Ginsenoside impedes proliferation and induces apoptosis of human osteosarcoma cells by down-regulating β -catenin 1

Abstract

BACKGROUND:

Osteosarcoma (OS) is the most commonly occurred primary bone malignancy with high incident rates among children and adolescents. In pharmacologic treatment, the drug ginsenoside has been shown to exert anticancer effects on several malignant diseases. The purpose of this research was to investigate the effect of ginsenoside on the apoptosis and proliferation of human OS MG-63 and Saos-2 cells by regulating the expression of $\beta$ -catenin.

METHODS:

Human OS MG-63 and Saos-2 cells were assigned into control group, and four groups with treatment by varying concentrations (12.5 $\mu$ g/mL, 25 $\mu$ g/mL, 50 $\mu$ g/mL and 100 $\mu$ g/mL) of ginsenoside, respectively. Cell growth after treatment was observed through cell slides. The proliferation rate of MG-63 and Saos-2 cells in each group was detected by CCK-8. After cell transfection at 48 h, cell cycle and cell apoptosis were detected by FITC-Annexin V staining and flow cytometry. The protein and mRNA expressions of $\beta$ -catenin, Cyclin D1, Bcl-2, Bax and cleaved caspase-3 were detected by RT-qPCR and western blot analysis.

RESULTS:

With increased exposure and concentration of ginsenoside, the cell density, total cell numbers and the absorbance of MG-63 and Saos-2 cells gradually decreased. FITC-Annexin V and FITC-Annexin V/PI staining demonstrated that the cell proportion at S phase decreased, whereas the total apoptotic rate of MG-63 and Saos-2 cells was increased. Furthermore, RT-qPCR and western blot analysis highlighted a gradual decrease in protein and mRNA expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1, while an elevation in those of Bax and cleaved caspase-3.

CONCLUSION:

The results of this study demonstrate that ginsenoside inhibits proliferation and promotes apoptosis of human OS MG-63 and Saos-2 cells by reducing the expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1 and increasing the expression of Bax and cleaved caspase-3.

1. Introduction

Osteosarcoma (OS) is the most common primary malignancy of bone in children and young adults, which is characterized by the emergence of primitively transformed cells in spindle shapes of mesenchymal origin and the formation of malignant osteoid or immature osteoid matrix [1]. OS occurs mostly in children and adolescent under 20 years old, comprising 2.4% of all malignant tumors in pediatric patients and approximately 20% of all primary bone cancers [2, 3]. Conventional treatment for patients with primary OS consists of a combination of surgery, neoadjuvant and adjuvant chemotherapy [4]. After the introduction of chemotherapy, the 5-year survival rate of OS patients increased to 65% from 20%, owing to the eradication of micro-metastases, even after surgical amputation [5]. The risk factors of OS include both environmental factors and non-environmental factors, such as remedial radiation, certain chemicals, Paget’s disease, genetic variants and several inherited cancer predisposition syndromes [6, 7]. However, the exact etiology of this disease still remains to be discovered. OS is highly aggressive and approximately 20% of patients have lung metastasis at the preliminary stage of diagnosis, whereby metastasis will occur in 40% of patients at of the late stage of this disease [8]. Therefore, a novel therapeutic method for OS remains to be a research of great value.

The health benefits of plants have been explored for years. One of significant discovery known as ginseng was discovered over 5,000 years ago in the hills of Manchuria in China, and has always been one of the most useful traditional medicines used over thousands of years in Korea, Japan and China [9]. Ginsenosides are a class of natural product steroid glycosides and triterpene saponins that are extracted from ginseng. They form the primary bioactive compositions of ginseng, which have been classified into three major types: panaxadiol, including Rb1, Rb2, Rg3, Rd, Rc, Rg3, Rh2 and Rs1); panaxatriol, including Rg1, Rg2, Re, Rf and Rh1; and oleanolic acid type ginsenosides, including Ro [10]. It has been demonstrated that ginsenosides can benefit the central nervous system in many ways, such as promoting neural survival, neurite growth and curing neurons from pathological states [11]. Plenty of natural antitumor products characterized by its high safety and efficacy can be extracted from ginsenosides. As such, ginsenosides are believed to be a potential therapeutic candidate in the treatments of many types of cancers [12]. It is revealed that ginsenosides exerts its anti-tumor effects on reducing cell proliferation, metastasis and increasing cell death in different kinds of cancer cells both in vitro and in vivo through modulating different signaling pathways. These pathways include Cyclin D1, B-cell lymphoma 2 (Bcl-2), and WNT, $\beta$ -catenin signaling pathways [13]. Therefore, in this study we aimed to explore the effect of ginsenoside on the proliferation and apoptosis of human OS MG-63 and Saos-2 cells by regulating the expression of $\beta$ -catenin.

