Licochalcone A Suppresses the Proliferation of Osteosarcoma Cells through Autophagy and ATM-Chk2 Activation

Abstract

Licochalcone A, a flavonoid extracted from licorice root, has been shown to exhibit broad anti-inflammatory, anti-bacterial, anticancer, and antioxidative bioactivity. In this study, we investigated the antitumor activity of Licochalcone A against human osteosarcoma cell lines. The data showed that Licochalcone A significantly suppressed cell viability in MTT assay and colony formation assay in osteosarcoma cell lines. Exposure to Licochalcone A blocked cell cycle progression at the G2/M transition and induced extrinsic apoptotic pathway in osteosarcoma cell lines. Furthermore, we found the Licochalcone A exposure resulted in rapid ATM and Chk2 activation, and high levels of nuclear foci of phosphorylated Chk2 at Thr 68 site in osteosarcoma cell lines. In addition, Licochalcone A exposure significantly induced autophagy in osteosarcoma cell lines. When Licochalcone A-induced autophagy was blocked by the autophagy inhibitor chloroquine, the expression of activated caspase-3 and Annexin V positive cells were reduced, and cell viability was rescued in Licochalcone A-treated osteosarcoma cell lines. These data indicate that the activation of ATM-Chk2 checkpoint pathway and autophagy may contribute to Licochalcone A-induced anti-proliferating effect in osteosarcoma cell lines. Our findings display the possibility that Licochalcone A may serve as a potential therapeutic agent against osteosarcoma.

Keywords: ATM-Chk2; Licochalcone A; autophagy; osteosarcoma.

Figure 1

Licochalcone A inhibits cell viability of osteosarcoma. (A) The chemical structure of Licochalcone A. (B) Licochalcone A inhibits osteosarcoma cell growth in a dose-dependent manner. MTT assays were performed with osteosarcoma HOS and MG-63 cells exposed to Licochalcone A (Lico A) in the indicated concentrations. Experiments were conducted with three biological replicates per treatment, and the values represent the mean ± SD. (*) p < 0.01 and (**) p < 0.001 as compared with the untreated cells. (C) Licochalcone A suppresses colony formation of osteosarcoma cell lines. HOS cells were plated in colony formation assays after treatment with Licochalcone A for 7 h. Five hundred cells were plated per dish. All experiments were performed in triplicate, and the figure above shows a representative example.

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.

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.

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.

Figure 5

Autophagy is induced in Licochalcone A -treated osteosarcoma cells. HOS cells and MG-63 cells were treated with indicated concentrations of Licochalcone A (Lico A) for 24 h. The treated cells were analyzed by Western blotting using the indicated antibodies (A), or were immunostained with LC3 antibody for autophagy formation (green color), phalloidin-iFluor 594 reagent for labeling actin filaments (red color), and 4′,6-diamidino-2-phenylindole (DAPI) for labeling nucleus (blue color) (B). The levels of actin were quantified and are shown below the blot in Western blotting.

Figure 6

Autophagy is involved in the apoptosis effect of Licochalcone A treatment. (A) Autophagy inhibitor, chloroquine, suppresses Licochalcone-induced caspase 3 activation. HOS cells were treated with indicated concentrations of Licochalcone A (Lico A) with or without chloroquine for 24 h. The treated cells were analyzed by Western blotting using the indicated antibodies. (B) Autophagy inhibitor chloroquine suppresses Licochalcone-induced apoptosis. HOS cells were treated with Licochalcone A (Lico A) (40 μM) with or without chloroquine for 24 h. To detect apoptosis, the HOS cells were stained with Annexin V and propidium iodide (PI), and analyzed using flow cytometry. Quantitative results of Annexin V positive cells are shown in the lower panels. (C) Autophagy inhibitor chloroquine rescues the anti-proliferative effect of Licochalcone A treatment. HOS cells were treated as described in (B). MTT assay was performed to determine cell viability in treated cells. Experiments were conducted with three biological replicates per treatment, and the values represent the mean ± SD. (*) p < 0.01 and (**) p < 0.001 as compared with the control group.