Anti-Invasive and Anti-Migratory Effects of Ononin on Human Osteosarcoma Cells by Limiting the MMP2/9 and EGFR-Erk1/2 Pathway

Abstract

Osteosarcoma is a common malignancy of the bone. Due to its high metastatic properties, osteosarcoma becomes the leading cause of cancer death worldwide. Ononin is an isoflavone glycoside known to have various pharmacological properties, including antioxidant and anti-inflammatory activities. In the present study, we aimed to investigate the efficacy of ononin on osteosarcoma cell migration, invasion, and the underlying mechanisms. The in vitro anti-tumorigenic and anti-migratory properties of ononin were determined by MTT, colony formation, invasion, and migration in MG-63 and U2OS osteosarcoma cell lines. The results were compared with the standard chemotherapeutic drug, doxorubicin (DOX), as a positive control. The dose-dependent manners of ononin treatment increased the expression of apoptosis and inhibition of cell proliferation through the EGFR-Erk1/2 signaling pathways. Additionally, ononin significantly inhibited the invasion and migration of human osteosarcoma cells. For consistency, we used the MG-63-xenograft mice model to confirm the in vivo anti-tumorigenic and anti-migratory efficacy of ononin by inhibiting the protein expressions of EGFR-Erk1/2 and MMP2/9. According to the histological study, ononin had no adverse effect on the liver and kidney. Overall, our findings suggested that ononin could be a potentially effective agent against the development and metastasis of osteosarcoma.

Keywords: inhibitory effects; invasion; migration; ononin; osteosarcoma.

Figure 1

Cultured cells were treated with (A) DOX (0.1–3 μM) or (B) ononin (0.1–3 μM) for 48 h. The cell viability was determined by the MTT assay. Data are presented as a percentage change from the control group and in Mean ± SEM, with n = 6. Statistical changes were clustered as significant *** where p < 0.001 as compared with the control group. (C) Cells were seeded into 6-well plate, and after 48 h of treatment, cells were treated with fresh DMEM for another 7 days. Ononin at 0.1 μM was labeled as -L, 0.3 μM was highlighted as m and 1 μM was shown as -H, respectively. Crystal violet was used to stain the colony formation assay.

Figure 2

(A) The viable cell population was displayed in the bottom left quadrant, the early apoptotic cells in the bottom right quadrant, and the late apoptotic cells in the top right quadrant of the dual parametric dot plots integrating annexin V-FITC and PI fluorescence. (B) The quantification analysis was analyzed by the program Flowjo v7.6. (C) The dual parametric dot plots combining caspase 3/7 and SYTOX AADvanced fluorescence showed activated caspase 3/7 in the bottom right quadrant (Q4). (D) The quantification analysis was conducted by the program Flowjo v7.6. Ononin at 0.1 μM was labeled as -L, 0.3 μM was highlighted as -M, and 1 μM was shown as -H. The values are expressed as the percentage of total cell number, n = 6, in Mean ± SEM. DOX (1 μM) was applied as a positive control.

Figure 3

(A) Transwell assay analysis of MG-63 or U2OS treated with ononin for migration activity. (B) Matrigel-coated transwell was used here for evaluating the anti-invasion potential of ononin on osteosarcoma cells. Ononin at 0.1 μM was labeled as -L, 0.3 μM was highlighted as -M, and 1 μM was shown as -H.

Figure 4

(A) Cultured MG-63 or (B) U2OS cells were incubated with DOX (1 μM) or ononin (0.1, 0.3, and 1 μM, labeled as -L, M, and –H) for 2 days, and the target proteins were detected by Western blot (left panel). The quantification of the target protein was calculated with a densitometer (right panel). The values are expressed as the fold of change (X basal), in Mean ± SEM, where n = 3. *** p < 0.01 was considered a significant result when compared to control, and ^### p < 0.001 was considered a significant result when compared to DOX group. Full Western blot images can be found at Supplementary Materials.

Figure 5

(A) Cultured MG-63 or (B) U2OS cells were incubated with DOX (1 μM) or ononin (0.1, 0.3, and 1 μM, labeled as -L, -M, and -H, respectively) for 2 days, and the target proteins were detected by Western blot (left panel). The quantification of the target protein was calculated with a densitometer (right panel). The values are expressed as the fold of changes (X basal), in Mean ± SEM, where n = 3. ** p < 0.01 and *** p < 0.0 1 were considered a significant result when compared to control, and ^## p < 0.01 or ^### p < 0.001 were considered significant results when compared to DOX group. Full Western blot images can be found at Supplementary Materials.

Figure 6

(A) Representative bioluminescence imaging of nude mice xenograft tumor after 42 days of treatment. The control group received PBS; the DOX group received DOX (2 mg/kg/i.p); the ononin group received different amounts of ononin (1, 3, and 10 mg/kg/i.p). (B) The mice’s body weight was recorded during the 42-day investigation. (C) On day 42, tumors were removed from mice. (D) After the studies, the tumor weight was calculated. (E) On day 42, the tumor size was calculated. The data were reported as Mean SEM, with n = 6. *** p < 0.001 was considered a significant result as compared to the control group; ^### p < 0.001 was considered a significant result as compared to the DOX group.

Figure 7

(A) Bioluminescence imaging of the separated lungs from each group of MG-63 xenograft nude mice. (B) Images of lungs from various groups and treatments. (C) The lung has been stained with HE. Ononin at 1, 3, and 10 mg/kg/i.p were shown as -L, -M, and -H, respectively.

Figure 8

The homogenized tumor tissue was subjected to the flow cytometer for analyzing the anti-osteosarcoma cell proliferation properties. (A) The viable cell population was displayed in the bottom left quadrant (Q3), the early apoptotic cells in the bottom right quadrant (Q4), and the late apoptotic cells in the top right quadrant (Q4) of the dual parametric dot plots integrating annexin V-FITC and PI fluorescence (Q2). (B) The quantification analysis was conducted by the program Flowjo v7.6. (C) The dual parametric dot plots combining caspase 3/7 and SYTOX AADvanced fluorescence showed activated caspase 3/7 in the bottom right quadrant (Q4). (D) The quantification analysis was conducted by the program Flowjo v7.6. The values are expressed as the percentage of total cell number, n = 6, in Mean ± SEM. Ononin at 1, 3, and 10 mg/kg/i.p were shown as -L, -M, and -H, respectively. *** p < 0.001 was considered a significant result when compared to control, and ^## p < 0.01 or ^### p < 0.001 were considered significant results when compared to DOX group.

Figure 9

The homogenized tumor cell was subjected to SDS-PAGE for determining the target genes (A,B) translational functions. GAPDH was used as a loading control. The quantification of the target protein was calculated with a densitometer (right panel). The values are expressed as the fold of changes (X basal), in Mean ± SEM, where n = 6. Ononin at 1, 3, and 10 mg/kg/i.p were shown as -L, -M, and -H, respectively. *** p < 0.001 was considered a significant result as compared with the control, and ^### p < 0.001 was considered a significant result as compared with the DOX group. Full Western blot images can be found at Supplementary Materials.

Figure 10

Biosafety of ononin in vivo. HE staining of (A) heart, (B) liver, and (C) kidney in MG-63 xenograft mice obtained from different groups. Ononin at 1, 3, and 10 mg/kg/i.p were shown as -L, -M, and -H, respectively.

Figure 11

Biochemical index of animals after ononin treatment. The levels of ALT and/or AST in different groups were determined, and values are expressed as the fold of changes (X basal), in Mean ± SEM, with n = 6.