2. Materials and methods

2.1 Cell culture and grouping

Human OS MG-63 cells, purchased from Shanghai Seymour biotechnology Development Corporation, and Saos-2 cells, purchased from Bei Na Biotechnology Co., Ltd. (Beijing, China), were incubated in an RPMI-1640 medium with 10% fetal bovine serum (FBS) (Gibco Company, Grand Island, NY, USA) at 37 ${}^{\circ }$ C with 5% CO ${}_2$ . Cells were sub-cultured and detached with 0.25% trypsin every 1–2 days. After the cell confluence reached approximately 60%, they were digested and treated with 0.25% trypsin to be collected for centrifugation at 1000 rpm for 5 min. After the removal of supernatant, the cell precipitation was washed using culture medium twice. After 5 minutes of centrifugation at 1000 rpm, cells were re-suspended in serum-free medium, counted, and diluted to produce a cell suspension of 1 $\times$ 10 ${}^8$ cells/mL. The cells with a density of 4 $\times$ 10 ${}^3$ were inoculated in 12-well plates, followed by the replacement of medium on the second day. Cells were then treated with different concentrations of ginsenoside (Ginsenoside Rh2 purchased from National Institute for the Control of Pharmaceutical and Biological Products). Human OS cells were assigned into control group and four groups respectively treated with 12.5 $\mu$ g/mL, 25 $\mu$ g/mL, 50 $\mu$ g/mL and 100 $\mu$ g/mL of ginsenoside. Cells were treated with ginsenoside once a day, and the control group was treated with normal saline. Cells were respectively collected at different time periods at 24 h, 48 h, and 72 h for later experiments.

2.2 Preparation of cell slides

Human OS cells collected at different time points were detached with 0.25% trypsin and then cultured in 6-well plates with coverslips at 37 ${}^{\circ }$ C, 5% CO ${}_2$ , followed by the addition of 2 mL of culture medium. The medium was removed on the following day and cells were washed three times with phosphate buffer saline (PBS) and fixed with 4 g/L of paraformaldehyde for 30 min. The plates were then rinsed thrice with PBS, and the gum coverslips surface without cells adhering to the glass slides followed by hematoxylin-eosin (HE) staining after leaving to dry for 1–2 days. The coverslips were then stained by hematoxylin for 10 min, rinsed with distilled water and differentiated with 0.5% hydrochloride for 5 seconds. Cells were rinsed with water for 5 seconds before and after a PBS rinse to allow the color to return to blue. The cells were then stained by eosin for 40 seconds and treated with 75%, 85%, 95% and 100% gradient ethanol for 2 min, respectively. Cells were sealed after xylene treatment for 1 min three times. The cell morphology was observed using an electron microscope.

2.3 Cell counting kit-8 (CCK-8) assay

Human OS cells collected at different time intervals were seeded into 96-well plates at the density of 2 $\times$ 10 ${}^4$ cells/mL with 100 $\mu$ L of cells in each well. After 24 h, cell viability was detected by CCK-8 assay immediately according to the following procedures: cells in each well were added with 10 $\mu$ L of CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) and cultured for 60 min. The absorbance (A) was detected by a microplate reader (Thermo Fisher Scientific, San Jose, California, USA) at 450 nm wavelength.

