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Journal of Traditional Chinese Medicine logoLink to Journal of Traditional Chinese Medicine
. 2024 Mar 5;44(2):251–259. doi: 10.19852/j.cnki.jtcm.2024.02.002

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

Junli CHANG 1, Fulai ZHAO 1, Xingyuan SUN 1, Xiaoping MA 1, Peng ZHAO 1, Chujie ZHOU 1, Binhao SHI 1, Wenchao GU 2, Yongjun WANG 1, Yanping YANG 1,✉
PMCID: PMC10927409  PMID: 38504531

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

1. INTRODUCTION

Osteosarcoma is a malignant bone tumor, which is most often occurred in children and adolescents, usually in the epiphyseal region of long bones.1,2 Osteosarcoma cells are aggressive and prone to early metastasis, especially in the lungs.3 Patients with metastatic osteosarcoma have a worse prognosis, and are more likely to be resistant to conventional therapies.4 Despite the improvements in treatment, the 5-year survival rate of osteosarcoma patients with metastasis is still only 30%-40%.5,6 Therefore, finding novel and effective strategies to inhibit osteosarcoma is urgent.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines, which can selectively induce cancer cell apoptosis with almost no side effect on normal cells.7,8 Although TRAIL is a therapeutic regimen with broad development prospects, the most osteosarcoma cells are resistant to TRAIL.9 It has been shown that TRAIL combined with chemotherapeutic drugs can reverse drug resistance of tumor cells and exert synergistic effects,10 and the combination of natural drugs with TRAIL can also significantly enhance the therapeutic effect of TRAIL.11,12

Polyphyllin I (PPI) is one of the bioactive components of the Traditional Chinese Medicine Chonglou (Rhizoma Paridis Chonglou). Recent studies have shown that PPI has a wide range of anti-tumor effects in various cancers,13,14 including inducing apoptosis, cell cycle arrest and autophagy, as well as anti-metastasis, anti-angiogenesis and sensitizing chemo-radiotherapies.

The Wnt/β-catenin signaling pathway, also called the canonical Wnt signaling pathway, is a conserved signaling axis participating in diverse physiological processes such as proliferation, differentiation, apoptosis, migration and invasion.15,⇓-17 Increasing evidence indicates that dysregulation of the Wnt/β-catenin cascade contributes to the development and progression of malignant tumors,18,19 including osteosarcoma.20 The glycogen synthase kinase 3β (GSK-3β) mediates the phosphorylation of β-catenin, promoting its ubiquitination and subsequent proteasomal degradation to inhibit the occurrence of certain cancers.21,⇓-23

Our previous work has confirmed that polyphyllin I (PPI), an effective component of Traditional Chinese Medicine Chonglou (Rhizoma Paridis Chonglou), suppresses human osteosarcoma growth by inactivation of Wnt/β-catenin pathway in vitro and in vivo.24,25 However, whether PPI can sensitize TRAIL in the prevention and treatment of osteosarcoma has not been reported, and it is achievable and necessary to explore the synergy and the possible mechanisms of PPI and TRAIL. Therefore, the present study aimed to investigate the synergistic effects of PPI combined with TRAIL on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway.

2. METHODS AND MATERIALS

2.1. Reagents

polyphyllin I was provided by the Institute for Drug Control (Shanghai, China, #111590) and dissolved in dimethyl sulfoxide (DMSO). TRAIL was obtained from PEPROTECH (Rosemont, IL, USA). Fluorescein isothiocyanate (FITC) Annexin V and propidium iodide (PI) were purchased from BD Biosciences (San Jose, CA, USA). The cell cycle detection kit was provided by the Key GENBioTECH (Nanjing, China, KGA512). Antibodies against Cleaved poly (adenosine diphosphate-ribose) polymerase (PARP) (#9542), Vimentin (5G3F10, #3390), N-Cadherin (#13116), C-Myc (D84C12, #5605), Cyclin B1 (#12231), cyclin-dependent kinases 1(CDK1) (#9116), glycogen synthase kinase-3β (GSK-3β) (27C10, #9315), phosphor-GSK-3β (#8213), active β-catenin (D13A1, #8814) and β-catenin (#6238) were purchased from Cell Signaling Technology (Danvers, MA, USA). Trypsin was purchased from Invitrogen (Rockville, MD, USA). Bio (#SML1046) and antibody against β-actin (#A2228) were from Sigma-Aldrich (St. Louis, MO, USA). Radio immuneoprecipitation assay (RIPA) Cell Lysis Buffer (#P0013B) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Matrigel (#356234) was provided by BD Biosciences (San Jose, CA, USA). Transwell chambers were from BD Biosciences (San Jose, CA, USA).

