OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2019.10561 OL-0-0-10561 Articles Ursolic acid suppresses the biological function of osteosarcoma cells PeiYi 1 ZhangYueyan 2 ZhengKe 1 ShangGuanning 1 WangYuming 1 WangWei 1 QiuEnduo 1 ZhangXiaojing 1 Department of Bone and Soft Tissue Surgery, Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute, Shenyang, Liaoning 110042, P.R. China Department of Clinical Pathology, Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute, Shenyang, Liaoning 110042, P.R. China Correspondence to: Dr Xiaojing Zhang, Department of Bone and Soft Tissue Surgery, Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute, 44 Xiaohe Road, Shenyang, Liaoning 110042, P.R. China, E-mail: zl_zhangxiaojing@163.com 09 2019 04 07 2019 18 3 2628 2638 15122017 29052019 Copyright © 2019, Spandidos Publications 2019

Osteosarcoma is a highly malignant tumour that occurs in adolescents. Upregulation or the constitutive activation of epidermal growth factor receptor (EGFR) is a hallmark of osteosarcoma. To investigate the effect of ursolic acid on the biological function of osteosarcoma, MTT assay was used to detect the effect of ursolic acid on the proliferation of HOS and MG63 cells, while flow cytometry was used to analyse the effect on the cell cycle and apoptosis. Transwell and Matrigel assays were used to detect the effect of ursolic acid on cell migration and invasion, respectively. Western blot analysis and reverse transcription-quantitative polymerase chain reaction were used to detect the effects of different concentrations of ursolic acid on EGFR signaling pathway-related proteins, cell cycle, apoptosis and cell migration-related proteins. After overexpression or silencing of EGFR, the effects of ursolic acid on EGFR pathway and cell biological function were subsequently detected, using the same methods. The present study identified that ursolic acid had inhibitory effects on the growth and metastatic ability of osteosarcoma cells by suppressing EGFR.

 ursolic acid osteosarcoma epidermal growth factor receptor signaling pathway Introduction

Osteosarcoma is a malignant bone tumour with a higher incidence in adolescents (1). Currently, the use of doxorubicin-based chemotherapeutics in combination with aggressive surgery remains the standard treatment for osteosarcoma (2). The poor survival rate is associated with the numerous pathological features of osteosarcoma, including high invasiveness, recurrent therapeutic resistance and multiple gene aberrations (3). Among these, gene dysregulation is an important factor in the tumourigenesis and progression of osteosarcoma (4).

Epidermal growth factor receptor (EGFR) is highly expressed in lung cancer, osteosarcoma, ovarian and a variety of other tumours and is involved in their development (2). Upregulation or the constitutive activation of EGFR is associated with tumourigenesis and more aggressive phenotypes in osteosarcoma (3,5,6). Certain important signalling pathways in tumour cells, including PI3K/Akt, Wnt/β-catenin and mitogen-activated protein kinase (MAPK), are activated by EGFR. Anti-EGFR is considered an effective therapeutic target against osteosarcoma (5).

Ursolic acid in its free form or in the form of a sugar glucoside can be found in ~7 families, 46 genera and 62 species of plants. A previous study has demonstrated that ursolic acid has a variety of pharmacological effects, including anti-inflammatory and immunosuppressant activities, and preventative effects against cartilage destruction (7). Moreover, ursolic acid has been safely used in the treatment of arthritis for various years (8). It has been reported that ursolic acid possesses anti-tumour effects and can regulate cell growth (9). Ursolic acid has many biological functions (10), can lower the blood pressure and presents anticancer properties (11). Additionally, ursolic acid may inhibit the growth of cells, promote the apoptosis of tumour cells and mediate the invasion and metastasis of tumour cells (7). However, there are few studies investigating the regulation of osteosarcoma by ursolic acid (12).

The present study suggested that ursolic acid could effectively suppress the growth and metastasis of HOS cells by decreasing the expression level and the activity of EGFR. Importantly, ursolic acid may be an effective drug in the treatment of osteosarcoma.

 Materials and methods <sec> <title>Cell culture

Human osteosarcoma cell lines HOS and MG63 were cultured with DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO2.

 MTT assays

A total of 1×104 HOS or MG63 cells were counted and plated in 96-well plates and following treatment with DMSO, 10, 20 or 30 µmol/l of ursolic acid, 0.01 ml of MTT solution (5 mg/ml in PBS) was added to each well at 0, 12, 24, 36 and 48 h. After a 4-h incubation at 37°C, the MTT solution was discarded. Following a 15-min incubation with DMSO used to dissolve the purple formazan, the optical densities at 490 nm were measured using a microplate reader (Bio-Rad Laboratories, Inc.). The inhibition rate of ursolic acid on cell proliferation was also detected following incubation of HOS and MG63 cells with 30–40 µmol/l of ursolic acid for 24 h using MTT. The half maximal inhibitory concentration (IC50) values were calculated and a linear fit curve was generated.