2.4 Flow cytometry

BD Annexin V-EGFP kit (Becton, Dickinson and Company, NJ, USA) was used to determine the proportion of cells in each group. Human OS cells that were collected after 48 h were seeded into 6-well plates at a concentration of 2 $\times$ 10 ${}^5$ cells/well. After 24 h, the supernatant was collected in a 5 mL tube and treated with 400 $\mu$ L trypsin. After cells became round, the supernatant was used for neutralization reaction. Cells were then centrifuged for 5 min at 2000 rpm, and the supernatant was removed afterwards. After rinsing the cells twice in 5% albumin from bovine serum (BSA), the cells were re-suspended with 300 $\mu$ L of 5% BSA and fixed with 700 $\mu$ L ethanol at $-$ 20 ${}^{\circ }$ C for 24 h. Cells were collected and then centrifuged at 2000 rpm for 5 min. The cells were stained with FITC-Annexin V in accordance with the following steps: cells were rinsed with 1 mL of PBS, re-suspended with 100 $\mu$ L of PBS followed by the addition of 1 $\mu$ L 10 mg/ $\mu$ L RNase A and incubated for 30 min at 37 ${}^{\circ }$ C. After staining with 300 $\mu$ L of 50 $\mu$ g/mL PI for 20 min, cells were tested by flow cytometry. For propidium iodide staining, cells were rinsed twice with 2% BSA with PBS. After removal of the supernatant, cells were collected and re-suspended with 500 $\mu$ L of Binding Buffer. The cells were then stained with 5 $\mu$ L of Annexin V-EGFP and 5 $\mu$ L of PI at room temperature for 5–15 min in a dark room. Cells were analyzed with BD FACS flow cytometry following the manufacturer’s directions (Becton, Dickinson and Company, NJ, USA).

2.5 Annexin V-FITC/PI flow cytometry

The transfected cells in each group collected after 48 h were seeded in 6-well plates and incubated with 5% CO ${}_2$ at 37 ${}^{\circ }$ C. After reaching 80% confluence, the medium was by a new RPMI-1640 culture medium. Cells were collected at a concentration of 1 $\times$ 10 ${}^5$ after digestion by the trypsin without ethylenediaminetetraacetic acid (EDTA) and re-suspended in 500 $\mu$ L of binding buffer. Cells were then mixed with 5 $\mu$ L of Annexin V-FITC and 5 $\mu$ L of Propidium Iodide (Nanjing KeyGen Biotech Co. Ltd. Nanjing, China), and reacted at room temperature for 5–15 min in a dark room. Flow cytometry and fluorescence activated cell sorting (FACS) (Becton, Dickinson and Company, NJ, USA) were employed to detect the number of cells that underwent apoptosis as well as to count the number of early and late apoptotic cells. This information was then used to calculate the apoptotic rate of cells (early apoptosis rate $+$ late apoptosis rate).

Table 1

Primer sequences for RT-qPCR

Gene	Sequence
$\beta$ -catenin	F: 5’-GCAGGCACT-3’
	R: 5’-CATGCGTGGCAG-3’
Bax	F: 5’-GGGACGAACTGGACAGTAACA-3’
	R: 5’-CCGCCACAAAGATGGTCAC-3’
CyclinD1	F: 5’-ATGCCAACCTCCTCAACGACC-3’
	R: 5’-TGGCACAGAGGGCAACGAAGG-3’
Bcl-2	F: 5’-GGAGAGTGCTGAAGATTG-3’
	R: 5’-ACTTCCTCTGTGATGTTGTA-3’
Cleaved	F: 5’-CAACATTTTTCAGAGGGGATCG-3’
caspase-3	R: 5’-GCATACTGTTTCAGCATGGCAC-3’
GAPDH	F: 5’-ACCACAGTCGATGCCATCAC-3’
	R: 5’-TCCACCACCCTGTTGCTGTA-3’

Note: F, forward; R, reverse; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 1.

Microscopic observation of MG-63 and Saos-2 cell growth ( $\times$ 100). A: characteristics of MG-63 cell number and density at different time points; B: characteristics of Saos-2 cell number and density at different time points.