2.2. Cell culture and treatment

The human osteosarcoma cells of MG-63 and U-2 OS cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). MG-63 and U-2 OS cells were cultured in Dulbecco minimum essential medium (DMEM), contained with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL), which were maintained at 37 ℃in a humidified atmosphere of 5% CO2. In this work, MG-63 and U-2 OS cells were treated with (200-600 nM) PPI, 50 ng/mL TRAIL or a combination (200-600 nM PPI plus 50 ng/mL TRAIL) for 24 h.

2.3. Cell viability assay

MG-63 and U-2 OS cells treated with various drugs for 24 h were harvested, the viability was determined by a Countstar automated cell counter, which uses the trypan blue (0.1 %) staining principle to calculate the living cells.

2.4. Observation of the osteosarcoma cell morphologies

MG-63 and U-2 OS cells were treated with PPI (400 nM), TRAIL (50 ng/mL) or their combination (400 nM PPI and 50 ng/mL TRAIL) for 24 h. The morphology, number and adherence of tumor cells were directly observed with inverted phase contrast microscope (Nikon, Tokyo, Japan).

2.5. Apoptosis assay

Cells were double-stained with Annexin V-FITC and PI Apoptosis Detection Kit. MG-63 and U-2 OS cells were treated with PPI (400 nM), TRAIL (50 ng/mL) or their combination (400 nM PPI and 50 ng/mL TRAIL) for 24 h. The harvested cells were washed twice with pre-cooled phosphate buffered solution (PBS), centrifuged at 500 g and 4 ℃ for 5 min, and incubated with 5 μL FITC-labeled Annexin-V at room temperature for 30 min in the dark, and then 5 μL PI (50 μg/mL) for 5 min on ice without light. The flow cytometry (BD Biosciences, San Jose, CA, USA) was used to detect the early apoptotic (Annexin V-FITC stained only) and the late apoptotic (Annexin V-FITC and propidium iodide double-stained) cells.

2.6. Cell cycle analysis

A cell cycle detection kit was used to analyze the cell cycle distribution according to the manufacturer's instructions. MG-63 and U-2 OS cells were treated with (400 nM) PPI, 50 ng/mL TRAIL or the combination (400 nM PPI and 50 ng/mL TRAIL) for 24 h. The cells was fixed with cold 70% ethanol overnight and then washed twice with cold PBS. The fixed cells were incubated with 40 μL of RNase A at 37 ℃ for 30 min, and then with 160 μL of propidium iodide at 4 ℃ for another 30 min in the dark. The flow cytometry (BD Biosciences, San Jose, CA, USA) was used to detect the cell cycle distribution.

2.7. Migration assay

The xCELLigence real time cell analysis DP system was used to determine the migration of MG-63 and U-2 OS cells. In each well of the designed CIM-Plate 16, put a total volume of 165 μL of 10% FBS-containing medium into the lower chamber, and 40 μL of FBS-free medium into the upper chamber. The pore size is 8 μm. And 100 μL (3 × 104 cells) of cell suspension was added with different concentration drugs (400 nM PPI, 50 ng/mL TRAIL or PPI and TRAIL combination). Then the cell migration was continuously monitored every 15 min for 24 h.

2.8. Transwell invasion assay

The invasive ability of MG-63 and U-2 OS cells was calculated by the number of cells that passed through a polycarbonate membrane (8 µm). The matrigel was diluted with serum-free DMEM medium at 1∶3. Then, the diluted Matrigel (20 μL) was added to the upper chamber. In the upper chamber, each well was seeded with 1 × 105 cells, while the lower chamber was added with 500 μL DMED medium supplemented with 10% FBS. After cultured for 24 h, cells were washed by PBS then fixed by paraformaldehyde for 20 min, stained with 0.1% crystal violet for 15 min, then removed the uninvaded cells in the upper chamber and washed twice with PBS, and then took pictures and counted cells number under an inverted phase contrast microscope.

2.9. Western blotting assay

The cells were washed twice with pre-cooled PBS and then lysed on ice with RIPA Cell Lysis Buffer for 30 min. Protein concentration was measured by the bicinchoninic acid (BCA) method. Cell extracts (30 µg protein) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred by electroblotting to polyvinylidene fluoride membranes. The primary antibodies were incubated at 4 ℃ overnight and the secondary antibodies were incubated at room temperature for 1 h. The ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA) and the software Image Lab version (Bio-Rad, Hercules, CA, USA) were used to acquire the images of the Western blots.

