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. 2018 May;15(5):6931-6940.
doi: 10.3892/ol.2018.8209. Epub 2018 Mar 8.

Combined application of Embelin and tumor necrosis factor-related apoptosis-inducing ligand inhibits proliferation and invasion in osteosarcoma cells via caspase-induced apoptosis

Hao Qian  1 , Yao Chen  1 , Tao Huang  1 , Tiemin Liu  2 , Xiucheng Li  1 , Guangjian Jiang  2 , Wei Zhang  1 , Shuo Cheng  1 , Pengcheng Li  1
Affiliations

Combined application of Embelin and tumor necrosis factor-related apoptosis-inducing ligand inhibits proliferation and invasion in osteosarcoma cells via caspase-induced apoptosis

Hao Qian et al. Oncol Lett. 2018 May.

Abstract

Embelin, as an inhibitor of the X-linked inhibitor of apoptosis protein (XIAP), may induce apoptosis in various types of cancer cells. The present study aimed to determine the effect of Embelin on the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of osteosarcoma cells. Embelin and TRAIL were applied to U2OS and MG63 cells, respectively or in combination. MTT was initially used to detect the difference in survival rates between the group receiving combined application of 100 ng/ml TRAIL and 20 µmol/l Embelin and the individual application groups. Light microscopic quantification was used to detect the morphology of the osteosarcoma cells in each group. Determination of cell apoptosis was subsequently performed using flow cytometry. The invasive ability of the cells was detected by a Transwell assay, prior to relative protein expression being determined by western blot analysis. Based on all the test data, it was revealed that the survival rates and the invasive ability were significantly lower following the combined application of 100 ng/ml TRAIL and 20 µmol/l Embelin than following the individual application of either (P<0.01). Additionally, upregulating expression of caspases, as well as death receptor 5, and downregulating expression of XIAP and matrix metalloproteinase 9 (MMP-9), had more significant effects in the combined group compared with the individual group and the control group. All these results suggested that Embelin may enhance TRAIL-induced apoptosis and inhibit the invasion of human osteosarcoma cells.

Keywords: Embelin; TRAIL; apoptosis; invasion; osteosarcoma.

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Figures

Figure 1.
Figure 1.
Viability of U2OS and MG63 cells, as measured by MTT after 12, 24 or 48. (A) The viability rate of U2OS cells treated with different concentrations of TRAIL. (B) The viability rate of MG63 cells treated with different concentrations of TRAIL. (C) The viability rate of U2OS cells treated with different concentration of Embelin. (D) The viability rate of MG63 cells treated with different concentration of Embelin. (E) The viability rate of U2OS cells treated with a combination of 100 ng/ml TRAIL and 20 µmol/l Embelin. (F) The viability rate of MG63 cells treated with a combination of 100 ng/ml TRAIL and 20 µmol/l Embelin. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. the control group; #P<0.01 vs. the TRAIL group or Embelin group. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
Figure 2.
Figure 2.
Morphological changes and fluorescent staining of U2OS and MG63 cells treated with different drugs after 48 h. (A) The morphological appearance of U2OS cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or treated with a combination of the two) under an inverted phase contrast microscope. (B) The morphological appearance of MG63 cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or treated with a combination of the two) under an inverted phase contrast microscope. (C) Fluorescent staining of U2OS cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or a combination of the two) under a fluorescence microscope. (D) Fluorescent staining of MG63 cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or a combination of the two) under a fluorescence microscope. Magnification, ×400. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
Figure 3.
Figure 3.
Morphological changes of U2OS and MG63 cells under the effects of chemotherapeutic agents after 48 h. (A) U2OS cells was co-treated with 100 ng/ml TRAIL and 20 µmol/l Embelin in the presence of 150 µM z-VAD-fmk or 150 µM z-IETD-fmk and images were captured under an inverted phase contrast microscope. (B) MG63 cells were co-treated with 100 ng/ml TRAIL and 20 µmol/l Embelin in the presence of 150 µM z-VAD-fmk or 150 µM z-IETD-fmk and images were captured under an inverted phase contrast microscope. Magnification, ×400. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
Figure 4.
Figure 4.
Invasion in U2OS and MG63 cells treated with different drugs after 24 h. (A) Crystal violet staining of the U2OS cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or treated with a combination of the two) that passed through the polycarbonate membrane under an inverted phase contrast microscope. (B) The crystal violet staining of the MG63 cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or treated with a combination of the two) that passed through the polycarbonate membrane under an inverted phase contrast microscope. (C) Quantification of U2OS cells that had attached to the basal side of the membrane. (D) Quantification of MG63 cells that had attached to the basal side of the membrane. Data are presented as the mean ± standard deviation of three independent experiments. Magnification, ×400. *P<0.05 vs. the control group. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
Figure 5.
Figure 5.
Apoptosis in U2OS and MG63 cells treated with different drugs after 24 h. The four groups of (A) U2OS and (B) MG63 cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or treated with a combination of the two) were incubated for 24 h. Cells stained with FITC-conjugated Annexin V and PI were analyzed by flow cytometric analysis. In the lower right quadrant, early apoptotic cells were observed, and the necrotic or late apoptotic cells were located in the upper right quadrant. The percentage of early apoptotic, late apoptotic and total apoptotic (C) U2OS and (D) MG63 cells (control, treated with 100 ng/ml TRAIL, treated with 20 µmol/l Embelin or treated with a combination of the two) for 24 h. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.01 vs. control group; #P<0.01 vs. TRAIL group or Embelin group. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; FITC, fluorescein isothiocyanate; PI, propidium iodide.
Figure 6.
Figure 6.
Western blot analysis of the expression of proteins after U2OS cells were cultured for 48 h. (A) The levels of caspase-3 (32 kDa), caspase-8 (18 kDa) and cleaved caspase-3 (17 kDa) were analyzed by western blot analysis. There was a marked increase in the expression of caspase-3, cleaved caspase-3, caspase-8 in the combined treatment group, P<0.01. (B) The levels of XIAP (55 kDa), MMP-9 (92 kDa) and DR5 (46 KDa) were analyzed by western blot analysis. There was a marked downregulation in the expression of XIAP and MMP-9 in the combined treatment group, P<0.01. However, there was a marked upregulation in the expression of DR5 in the combination group, P<0.01. (C) Quantification of relative protein expression. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.01 vs. control group; #P<0.01 vs. TRAIL group or Embelin group. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis protein; MMP-9, matrix metalloproteinase 9; DR5, death receptor 5.
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