2.6 Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from 1 $\times$ 10 ${}^7$ cells that had been collected at 48 h from each group using an miRNeasy Mini Kit (IAGEN, GmbH, Germany). Total RNA reverse transcription was performed in a total reaction system of 15 $\mu$ L using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Inc., CA, USA). The reaction conditions were set according to the following: 16 ${}^{\circ }$ C for 30 min, 42 ${}^{\circ }$ C for 30 min and 85 ${}^{\circ }$ C for 15 min. RT-qPCR detection was performed using a TaqMan Universal PCR kit (Applied Biosystems, Inc., CA, USA). The reaction systems were consisted of 20 $\mu$ L and the reaction conditions were as follows: at 95 ${}^{\circ }$ C for 10 min, 95 ${}^{\circ }$ C for 15 seconds, and 60 ${}^{\circ }$ C for 1 min with a total of 40 cycles. RT-qPCR was performed using ABI 7500 (Applied Biosystems, Inc., CA, USA). The total mRNA of each gene was detected in triplicates. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference. Primer sequences are shown in Table 1. Relative quantification was performed by the 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method.

Figure 2.

Ginsenoside suppresses the proliferation of MG-63 and Saos-2 cells. A: proliferation of MG-63 cells treated with different concentration of ginsenoside at 24 h, 48 h and 72 h; B: proliferation of Saos-2 cells treated with different concentration of ginsenoside at 24 h, 48 h and 72 h; a, compared with the control group, $p<$ 0.05; b, compared with 24 h, $p<$ 0.05; c, compared with 48 h, $p<$ 0.05; d, compared with 12.5 $\mu$ g/mL, $p<$ 0.05; e, compared with 25 $\mu$ g/mL, $p<$ 0.05; f, compared with 50 $\mu$ g/mL, $p<$ 0.05; OD, optical density.

2.7 Western blot analysis

The transfected cells in each group collected after 48 h were rinsed with PBS and then treated by lysis buffer with an appropriate protease inhibitor. After shock treatment for 5 min at 4 ${}^{\circ }$ C, the supernatant was collected after centrifugation at 12000 $\times$ g for 10 min at 4 ${}^{\circ }$ C. The concentration of protein was determined by a Protein Quantification kit. The supernatant was added with 6 $\times$ buffer for sample preparation, boiled and stored at $-$ 20 ${}^{\circ }$ C. Protein samples (50 $\mu$ g) were prepared and separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. After blocking with milk, the membrane was diluted with a proportion of 1:200, followed by incubation with mouse anti-human monoclonal antibodies of $\beta$ -catenin (05-665-AF647), Cyclin D1 (CC12-100UGCN), Bax (AB2915), Bcl-2 (MABC74), cleaved caspase-3 (CST #9661) and GAPDH (SF-141) overnight at 4 ${}^{\circ }$ C. The membrane was washed by a mixture of tris-buffered saline and Polysorbate 20 (TBST) four times (10 min per wash). Following the membrane was incubated with IRDyeTM 800DX-labled goat anti-mouse immunoglobulin G (IgG) (1:10000) at room temperature for 1 h, and then, washed by TBST four times (10 min per wash). Finally, the band was developed using substrates. Antibodies were purchased from American Millipore Company. Quantification of protein bands was obtained by analytical processing using LabWorks Image Acquisition and Analysis Software (UVP, Inc., Upland, CA, USA).

2.8 Statistical analysis

SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used for data analysis. The measurement data was expressed as a mean $\pm$ standard deviation. Data comparison between two groups was conducted by $t$ test. One-way analysis of variance (ANOVA) was used to compare data among multiple groups (before analysis, homogeneity of variances was conducted). The pairwise comparison on mean values among mul- tiple groups was conducted using least significant difference (LSD)- $t$ test. Values of $p<$ 0.05 was considered statistically significant.

3. Results

3.1 Ginsenoside reduces the total cell numbers and cell density

The potential effects of ginsenoside on the growth of MG63 and Saos-2 cells were evaluated. As shown in Fig. 1, there were no significant differences in the number of MG63 and Saos-2 cells in the control group at different time points of incubation. After 24 h, the number of MG63 and Saos-2 cells in the ginsenoside-treated groups was lower than those in the control group. However, after 48 h and 72 h, the number of MG63 and Saos-2 cells decreased significantly in the ginsenoside-treated groups compared with the control group. We noted that the number of cells at 72 h incubation period time interval was lower than that at 48 h. Therefore, we conclude that a higher concentration of ginsenoside resulted in decreased cellular growth.