2.10. Statistical analysis

The mean ± standard deviation ($\bar{x}±s$) were calculated for each group. The components were analyzed by one-way analysis of variance (GraphPad Software, Boston, MA, USA). P < 0.05 was considered statistically significant.

3. RESULTS

3.1. PPI sensitizes TRAIL-inhibited osteosarcoma cell viability

Recent studies have confirmed that combined chemotherapy enhance the sensitivity of cancer cells to TRAIL and reduce its drug resistance.26,27 To determine the synergetic effect of TRAIL and PPI on osteosarcoma cells, the MG-63 and U-2 OS cells were first treated with different concentrations of TRAIL (12.5, 25, 50, 100, 200 ng/mL) or PPI (200，400, 600 nM) for 24 h. TRAIL showed no inhibitory effect on the viabilities of MG-63 and U-2 OS cells at a dosage lower than 50 ng/mL (Table 1). Meanwhile, the effective concentration of PPI on the inhibition of MG-63 and U-2 OS cell viabilities is initiated from 600 nM (Table 2). However, the combination of 400 nM PPI with 50 ng/mL TRAIL significantly inhibited MG-63 and U-2 OS cell viabilities (P < 0.01), which were fundamentally significant than 400 nM PPI or 50 ng/mL TRAIL only, indicating the synergic efficacy of TRAIL with PPI (Table 3) in the inhibition of osteosarcoma cell viability.

Table 1.

Change of viability in MG-63 and U-2 OS cells were treated with various concentrations of TRAIL for 24 h (%,$\bar{x}±s$)

Group n MG-63 U-2 OS
TRAIL-Ctrl 3 97.32±0.61 97.06±0.17
TRAIL-12.5 ng/mL 3 98.99±0.39 98.51±0.97
TRAIL-25 ng/mL 3 98.33±0.58 97.99±0.59
TRAIL-50 ng/mL 3 84.58±3.71 85.99±0.65
TRAIL-100 ng/mL 3 40.72±5.56a 67.78±4.25a
TRAIL-200 ng/mL 3 17.96±2.98a 38.62±0.39a
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Notes: Ctrl: treated with PBS; 12.5 ng/mL: treated with 12.5 ng/mL TRAIL; 25 ng/mL: treated with 25 ng/mL TRAIL; 50 ng/mL: treated with 50 ng/mL TRAIL; 100 ng/mL: treated with 100 ng/mL TRAIL; 200 ng/mL: treated with 200 ng/mL TRAIL. Cells were treated with various concentrations of TRAIL for 24 h. TRAIL: tumor necrosis factor-related apoptosis-inducing ligand. The components were analyzed by one-way analysis of variance. aP < 0.001 versus the control group.

Table 2.

Change of viability in MG-63 and U-2 OS cells were treated with various concentrations of PPI for 24 h (%, $\bar{x}±s$)

Group n MG-63 U-2 OS
PPI-Ctrl 3 95.18±0.19 98.52±0.12
PPI-200 nM 3 91.66±1.62 97.32±0.18
PPI-400 nM 3 88.27±0.89 90.64±1.39
PPI-600 nM 3 72.03±3.51a 80.52±0.79a
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Notes: Ctrl: treated with PBS; 200 nM: treated with 200 nM PPI; 400 nM PPI: treated with 400 nM PPI; 600 nM PPI: treated with 600 nM PPI. Cells were treated with various concentrations of PPI for 24 h. PPI: polyphyllin I. The components were analyzed by one-way analysis of variance. aP < 0.05 versus the control group.

Table 3.

Change of viability in MG-63 and U-2 OS cells (%,$\bar{x}±s$)

Group n MG-63 U-2 OS
Ctrl 3 95.18±0.19 98.52±0.12
PPI 3 88.27±0.89 90.64±1.39
TRAIL 3 88.55±0.66 87.52±1.53
PPI+TRAIL 3 48.18±2.58abc 55.78±1.81ade
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Notes: 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. PPI: polyphyllin I; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand. The components were analyzed by one-way analysis of variance. 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.

