotting (B). Experiments were conducted with three biological replicates per condition, and the values represent the mean ± SD.

Cell cycle distribution of HOS cells or MG-63 cells treated with Licochalcone A (30 μM, a concentration close to IC₅₀ at 24 h for both cell lines) for different time points were analyzed by flow cytometry. The results showed that a significant accumulation of 4N cells (G2/M phase cells) was induced in Licochalcone A-treated HOS cells (Figure 3A) and MG-63 cells (Figure 3B). Furthermore, we evaluated the expression of proteins that regulate the G2/M phase transition by Western blot assay. The data showed that the protein level of phospho-cdc2, cdc2, and Cdc25C were decreased in treated both cell lines, but Cyclin B1 protein levels had no apparent change in HOS cells and was decreased in MG-63 cells (Figure 3C), indicating Licochalcone A induces G2/M phase arrest of osteosarcoma cell lines.

Open in a separate window

Figure 3

Licochalcone A induces G2/M phase arrest in osteosarcoma cell lines. HOS cells (A) or MG-63 cells (B) were treated with Licochalcone A (30 μM), and harvested in the indicated time points. The cells were stained with propidium iodide and analyzed by flow cytometer. 2n corresponds to G1 phase cells and 4n corresponds to the G2/M phase cells. The ration of G2/M to G1 phase cells were showed in right panel. Experiments were conducted with three biological replicates per condition, and the values represent the mean ± SD. (C) Licochalcone A decreases the expression of p-cdc2, cdc2, and cdc25c in osteosarcoma cell lines. HOS cells or MG-63 cells were treated with Licochalcone A (30 μM), and harvested in the indicated time points. The treated cells were analyzed by Western blotting using the indicated antibodies.

2.3. Activation of Chk2 and ATM in Response to Licochalcone A

To identify the mechanism involving in Licochalcone A-induced G2/M phase arrest in osteosarcoma cells, we tested the activation of ATM-Chk2 and ATR-Chk1, the primary pathway for activating G2/M checkpoint. The data showed that the treatment with Licochalcone A induced rapid phosphorylation of Chk2 (at threonine 68) and ATM (at serine 1981) (Figure 4A), but did not induce Chk1 phosphorylation (at serine 345) (data not shown). Interestingly, we observed the threonine 68-phosphorylated form of Chk2 formed distinct nuclear foci in response to Licochalcone A treatment in HOS cells (Figure 4B). These data suggest that ATM-Chk2 pathway may contribute to Licochalcone A-induced G2/M phase arrest in osteosarcoma cells. Recently, it has been reported that oxidative stress can activate ATM [26]. We examined the cellular redox status following Licochalcone A treatment in osteosarcoma MG-63 cells using 2′,7′-dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that measures reactive oxygen species (ROS) within the cell. The data showed that ROS levels were significantly elevated in Licochalcone A-treated cells. Thus, we propose oxidative stress to be involved in Licochalcone A-mediated activation of ATM.

Open in a separate window

Figure 4

Activation of Chk2 and ATM in response to Licochalcone A. (A) ATM-Chk2 pathway is activated in Licochalcone A -treated HOS cells. HOS cells or MG-63 cells were treated with Licochalcone A (30 μM), and harvested in the indicated time points. The treated cells were analyzed by Western blotting using the indicated antibodies. The levels of p-Chk2 (Thr 68) and p-ATM (Ser 1981) were quantified and are shown below each blot. (B) Licochalcone A induces phospho-Chk2 T68 foci. HOS cells were treated with Licochalcone A (30 μM), and 2 h later were fixed with formaldehyde, permealized with Triton X-100, and then immunostained with antibody to phospho-Chk2 T68 (green color) and 4′,6-diamidino-2-phenylindole (DAPI) for labeling nucleus (blue color). (C) Licochalcone A enhances reactive oxygen species (ROS) generation. MG-63 cells were treated with Licochalcone A (Lico) for 2 h. Intracellular ROS was analyzed by flow cytometry using 2′,7′-dichlorofluorescin diacetate (DCFDA) staining and is shown as median fluorescence intensity. Mean ± SD. is plotted for 3 replicates from each condition.

4.2. Colony Formation Assay

HOS cells were briefly treated with Licochalcone A at various concentrations (0, 10, 20, and 40 μM). Once the expression of proteins that regulate the G2/M phase transition such as Cdc25C were decreased, starting at 6–8 h after Licochalcone A treatment (Figure 3C), the treated cells were washed by PBS three times, trypsinized, plated and maintained onto 35 mm dishes (500 cells/dish) with drug-free complete medium and cultured for another 10–14 days to allow colony formation. Colonies were fixed in 70% ethanol and stained by 1% crystal violet solution before counting.

4.3. Cell Viability Assay

The cytotoxic activity of Licochalcone A was tested using the MTT assay. The human osteosarcoma HOS and MG-63 cells were seeded in 24-well plates for overnight. The cells were treated with different concentrations of Licochalcone A for 24 h or 48 h. At the end of the assay time, the cells was incubated with 15 µL of MTT solution (5 mg/mL) (Invitrogen, Carlsbad, CA, USA) for 2 h at 37 °C. After removing the cultured medium, 200 µL of dimethyl sulfoxide (DMSO) was added to each well. Absorbance at 590 nm of the dissolved formazan product was read on an automated microplate spectrophotometer (Thermo Multiskan SPECTRUM, Thermo Fisher Scientific, Waltham, MA, USA). The half maximal inhibitory concentration (IC₅₀) for HOS and MG-63 cells were calculated by the “Forecast” function in Microsoft Excel.