Table 2

Cell apoptosis detected in each group by CCK-8 at different time points

	Group	24 h		48 h		72 h
MG63	Control group	1.03	$\pm$ 0.22	2.74	$\pm$ 0.68 ${}^{\text{b}}$	4.26	$\pm$ 0.63 ${}^{\text{bc}}$
	12.5 $\mu$ g/mL ginsenoside	2.56	$\pm$ 0.40 ${}^{\text{a}}$	4.83	$\pm$ 0.61 ${}^{\text{ab}}$	7.38	$\pm$ 0.69 ${}^{\text{abc}}$
	25 $\mu$ g/mL ginsenoside	4.40	$\pm$ 0.21 ${}^{\text{ad}}$	7.38	$\pm$ 0.74 ${}^{\text{abd}}$	9.24	$\pm$ 0.37 ${}^{\text{abcd}}$
	50 $\mu$ g/mL ginsenoside	6.89	$\pm$ 0.53 ${}^{\text{ade}}$	10.11	$\pm$ 0.87 ${}^{\text{abde}}$	12.69	$\pm$ 0.60 ${}^{\text{abcde}}$
	100 $\mu$ g/mL ginsenoside	9.26	$\pm$ 0.67 ${}^{\text{adef}}$	12.52	$\pm$ 0.94 ${}^{\text{abdef}}$	15.38	$\pm$ 0.68 ${}^{\text{abcdef}}$
Saos-2	Control group	1.76	$\pm$ 0.17	3.32	$\pm$ 0.21 ${}^{\text{b}}$	5.00	$\pm$ 0.25 ${}^{\text{bc}}$
	12.5 $\mu$ g/mL ginsenoside	3.13	$\pm$ 0.32 ${}^{\text{a}}$	4.89	$\pm$ 0.22 ${}^{\text{ab}}$	7.04	$\pm$ 0.38 ${}^{\text{abc}}$
	25 $\mu$ g/mL ginsenoside	4.75	$\pm$ 0.41 ${}^{\text{ad}}$	8.20	$\pm$ 0.47 ${}^{\text{abd}}$	9.33	$\pm$ 0.39 ${}^{\text{abcd}}$
	50 $\mu$ g/mL ginsenoside	7.11	$\pm$ 0.52 ${}^{\text{ade}}$	11.13	$\pm$ 0.88 ${}^{\text{abde}}$	13.44	$\pm$ 0.74 ${}^{\text{abcde}}$
	100 $\mu$ g/mL ginsenoside	8.67	$\pm$ 0.50 ${}^{\text{adef}}$	12.90	$\pm$ 0.68 ${}^{\text{abdef}}$	17.52	$\pm$ 0.98 ${}^{\text{abcdef}}$

Note: a, compared with the control group, $p<$ 0.05; b, compared with 24 h, $p<$ 0.05; c, compared with 48 h, $p<$ 0.05; d, compared with 12.5 $\mu$ g/mL, $p<$ 0.05; e, compared with 25 $\mu$ g/mL, $p<$ 0.05; f, compared with 50 $\mu$ g/mL, $p<$ 0.05; CCK-8, cell counting kit-8.

Figure 3.

Ginsenoside shortens the cell cycle of MG-63 and Saos-2 cells. A: flow chart of MG-63 cell cycle in response to the treatment of different concentration of ginsenoside; B: histogram of MG63 cell cycle in response to the treatment of different concentration of ginsenoside; C: flow chart of Saos-2 cell cycle in response to the treatment of different concentration of ginsenoside; D: histogram of Saos-2 cell cycle in response to the treatment of different concentration of ginsenoside; a, compared with the control group, $p<$ 0.05; b, compared with 12.5 $\mu$ g/mL, $p<$ 0.05; c, compared with 25 $\mu$ g/mL, $p<$ 0.05; d, compared with 50 $\mu$ g/mL, $p<$ 0.05.

Figure 4.