3.2. PPI sensitizes TRAIL-induced osteosarcoma cell apoptosis

Our previous studies have identified that PPI induces osteosarcoma cell apoptosis.24,25 Here we intended to determine the potential synergetic effects of PPI and TRAIL on osteosarcoma cell apoptosis. MG-63 and U-2 OS cells were treated with 400 nM PPI or 50 ng/mL TRAIL or their combination (400 nM PPI with 50 ng/mL TRAIL) for 24 h. Apoptosis was determined by flow cytometry. As shown in Figure 1A, 400 nM PPI or 50 ng/mL TRAIL only did not significantly induce the apoptosis of MG-63 and U-2 OS cells, however, the combination of 400 nM PPI with 50 ng/mL TRAIL statistically significantly promoted the apoptosis of MG-63 and U-2 OS cells (P < 0.001) (Figure 1A，Table 4). The cell morphology observed by the microscope showed that the number of floating round cells was increased significantly in the combination group (Figure 1B). Cleaved PARP, the activated apoptosis-related protein PARP, is an apoptotic effector protein. The expression of Cleaved PARP was detected by Western blot assay, which showed that PPI (200-600 nM) dose dependently enhanced TRAIL (50 ng/mL) induced Cleaved PARP in MG-63 and U-2 OS cell lines (Figure 1C), which further confirmed the results of flow cytometry. These data suggest that PPI dose dependently sensitizes the ability of TRAIL to induce the apoptosis of osteosarcoma cells.

Figure 1. PPI sensitizes TRAIL-induced osteosarcoma cells apoptosis.

Figure 1

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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.

Table 4.

Statistics of apoptosis rates (%,$\bar{x}±s$)

Group n MG-63 U-2 OS
Ctrl 3 3.81±0.66 4.16±0.16
PPI 3 30.4±1.08 16.81±0.19
TRAIL 3 13.6±0.58 9.93±0.12
PPI+TRAIL 3 79.48±1.05abc 42.17±0.14abc
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Notes: 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. PPI: polyphyllin I; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand. The components were analyzed by one-way analysis of variance. aP < 0.001 versus the control group; bP < 0.001 versus the PPI group; cP < 0.001 versus the TRAIL group.

3.3. PPI sensitizes TRAIL-induced cell cycle arrest in osteosarcoma cells

The potential synergetic effects of PPI and TRAIL on the cell cycle of osteosarcoma cells were determined by flow cytometry. The combined application of PPI (400 nM) and TRAIL (50 ng/mL) significantly decreased the cell numbers in the G0/G1 phase and significantly increased the cell numbers in the G2/M phase, indicating a G2/M phase arrest (Figure 2A). Subsequently, we detected the expressions of the cell cycle-related proteins, including C-Myc, Cyclin B1 and CDK1, which revealed that the decrease of these protein expressions was most significant in the combination group versus the control group, as well as the PPI or TRAIL only group with a dose dependent manner (Figure 2B). These data suggested that PPI and TRAIL had synergistic effects in cell cycle arrest.

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

Figure 2

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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.

3.4. PPI sensitizes TRAIL-induced inhibition of osteosarcoma cell migration and invasion

Metastasis is considered be one of the criteria to judge the malignant degree of tumors. Epithelial-mesenchymal transition (EMT) is an important point during tumor metastasis, also plays a critical role in the migration and invasion of osteosarcoma cells. To observe whether the combination of PPI and TRAIL enhances TRAIL-induced migration and invasion of osteosarcoma cells, xCELLigence RTCA DP system and transwell assays were utilized to examine the ability of migration and invasion in MG-63 and U-2 OS cells. Compared with the PPI or the TRAIL group, the migration ability was the weakest in the combination of PPI and TRAIL group (Figure 3A). Compared with the other three groups, the invasion cell number was the lowest in the PPI and TRAIL combination group, and the difference was statistically significant (P < 0.01) (Table 5). Further, detecting the expressions of EMT-associated proteins in MG-63 and U-2 OS cells by Western blotting, it was identified that the inhibitory effect on Vimentin and N-cadherin expressions in the PPI and TRAIL combination group is stronger than PPI or TRAIL only (Figure 3B). These data suggest the synergistic effects of PPI and TRAIL on inhibiting the migration and invasion of osteosarcoma cells.

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

Figure 3

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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.

Table 5.

Statistics of invasion cell numbers ($\bar{x}±s$)

Group n MG-63 U-2 OS
Ctrl 3 449±36 659±48
PPI 3 201±6 297±18
TRAIL 3 245±13 367±27
PPI+TRAIL 3 104±7abc 178±17acd
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Notes: 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. PPI: polyphyllin I; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand. The components were analyzed by one-way analysis of variance. aP < 0.001 versus the control group; bP < 0.001, dP < 0.01 versus the PPI group; cP < 0.001 versus the TRAIL group.