4.4. Cell Cycle Analysis

The cells were washed by PBS two times, trypsinized, fixed with ice-cold 100% ethanol and kept on −20 °C for overnight. Cells were rehydrated with cold PBS, and then resuspended in PBS with propium iodine (40 µg/mL) (#P4170; Sigma-Aldrich, St. Louis, MO, USA) and Ribonuclease A (0.2 µg/mL) at room temperature for 30 min in the dark. The content of DNA in each sample were analyzed by a Cytomics^TM FC500 flow cytometer (Beckman Coulter; Brea, CA, USA).

4.5. Apoptosis Assay

Licochalcone A-induced apoptosis in HOS cells was determined by flow cytometry using the Annexin-V-FITC staining kit (Becton Dickinson, San Jose, CA, USA) according to the manufacturer’s protocol. Briefly, the treated cells were trypsinized, and washed twice by cold PBS. The cells were incubated with 100 μL of 1× binding buffer with 5 μL of FITC Annexin V and 5 μL of propidium iodide for 15 min at room temperature (RT) in the dark. After incubation, 400 μL of 1× binding buffer was added to each tube, and the fluorescence was measured by a Cytomics^TM FC500 flow cytometer (Beckman Coulter, Miami, FL, USA).

4.6. Immunofluorescence Assays

For immunofluorescence analysis, cells were grown on glass coverslips and fixed in a 4% formaldehyde solution for 15 min at room temperature. After rinsing three times with PBS for 5 min each, the cells were blocked in blocking buffer (1XPBS, 5% normal serum, 0.3% Triton X-100) for 60 min at room temperature. After removing blocking solution, the primary antibodies were diluted in antibody dilution buffer (1XPBS, 1% BSA, 0.3% Triton X-100), and incubated on cells overnight at 4 °C. The coverslips were washed with PBS three times and incubated in fluorescent-dye conjugated secondary antibody diluted in antibody dilution buffer for 2 h at room temperature. The cells were washed with PBS three times and counterstained with DAPI. The cells were examined and photographed by immunofluorescence microscopy. The primary antibodies was used in this assay included Phospho-Chk2 (Thr68) (#2197; Cell Signaling, Danvers, MA, USA), LC3A/B (#12741; Cell Signaling, Danvers, MA, USA). Phalloidin-iFluor 594 reagent (ab176757; Abcam, Cambridge, UK) were used for labeling actin filaments

4.7. Western Blot Analysis

Whole cell lysates were produced using TEGN buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 420 mM NaCl, 10% glycerol, and 0.5% Nonidet P-40) containing proteases inhibitor cocktail (Roche, Mannheim, Germany), phosphatase inhibitors (Roche, Mannheim, Germany), and 1 mM dithiothreitol (DTT). For Western blotting, the cell lysates were boiled in protein sample buffer (2 Mβ-mercaptoethanol, 12% sodium dodecyl sulfate (SDS), 0.5 M Tris, pH 6.8, 0.5 mg/mL bromophenol blue, and 30% glycerol). The samples were analyzed by 8–12% SDS-polyacrylamide gel electrophoresis (PAGE). The primary antibodies were listed as following: cleaved caspase-3 (#9661; Cell Signaling, Danvers, MA, USA), cleaved caspase-8 (#9496; Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9508; Cell Signaling, Danvers, MA, USA), Bcl-2 (#9258; Cell Signaling, Danvers, MA, USA), XIAP (#2042; Cell Signaling, Danvers, MA, USA), Survivin (#2808; Cell Signaling, Danvers, MA, USA), GAPDH (#2118; Cell Signaling, Danvers, MA, USA), actin (A2066; Sigma-Aldrich, St. Louis, MO, USA), PARP (#9542; Cell Signaling, Danvers, MA, USA), Bax (#2772; Cell Signaling, Danvers, MA, USA), Phospho-cdc2 (Tyr15) (#4539; Cell Signaling, Danvers, MA, USA), Phospho-Chk2 (Thr68) (#2197; Cell Signaling, Danvers, MA, USA), LC3A/B (#12741; Cell Signaling, Danvers, MA, USA), cdc2 (06-923SP; Millipore, Temecula, CA, USA), p-ATM (Ser1981) (sc-47739; Santa Cruz, CA, USA), ATM (GTX70103; GeneTex, Irvine, CA, USA), Chk2 (#05-649; EMD Millipore, Temecula, CA, USA).

4.8. Detection of Intracellular ROS

Cellular ROS levels were measured in live cells by 2′,7′-dichlorofluorescin diacetate (DCFDA) (#ab113851, Abcam, Cambridge, UK). After treatment with Licochalcone A, the cells were washed with serum free media, and incubate at 37 °C incubator for 30min with 10 μM DCFDA in serum free media. Finally, the cells were trypsinized and suspended in phosphate-buffered saline (PBS). The fluorescence was detected by a Cytomics™ FC500 flow cytometer (Beckman Coulter, Miami, FL, USA).