Ginsenoside elevates mRNA expressions of Bax and cleaved caspase-3 and reduces that of $\beta$ -catenin, Bcl-2, and Cyclin D1 in MG-63 and Saos-2 cells. A: mRNA expressions of $\beta$ -catenin, Bcl-2, Bax, cleaved caspase-3 and Cyclin D1 in MG63 cells treated with different concentration of ginsenoside; B: mRNA expressions of $\beta$ -catenin, Bcl-2, Bax, cleaved caspase-3 and Cyclin D1 in Saos-2 cells treated with different concentration of ginsenoside; a, compared with the control group, $p<$ 0.05; b, compared with 12.5 $\mu$ g/mL, $p<$ 0.05; c, compared with 25 $\mu$ g/mL, $p<$ 0.05; d, compared with 50 $\mu$ g/mL, $p<$ 0.05; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2.

Figure 5.

Ginsenoside increases protein expressions of Bax and cleaved caspase-3 while lowering that of $\beta$ -catenin, Bcl-2, and Cyclin D1 in MG-63 and Saos-2 cells. A: protein bands of $\beta$ -catenin, Bax, cleaved caspase-3, Bcl-2 and Cyclin D1 in MG-63 cells treated with different concentration of ginsenoside; B: protein expressions of $\beta$ -catenin, Bax, cleaved caspase-3, Bcl-2 and Cyclin D1 in MG-63 cells treated with different concentration of ginsenoside; C: protein bands of $\beta$ -catenin, Bax, cleaved caspase-3, Bcl-2 and Cyclin D1 in Saos-2 cells treated with different concentration of ginsenoside; D: protein expressions of $\beta$ -catenin, Bax, cleaved caspase-3, Bcl-2 and Cyclin D1 in Saos-2 cells treated with different concentration of ginsenoside; a, compared with the control group, $p<$ 0.05; b, compared with 12.5 $\mu$ g/mL, $p<$ 0.05; c, compared with 25 $\mu$ g/mL, $p<$ 0.05; d, compared with 50 $\mu$ g/mL, $p<$ 0.05; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2.

3.2 Ginsenoside inhibits the proliferation of MG-63 and Saos-2 cells

To elucidate the effects of ginsenoside on the proliferation of MG-63 and Saos-2 cells, CCK-8 assay was performed. As shown in Fig. 2, the absorbance of MG-63 and Saos-2 cells of all the ginsenoside-treated groups was significantly lower than that in the control group, suggesting that the proliferation was significantly inhibited (all $p<$ 0.05). Meanwhile, the proliferation rate of MG-63 and Saos-2 cells in the ginsenoside-treated groups at 48 h was lower than that at 24 h. Moreover, the proliferation at 72 h was significantly lower than all other time intervals (all $p<$ 0.05). On the other hand, during the same time interval of ginsenoside drug exposure, the absorbance of cells in the ginsenoside groups decreased with the increase in concentration of ginsenoside (all $p<$ 0.05). These findings demonstrated that ginsenoside was able to reduce cell proliferation of OS cells.

3.3 Ginsenoside induces cell cycle arrest of MG-63 and Saos-2 cells

The effects of ginsenoside on the cell cycle of MG-63 and Saos-2 cells were investigated (Fig. 3). MG-63 and Saos-2 cell proportions at the S phase in the ginsenoside-treated groups significantly decreased compared with the control group (all $p<$ 0.05). MG-63 and Saos-2 cells that treated with different concentrations of ginsenoside produced a correlation whereby the higher the concentrations of ginsenoside, resulting in lower proportions of cells present in the S phase (all $p<$ 0.05). Thus, treatment by ginsenoside shortens the cell cycle of OS cells.

3.4 Ginsenoside promotes apoptosis of MG-63 and Saos-2 cells

Flow cytometry was use to evaluate the effects of ginsenoside on apoptosis of MG-63 and Saos-2 cells. The results are shown in Table 2. After 48 h, the total apoptotic rate of each ginsenoside-treated group significantly increased compared with the control group (all $p<$ 0.05). The apoptotic rate of MG-63 and Saos-2 cells treated with different concentrations of ginsenoside at the 48 h time interval was significantly higher than that of the 24 h time interval. We also noted that of the apoptotic rate of MG-63 and Saos-2 cells at the 72 h was significantly higher than that during the 24 h and 48 h, respectively (all $p<$ 0.05). As the concentration of ginsenoside increased, the total apoptotic rate of MG-63 and Saos-2 cells also increased (all $p<$ 0.05). The above results suggested that ginsenoside was capable of inducing OS cell apoptosis.