3.5. Combination of PPI and TRAIL inhibits the progression of osteosarcoma by downregulating the Wnt/β-catenin signaling pathway

Wnt/β-catenin signaling pathway plays an important role in osteosarcoma development, including cell proliferation, apoptosis, cell cycle regulation, and EMT. Our previous work identified that β-catenin was up-regulated in osteosarcoma patients.25 To confirm whether the combined inhibition of osteosarcoma cells by PPI and TRAIL were regulated by Wnt/β-catenin signaling, we detected the expressions of Wnt/β-catenin signaling pathway associated proteins by Western blotting. It has been found that PPI and TRAIL synergistically inhibit the expressions of active β-catenin and phosphorylated glycogen synthase kinase-3β (p-GSK-3β) (Figure 4A).

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

Figure 4

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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.

To verify the synergistic effect of PPI with TRAIL in the treatment of osteosarcoma is achieved by specifically regulating the Wnt/β-catenin signaling pathway, MG-63 and U-2 OS cells were pretreated with BIO (a small molecule inhibitor of GSK-3β, that is a Wnt/β-catenin pathway specific activator), and then treated cells with 400 nM PPI, 50 ng/mL TRAIL or 400 nM PPI combined with 50 ng/mL TRAIL, respectively. Western blotting analysis indicated that the active-β-catenin expression in the BIO + PPI + TRAIL group was higher than the single or combination groups, but lower than the BIO group (Figure 4B), indicating that PPI and TRAIL synergistically inhibited Wnt/β-catenin signaling pathway activity was partially rescued by BIO. Our study suggested that PPI combined with TRAIL plays a synergistic role in inhibiting the growth of osteosarcoma cells, at least partly by inhibiting the activity of the Wnt/ β-catenin signaling pathway.

4. DISCUSSION

Osteosarcoma is the most common malignant bone tumor in children and adolescents and is prone to metastasis.6 Current treatments include surgery, chemo-radiotherapy, or their combination, but there has been no significant improvement in survival rates for patients with osteosarcoma over the past few decades. Besides, osteosarcoma often produces multidrug resistance, which brings great difficulties for treatment.

PPI is one of the bioactive components of the Traditional Chinese Medicine Chonglou (Rhizoma Paridis Chonglou), which shows obvious anti-cancer effects in various cancers, including osteosarcoma.24

TRAIL selectively induces cancer cell apoptosis without obvious side effect on most normal cells, thus it has great potential in cancer therapy.28 However, most cancers are not sensitive to TRAIL, which really limits its application.

Recent studies have reported that the efficacy of TRAIL is improved by combination with other anticancer drugs. Celecoxib sensitized TRAIL-resistant MG-63 cells to TRAIL-induced apoptosis through downregulation of cellular B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein, caspase-8 and caspase-3.29 Embelin enhanced TRAIL-induced apoptosis by downregulating expressions of x-linked inhibitor of apoptosis protein and matrix metalloproteinase 9 in osteosarcoma cells.30 These studies demonstrated that combination therapy with TRAIL and anticancer drugs significantly inhibited cell viability and induced apoptosis compared with single drug treatment.

In this study, TRAIL combined with PPI has been found to synergistically inhibit the growth of osteosarcoma cells and induce osteosarcoma cell apoptosis, while the single drug treatment effect was not significant. To our knowledge, this is the first study to investigate the combined effect of TRAIL and PPI on osteosarcoma.

Meanwhile, cleaved-PARP, which is well known to be the most important indicator of apoptosis, 31 was found to be significantly upregulated in the combined treatment group than in the control group and the individual treatment groups. Our current work also revealed that cells in the G2/M phase was blocked by down-regulation of cell cycle-related proteins, including C-Myc, CyclinB1 and CDK1, which was also more obvious in the combined treatment group. These results provide further evidences that combination therapy is more effective than single drug therapy in cancer treatment.

In addition, osteosarcoma is prone to pulmonary metastasis, leading to a great challenge for treatment.32 EMT is widely involved in and promotes osteosarcoma metastasis.33,34 Therefore, inhibiting the EMT process of osteosarcoma is a potential therapeutic strategy. We investigated the effect of the combined application of PPI and TRAIL on the EMT of MG-63 and U-2 OS cells, which indicated that the combination of PPI and TRAIL had a greater effect on the migration and invasion of MG-63 and U-2 OS cells than PPI or TRAIL used individually. Simultaneously, we found that the expression levels of N-cadherin and vimentin, the EMT markers, were significantly decreased in combined group than in individual group and control group. These results suggested that the combination of TRAIL and PPI may be more effective than single agents in the treatment of osteosarcoma metastasis.

