Polyphyllin I enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth downregulating the Wnt/β-catenin pathway

Abstract

Objective: To investigate the synergistic effects of polyphyllin I (PPI) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway.

Methods: Cell viability, apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays. The morphology of cancer cells was observed with inverted phase contrast microscope. The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays. The expressions of poly (adenosine diphosphate-ribose) polymerase, C-Myc, Cyclin B1, cyclin-dependent kinases 1, N-cadherin, Vimentin, Active-β-catenin, β-catenin, p-glycogen synthase kinase 3β (GSK-3β) and GSK-3β were determined by Western blotting assay.

Results: PPI sensitized TRAIL-induced decrease of viability, migration and invasion, as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells. The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/β-catenin signaling pathway.

Conclusion: The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Keywords: Wnt signaling pathway; beta-catenin; osteosarcoma; polyphyllin I; tumor necrosis factor-related apoptosis-inducing ligand.

Figure 1. PPI sensitizes TRAIL-induced osteosarcoma cells apoptosis

MG-63 and U-2 OS cells were treated with 400 nM PPI, 50 ng/mL TRAIL or a combination (400 nM PPI plus 50 ng/mL TRAIL) for 24 h, then detected the apoptosis by flow cytometry. A: representative images of apoptosis. A1: control group of MG-63 cell lines; A2: PPI group of MG-63 cell lines; A3: TRAIL group of MG-63 cell lines; A4: PPI + TRAIL group of MG-63 cell lines; A5: control group of U-2 OS cell lines; A6: PPI group of U-2 OS cell lines; A7: TRAIL group of U-2 OS cell lines; A8: PPI + TRAIL group of U-2 OS cell lines. B: morphological images under an inverted phase contrast microscope (magnification, × 40). B1: control group of MG-63 cell lines; B2: PPI group of MG-63 cell lines; B3: TRAIL group of MG-63 cell lines; B4: PPI+TRAIL group of MG-63 cell lines; B5: control group of U-2 OS cell lines; B6: PPI group of U-2 OS cell lines; B7: TRAIL group of U-2 OS cell lines; B8: PPI + TRAIL group of U-2 OS cell lines. C: expressions of apoptosis-related proteins (total PARP and cleaved-PARP) were detected by Western blotting analysis. The β-actin was used as a loading control for Western blotting analysis. 1, 9: control group; 2, 10: 200 nM PPI group; 3, 11: 400 nM PPI group; 4, 12: 600 nM PPI group; 5, 13: 50 ng/mL TRAIL group; 6, 14: 200 nM PPI + 50 ng/mL TRAIL group; 7, 15: 400 nM PPI+50 ng/mL TRAIL group; 8, 16: 600 nM PPI + 50 ng/mL TRAIL group. Ctrl: treated with PBS; PPI: treated with 400 nM PPI; TRAIL: treated with 50 ng/mL TRAIL; PPI+TRAIL: treated with 400 nM PPI plus 50 ng/mL TRAIL. Cells were treated for 24 h. FITC: fluorescein isothiocyanate; PPI: polyphyllin I; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand; PARP: poly (adenosine diphosphate-ribose) polymerase.

Figure 2. PPI and TRAIL synergistically arrest the cell cycle of osteosarcoma cells at G₂/M