3.5 Ginsenoside increases mRNA expressions of Bax and cleaved caspase-3 while decreasing that of $\beta$ -catenin, Bcl-2, and Cyclin D1 in MG-63 and Saos-2 cells

The effects of ginsenoside on the mRNA expressions of $\beta$ -catenin, Bcl-2, Bax, cleaved caspase-3 and Cyclin D1 in MG-63 and Saos-2 cells were evaluated by RT-qPCR. After 48 h, the mRNA expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1 in the ginsenoside-treated groups were significantly lower, whereas that of Bax and cleaved caspase-3 was significantly higher compared with that in the control group (all $p<$ 0.05). As the concentration of ginsenoside increased, we observe a decrease in the mRNA expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1. while concentrations of Bax and cleaved caspase-3 significantly increased (all $p<$ 0.05) (Fig. 4). Altogether, ginsenoside can elevate mRNA expressions of Bax and cleaved caspase-3 while reducing that of $\beta$ -catenin, Bcl-2, and Cyclin D1 in OS cells.

3.6 Ginsenoside increases protein expressions of Bax and cleaved caspase-3 while decreasing that of $\beta$ -catenin, Bcl-2, and Cyclin D1 in MG-63 and Saos-2 cells

The effects of ginsenoside on the mRNA expressions of $\beta$ -catenin, Bcl-2, Bax, cleaved caspase-3 and Cyclin D1 in MG-63 and Saos-2 cells were evaluated by western blot analysis. After 48 h, protein expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1 in ginsenoside-treated groups significantly decreased, while that of Bax and cleaved caspase-3 significantly increased compared with the control group (all $p<$ 0.05). In groups treated with different concentrations of ginsenoside, an increase in the concentration of ginsenoside produced a decrease in protein expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1. In contrast, this produced a significant increase in Bax and cleaved caspase-3 protein concentration (all $p<$ 0.05) (Fig. 5). It was demonstrated that ginsenoside could stimulate protein expressions of Bax and cleaved caspase-3 while inhibiting that of $\beta$ -catenin, Bcl-2, and Cyclin D1 in OS cells.

4. Discussion

OS is a common primary bone cancer characterized by intermittent and varying intensity pain in the lower femur or below the knee with poor prognosis [14]. In this study, the effects of ginsenoside on the apoptosis and proliferation of human OS MG-63 and Saos-2 cells by regulating the expressions of $\beta$ -catenin, Bcl-2, Cyclin D1, cleaved caspase-3 and Bax were studied. Our results demonstrate that ginsenoside was able to help accelerate cell apoptosis and control the proliferation of human OS MG-63 and Saos-2 cells by decreasing the mRNA and protein expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1 while increasing those of Bax and cleaved caspase-3.

According to the results of cell slides and CCK-8, ginsenoside inhibits the proliferation of human OS MG-63 and Saos-2 cells. The total cell number, density and absorbance of MG-63 and Saos-2 cells treated with ginsenoside were found to be decreased as concentration of ginsenoside increased. In addition, we also found that an increase in duration of cellular exposure to ginsenoside also resulted in further reduction of cell. Ginsenoside, found in a traditional Chinese medicine ginseng, has been proven to contain antitumor effects in many cancers, such as anti-metastasis, anti-angiogenesis, anti-proliferation and enhancement of chemotherapeutic susceptibility [15]. A study supports our findings that also demonstrated that ginsenoside was capable of inhibiting the proliferation and migration of human OS MG-63 and Saos-2 cells in a concentration- and time-dependent manner [16]. Another study revealed that ginsenoside has cytotoxic effects on human OS MG-63 cells, and inhibits cell proliferation by regulating the mitochondrial pathway, in which ginsenoside Rf decreased the ratio of Bcl-2 to Bax and the transmembrane potential of mitochondrial, leading to cytochrome $c$ release and caspase-9 and caspase-3 activation [12]. It is consistent with another study that reported that the proliferation of prostate cancer cells was also inhibited by ginsenoside with the concentration of 50% [17].