Dysregulation of the Wnt/β-catenin signaling pathway affects the downstream gene expression and is associated with many cancers, including osteosarcoma.35,⇓-37 Our previous study has shown that PPI suppressed human osteosarcoma growth by inactivation of Wnt/β-catenin signaling.25 The sensitivity of melanoma to TRAIL has been reported to be regulated by the activity change of Wnt/β-catenin signaling pathway.38 Therefore, we also examined the effect of PPI combined with TRAIL on Wnt/β-catenin signaling pathway in this study to investigate the mechanism of PPI enhanced TRAIL-induced inhibition of human osteosarcoma cell growth. Our results showed that the expression of p-GSK-3β (Ser-9 site) was significantly lower in combined group than in the individual group and the control group, indicating that the GSK-3β activity was activated. Meanwhile, the active-β-catenin (non-phosphorylated-β-catenin) was also significantly downregulated in the combined group than in the individual group and the control group, which was consistent with activated GSK-3β. Since the activated GSK-3β mediates β-catenin phosphorylation and results in a reduced non-phos-phorylated β-catenin, which promotes its ubiquitination and subsequent proteasomal degradation,39,40 and then inhibits the nucleus translocation of β-catenin to reduce the cancer occurrence.41,⇓,⇓-44 These results suggested that the synergistic effect of PPI and TRAIL was mediated via the inactivation of Wnt/β-catenin pathway. Furthermore, the BIO (a small molecule inhibitor of GSK-3β, that is a Wnt/β-catenin pathway specific activator45) was found to offset the synergistic effect of PPI combined with TRAIL. Thus, our findings suggest that the combination of PPI and TRAIL exerts a synergistic effect on inhibiting the growth of osteosarcoma via downregulating the Wnt/β-catenin signaling pathway.

In conclusion, the combination of PPI and TRAIL plays a synergistic role in promoting apoptosis and cell cycle arrest, as well as inhibiting the migration and invasion of osteosarcoma cells by downregulating the activity of the Wnt/β-catenin signaling pathway. Our findings may throw light on osteosarcoma treatment and bring hope to the clinical management of the osteosarcoma patients. However, drug efficacy test in vitro may be different from that in vivo given the impact of human pharmacokinetics. Therefore, further in vivo investigations may need to clarify the molecular mechanisms underlying the present findings.