MG-63 and U-2 OS cells were treated with 400 nM PPI, 50 ng/mL TRAIL or a combination (400 nM PPI plus 50 ng/mL TRAIL) for 24 h, then detected the cell cycle by flow cytometry. A: percent distribution of specific phases in the cell cycle. A1: ratio of G₀/G₁, S and G₂/M period of MG-63 cell; A2: ratio of G₀/G₁, S and G₂/M period of U-2 OS cell. B: expression levels of key proteins involved in cell cycle (C-Myc, Cyclin B1 and CDK1) were detected by Western blotting analysis. The β-actin was used as a loading control for Western blotting analysis. 1, 9: control group; 2, 10: 200 nM PPI group; 3, 11: 400 nM PPI group; 4, 12: 600 nM PPI group; 5, 13: 50 ng/mL TRAIL group; 6, 14: 200 nM PPI + 50 ng/mL TRAIL group; 7 and 15: 400 nM PPI + 50 ng/mL TRAIL group; 8, 16: 600 nM PPI + 50 ng/mL TRAIL group. Ctrl: treated with PBS; PPI: treated with 400 nM PPI; TRAIL: treated with 50 ng/mL TRAIL; PPI + TRAIL: treated with 400 nM PPI plus 50 ng/mL TRAIL. Cells were treated for 24 h. G₀/G₁: gap0/gap1, the gap time from the completion of mitosis to the start of DNA replication; S: synthesis phase, the period of DNA replication; G₂/M: gap2/mitosis, the ratio of the period from the completion of DNA replication to the beginning of mitosis and mitosis; CDK1: cyclin-dependent kinases 1; PPI: polyphyllin I; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand. Data are presented as the mean ± standard deviation of three independent experiments. ^aP < 0.001 versus the control group; ^bP < 0.001, ^dP < 0.01 versus the PPI group; ^cP < 0.001, ^eP < 0.01 versus the TRAIL group.

Figure 3. PPI sensitizes TRAIL-inhibited osteosarcoma cell migration and invasion

A: migration of MG-63 and U-2 OS cells were assessed by the xCELLigence RTCA DP system respectively, as described in Materials and Methods. A1: MG-63; A2: U-2 OS. B: The expressions of proteins involved in EMT (N-cadherin and vimentin) were detected by Western blotting analysis. The β-actin was used as a loading control for Western blotting analysis. 1, 9: control group; 2, 10: 200 nM PPI group; 3, 11: 400 nM PPI group; 4, 12: 600 nM PPI group; 5, 13: 50 ng/mL TRAIL group; 6, 14: 200 nM PPI + 50 ng/mL TRAIL group; 7, 15: 400 nM PPI + 50 ng/mL TRAIL group; 8, 16: 600 nM PPI + 50 ng/mL TRAIL group. Ctrl: treated with PBS; PPI: treated with 400 nM PPI; TRAIL: treated with 50 ng/mL TRAIL; PPI + TRAIL: treated with 400 nM PPI plus 50 ng/mL TRAIL. Cells were treated for 24 h. RTCA: real time cell analysis; EMT: epithelial-mesenchymal transition; PPI: polyphyllin I; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand.

Figure 4. Combination of PPI and TRAIL inhibits osteosarcoma development by downregulating the activity of the Wnt/β-catenin signaling pathway

A: MG-63 and U-2 OS cells were exposed to different concentrations of PPI, 50 ng/mL TRAIL or their combination for 24 h. The expression levels of active-β-catenin, p-GSK-3β and total GSK-3β proteins were detected by Western blotting analysis. 1, 9: control group; 2, 10: 200 nM PPI group; 3, 11: 400 nM PPI group; 4, 12: 600 nM PPI group; 5, 13: 50 ng/mL TRAIL group; 6, 14: 200 nM PPI+50 ng/mL TRAIL group; 7, 15: 400 nM PPI + 50 ng/mL TRAIL group; 8, 16: 600 nM PPI + 50 ng/mL TRAIL group. B: MG-63 and U-2 OS cells pretreated with 4 μM BIO (the specific GSK-3β inhibitor that is a Wnt/β-catenin pathway activator) for 24 h were exposed to 400 nM PPI, 50 ng/mL TRAIL or a combination (400 nM PPI plus 50 ng/mL TRAIL) for 24 h. The expression levels of active-β-catenin, p-GSK-3β and total GSK-3β proteins were detected by Western blotting analysis. 1, 7: control group; 2, 8: 4 μM BIO group; 3, 9: 400 nM PPI group; 4, 10: 50 ng/mL TRAIL group; 5, 11: 400 nM PPI + 50 ng/mL TRAIL group; 6, 12: 4 μM BIO + 400 nM PPI + 50 ng/mL TRAIL group. The β-actin was used as a loading control for Western blotting analysis. GSK-3β: glycogen synthase kinase 3β; PPI: polyphyllin I; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand; BIO: 6-Bromoindirubin-3'-oxime.