FITC-Annexin V and FITC-Annexin V/PI staining showed that ginsenoside promoted the apoptosis of human OS MG-63 and Saos-2 cells. With an increase in ginsenoside concentration, we observed a decrease of MG-63 and Saos-2 cells in the S phase as well as an increase in the total apoptosis rate. Ginsenoside Rh2 is a potential active metabolite of ginseng and has been shown to have an anti-tumor effect on a wide range of cancers [18]. It exerts its anti-tumor effects by inducing apoptosis and G1 cell cycle arrest in cancer cells such as human lung adenocarcinoma A549 cells [19]. Additionally, Rh2 is observed to arrest HepG2 (hepatocellular carcinoma cells) and HepG2- $\beta$ -catenin cells in G0/G1 phase and subsequently promote apoptosis of liver cancer cells [20]. Ginsenoside has also been found to promote apoptosis in human OS MG-63 cells through the caspase cascade in a dose-dependent manner. This pathway is one of the key steps during cell apoptosis process initiated by the mitochondrial pathway through cytochrome $c$ in the intermembrane space [21]. Our findings were consistent with another study reporting that ginsenoside exerts its antitumor effects by regulating many signaling pathways including modulation of cell death mediator caspases [13]. Moreover, ginsenoside is observed to stimulate the apoptosis of human OS MG-63 and Saos-2 cells in a dose-dependent manner [16].

RT-qPCR and western blot analysis indicated that ginsenoside inhibits protein and mRNA expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1 while increasing those of Bax and cleaved caspase-3. Our results are in line with another study that highlights how ginsenoside regulates the mRNA and protein expressions of $\beta$ -catenin, Bcl-2, Cyclin D1, cleaved caspase-3 and Bax in thyroid follicular neoplasm cells [22]. As ginsenoside concentrations increase, mRNA and protein levels of $\beta$ -catenin, Bcl-2 and Cyclin D1 gradually decreased while those of Bax and cleaved caspase-3 increased. $\beta$ -catenin is a protein that is involved in regulation and cell-cell adhesion and gene transcription, it plays a central role in cell junctions and its effects on activating cancers depend on the signaling pathways that involves several transcriptional regulators [23]. It is revealed that ginsenoside degrades mRNA and protein expression of $\beta$ -catenin by activating Gsk-3 $\beta$ [24]. As a key regulator of cell cycle progression, Cyclin D1 exerts its functions through regulating cyclin-dependent kinases (CDK) 4 or 6 [25]. The change in Cyclin D1 concentration is most likely caused by the activation of the EGFR-Akt pathway [26]. Many oncogenes promote proliferation, however Bcl-2 functions to prevent apoptosis from occurring, whereby ginsenoside degrades the mRNA and protein expressions of Bcl-2 to improve the apoptotic pathways of the cancer cells, leading to cell death [27]. On the contrary, Bax is a pro-apoptotic protein and that increased mRNA and protein expressions of Bax can promote cell apoptosis followed by an apoptotic stimulus [28]. It has been demonstrated that ginsenoside is able to increase mRNA and protein expressions of Bax by causing a dramatic and rapid translocation of Bax protein, consequently initiating the caspase activation and mitochondrial cytochrome c release [29]. Cleaved caspase-3, a key executor in apoptosis, is involved in the growth stimulation [30]. A recent study shows p21 overexpression sensitizes osteosarcoma U2OS cells to cisplatin by activating cleaved caspase-3 cascade [31]. Ginsenoside treatment causes up-regulation of Bax and down-regulation of Bcl-2 and Cyclin B1, the activation of cleaved caspase-3 in MG-63 cells [12].

In conclusion, our study provided evidence that ginsenoside was able to promote the apoptosis of human OS MG-63 and Saos-2 cells and inhibit their proliferation by decreasing mRNA and protein expressions of $\beta$ -catenin, Bcl-2 and Cyclin D1 while increasing those of Bax and cleaved caspase-3. These findings might offer a new guidance for the treatments of human OS. However, there are different kinds of ginsenosides that we did not study the specific mechanism of. In addition, whether the changes in mRNAs or proteins are the main reason for the response of OS cells to ginsenoside still remains to be clarified. Therefore, further studies will need to be conducted in the future to provide a deeper understanding and knowledge of the effect of ginsenosides on the treatment of human OS.