REFERENCES

  • 1. Saraf AJ, Fenger JM, Roberts RD. . Osteosarcoma: accelerating progress makes for a hopeful future. Front Oncol 2018; 8: 4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Ligon JA, Choi W, Cojocaru G, et al. . Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes. J Immunother Cancer 2021; 9: e001772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Miwa S, Shirai T, Yamamoto N, et al. . Current and emerging targets in immunotherapy for osteosarcoma. J Oncol 2019; 2019: 7035045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Tsukamoto S, Errani C, Angelini A, Mavrogenis AF. . Current treatment considerations for osteosarcoma metastatic at presentation. Orthopedics 2020; 43: e345-8. [DOI] [PubMed] [Google Scholar]
  • 5. Anderson ME. . Update on survival in osteosarcoma. Orthop Clin North Am 2016; 47: 283-92. [DOI] [PubMed] [Google Scholar]
  • 6. Eaton BR, Schwarz R, Vatner R, et al. . Osteosarcoma. Pediatr Blood Cancer 2021; 68: e28352. [DOI] [PubMed] [Google Scholar]
  • 7. Gamie Z, Kapriniotis K, Papanikolaou D, et al. . TNF-related apoptosis-inducing ligand (TRAIL) for bone sarcoma treatment: Pre-clinical and clinical data. Cancer Lett 2017; 409: 66-80. [DOI] [PubMed] [Google Scholar]
  • 8. Zhao J, Lu Y, Shen HM. . Targeting p53 as a therapeutic strategy in sensitizing TRAIL-induced apoptosis in cancer cells. Cancer Lett 2012; 314: 8-23. [DOI] [PubMed] [Google Scholar]
  • 9. Deng X, Yi X, Huang D, et al. . ROCK2 mediates osteosarcoma progression and TRAIL resistance by modulating O-GlcNAc transferase degradation. Am J Cancer Res 2020; 10: 781-98. [PMC free article] [PubMed] [Google Scholar]
  • 10. Singh D, Prasad CB, Biswas D, et al. . TRAIL receptors are differentially regulated and clinically significant in gallbladder cancer. Pathology 2020; 52: 348-58. [DOI] [PubMed] [Google Scholar]
  • 11. Moon MH, Jeong JK, Seo JS, et al. . Bisphosphonate enhances TRAIL sensitivity to human osteosarcoma cells via death receptor 5 upregulation. Exp Mol Med 2011; 43: 138-45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Shankar S, Ganapathy S, Srivastava RK. . Sulforaphane enhances the therapeutic potential of TRAIL in prostate cancer orthotopic model through regulation of apoptosis, metastasis, and angiogenesis. Clin Cancer Res 2008; 14: 6855-66. [DOI] [PubMed] [Google Scholar]
  • 13. Tian Y, Gong GY, Ma LL, Wang ZQ, Song D, Fang MY. . Anti-cancer effects of polyphyllin I: an update in 5 years. Chem Biol Interact 2020; 316: 108936. [DOI] [PubMed] [Google Scholar]
  • 14. Wang Q, Zhou X, Zhao Y, et al. . Polyphyllin I ameliorates collagen-induced arthritis by suppressing the inflammation response in macrophages through the NF-κB pathway. Front Immunol 2018; 9: 2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Li K, Zhang J, Tian Y, et al. . The Wnt/β-catenin/VASP positive feedback loop drives cell proliferation and migration in breast cancer. Oncogene 2020; 39: 2258-74. [DOI] [PubMed] [Google Scholar]
  • 16. Zhang LN, Zhao L, Yan XL, Huang YH. . Loss of G3BP1 suppresses proliferation, migration, and invasion of esophageal cancer cells via Wnt/β-catenin and PI3K/AKT signaling pathways. J Cell Physiol 2019; 234: 20469-84. [DOI] [PubMed] [Google Scholar]
  • 17. Zhang Y, Wang X. . Targeting the Wnt/β-catenin signaling pathway in cancer. J Hematol Oncol 2020; 13: 165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. He S, Tang S. . WNT/β-catenin signaling in the development of liver cancers. Biomed Pharmacother 2020; 132: 110851. [DOI] [PubMed] [Google Scholar]
  • 19. Gajos-Michniewicz A, Czyz M. . WNT signaling in melanoma. Int J Mol Sci 2020; 21: 4852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Liu Y, Liu YZ, Zhang RX, et al. . Oridonin inhibits the proliferation of human osteosarcoma cells by suppressing Wnt/β-catenin signaling. Int J Oncol 2014; 45: 795-803. [DOI] [PubMed] [Google Scholar]
  • 21. Nusse R, Clevers H. . Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell 2017; 169: 985-99. [DOI] [PubMed] [Google Scholar]
  • 22. Wiese KE, Nusse R, van Amerongen R. . Wnt signalling: conquering complexity. Development 2018; 145: dev165902. [DOI] [PubMed] [Google Scholar]
  • 23. Ono M, Yin P, Navarro A, et al. . Paracrine activation of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth. Proc Natl Acad Sci USA 2013; 110: 17053-58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Chang J, Wang H, Wang X, et al. . Molecular mechanisms of polyphyllin I-induced apoptosis and reversal of the epithelial-mesenchymal transition in human osteosarcoma cells. J Ethnopharmacol 2015; 170: 117-27. [DOI] [PubMed] [Google Scholar]
  • 25. Chang J, Li Y, Wang X, et al. . Polyphyllin I suppresses human osteosarcoma growth by inactivation of Wnt/β-catenin pathway in vitro and in vivo. Sci Rep 2017; 7: 7605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Gallego-Lleyda A, De Miguel D, Anel A, Martinez-Lostao L. . Lipid nanoparticles decorated with TNF-related aptosis-inducing ligand (TRAIL) are more cytotoxic than soluble recombinant TRAIL in sarcoma. Int J Mol Sci 2018; 19: 1449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Kretz AL, von Karstedt S, Hillenbrand A, et al. . Should we keep walking along the trail for pancreatic cancer treatment? Revisiting TNF-related apoptosis-inducing ligand for anticancer therapy. Cancers (Basel) 2018; 10: 77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Matsui TA, Sowa Y, Yoshida T, et al. . Sulforaphane enhances TRAIL-induced apoptosis through the induction of DR5 expression in human osteosarcoma cells. Carcinogenesis 2006; 27: 1768-77. [DOI] [PubMed] [Google Scholar]
  • 29. Liu G, Yu MY, Huang X, et al. . Synergistic effect of celecoxib in tumor necrosis factor‑related apoptosis‑inducing ligand treatment in osteosarcoma cells. Mol Med Rep 2014; 10: 2198-202. [DOI] [PubMed] [Google Scholar]
  • 30. Qian H, Chen Y, Huang T, et al. . Combined application of embelin and tumor necrosis factor-related apoptosis-inducing ligand inhibits proliferation and invasion in osteosarcoma cells via caspase-induced apoptosis. Oncol Lett 2018; 15: 6931-40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Li B, Chen R, Chen L, et al. . Effects of DDIT4 in Methamphetamine-induced autophagy and apoptosis in dopaminergic neurons. Mol Neurobiol 2017; 54: 1642-60. [DOI] [PubMed] [Google Scholar]
  • 32. Meazza C, Scanagatta P. . Metastatic osteosarcoma: a challenging multidisciplinary treatment. Expert Rev Anticancer Ther 2016; 16: 543-56. [DOI] [PubMed] [Google Scholar]
  • 33. Jiang X, Zhang Z, Song C, et al. . Glaucocalyxin a reverses EMT and TGF-β1-induced EMT by inhibiting TGF-β1/Smad2/ 3 signaling pathway in osteosarcoma. Chem Biol Interact 2019; 307: 158-66. [DOI] [PubMed] [Google Scholar]
  • 34. Shen S, Huang K, Wu Y, et al. . A miR-135b-TAZ positive feedback loop promotes epithelial-mesenchymal transition (EMT) and tumorigenesis in osteosarcoma. Cancer Lett 2017; 407: 32-44. [DOI] [PubMed] [Google Scholar]
  • 35. Ghosh N, Hossain U, Mandal A, Sil PC. . The Wnt signaling pathway: a potential therapeutic target against cancer. Ann N Y Acad Sci 2019; 1443: 54-74. [DOI] [PubMed] [Google Scholar]
  • 36. Vijayakumar S, Liu G, Rus IA, et al. . High-frequency canonical Wnt activation in multiple sarcoma subtypes drives proliferation through a TCF/β-catenin target gene, CDC25A. Cancer Cell 2011; 19: 601-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Zhang W, Duan N, Zhang Q, et al. . DNA methylation mediated down-regulation of miR-370 regulates cell growth through activation of the Wnt/β-catenin signaling pathway in human osteosarcoma cells. Int J Biol Sci 2017; 13: 561-73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Zimmerman ZF, Kulikauskas RM, Bomsztyk K, Moon RT, Chien AJ. . Activation of Wnt/β-catenin signaling increases apoptosis in melanoma cells treated with trail. PLoS One 2013; 8: e69593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Danieau G, Morice S, Rédini F, Verrecchia F, Royer BB. . New insights about the Wnt/β-catenin signaling pathway in primary bone tumors and their microenvironment: a promising target to develop therapeutic strategies. Int J Mol Sci 2019; 20: 3751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Hsieh CH, Hsu HH, Shibu MA, et al. . Down-regulation of β-catenin and the associated migration ability by Taiwanin C in arecoline and 4-NQO-induced oral cancer cells via GSK-3β activation. Mol Carcinog 2017; 56: 1055-67. [DOI] [PubMed] [Google Scholar]
  • 41. Cai Y, Mohseny AB, Karperien M, Hogendoorn PC, Zhou G, Cleton-Jansen AM. . Inactive Wnt/beta-catenin pathway in conventional high-grade osteosarcoma. J Pathol 2010; 220: 24-33. [DOI] [PubMed] [Google Scholar]
  • 42. Clevers H. . Wnt/beta-catenin signaling in development and disease. Cell 2006; 127: 469-80. [DOI] [PubMed] [Google Scholar]
  • 43. Sha L, Ma D, Chen C. . Exosome-mediated Hic-5 regulates proliferation and apoptosis of osteosarcoma via Wnt/β-catenin signal pathway. Aging (Albany NY) 2020; 12: 23598-608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Pai SG, Carneiro BA, Mota JM, et al. . Wnt/beta-catenin pathway: modulating anticancer immune response. J Hematol Oncol 2017; 10: 101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Yadav PS, Feng S, Cong Q, Kim H, Liu Y, Yang Y. . Stat3 loss in mesenchymal progenitors causes Job syndrome-like skeletal defects by reducing Wnt/β-catenin signaling. Proc Natl Acad Sci USA 2021; 118: e2020100118. [DOI] [PMC free article] [PubMed] [Google Scholar]

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