 Cell cycle analysis using flow cytometry

HOS cells were treated with DMSO, 10, 20 or 30 µmol/l ursolic acid for 24 h. After washing with PBS twice, cells were suspended in propidium iodide and analysed using a MoFlo XDP flow cytometer (Beckman Coulter, Inc.). The analysis of data was using FlowJo software v10.0.7.2 (FlowJo LLC).

 Transwell assay

The assays were performed using Transwell chambers, with or without Matrigel. A total of 1×105 HOS or MG63 cells treated with DMSO, 10, 20 or 30 µmol/l ursolic acid for 24 h were cultivated on the upper chamber with DMEM, and DMEM containing 10% bovine serum was added to the lower chamber. After 24 h incubation in a humidified incubator at 37°C with 5% CO2, the medium was removed from the upper chamber. The non-invaded cells on the upper side of the chamber were scraped off with a cotton swab. The cells that had migrated into the pores of the inserted filter were stained with trypan blue for 20 min at room temperature. The numbers of cells were counted in three randomly selected visual fields, from the central and peripheral portion of the filter using an inverted microscope (×200 magnification; Leica DM2500M; Leica Microsystems GmbH). Each assay was repeated three times.

 Cell apoptosis

Apoptosis of HOS cells treated with DMSO, 10, 20 or 30 µmol/l ursolic acid for 24 h was detected with an Annexin-V-FITC apoptosis detection kit (Hanbio Biotechnology Co., Ltd.). In brief, cells were washed twice with cold PBS and suspended with binding buffer. Cells were incubated with 5 µl Annexin-V-FITC and 10 µl propidium iodide in the dark at room temperature for 15 min. Finally, binding buffer was added to each tube and the rate of apoptosis was measured using a flow cytometer within 1 h. Cells analysed using a MoFlo XDP flow cytometer (Beckman Coulter, Inc.). The analysis of data was using FlowJo software v10.0.7.2 (FlowJo LLC).

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

Total RNA was extracted with TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) from HOS or MG63 cells. A transcription kit (Beyotime Institute of Biotechnology,) was used for the RT of RNA into cDNA using the following conditions: 37°C for 15 min and 85°C for 5 sec. RT-qPCR was subsequently performed using a SYBR® Green Real-time PCR master mix (Toyobo Life Science,). RT-qPCR was performed using MX3000P real-time PCR instrument according to the manufacturer's protocol (Beyotime Institute of Biotechnology), using the following conditions: 94°C for 5 min, then 30 cycles of 94°C for 30 sec, 58°C for 30 sec, and elongation at 72°C for 10 sec. β-actin was used as the internal control. The expression of miRNAs was detected using a miRcute miRNA qPCR detection kit (SYBR Green; Tiangen Biotech Co., Ltd.) (13). Primer sequences are as follows: EGFR forward, 5′-CTAAGATCCCGTCCATCGCC-3′ and reverse, 5′-GGAGCCCAGCACTTTGATCT-3′; cyclin D1 forward, 5′-GATCAAGTGTGACCCGGAC-3′ and reverse, 5′-AGAGATGGAAGGGGGAAAGA-3′; CDK4 forward, 5′-GCGTGAGGGTCTCCCTTGAT-3′ and reverse, 5′-CAGTCGCCTCAGTAAAGCCA-3′; CDK6 forward, 5′-TCCCCAGAGTCTGATTACCT-3′ and reverse, 5′-ACGATTACATAGCCTCTGCC-3′; caspase 3 forward, 5′-ATGGAGAACAATAAAACCT-3′ and reverse, 5′-CTAGTGATAAAAGTAGAGTTC-3′; Bcl-2 forward, 5′-CGACGACTTCTCCCGCCGCTACC-3′ and reverse, 5′-CCGCATGCTGGGGCCGTACAG-3′; Bax forward, 5′-TCCACCAAGAAGCTGAGCGAG-3′ and reverse, 5′-GTCCAGCCCATGATGGTTC-3′; matrix metallopeptidase 2 (MMP2) forward, 5′-CTATTCTGCCAGCACTTTGG-3′ and reverse, 5′-CAGACTTTGGTTCTCCAAC-3′; β-actin forward, 5′-GAGAGGGAAATCGTGCGTGAC-3′ and reverse, 5′-CATCTGCTGGAAGGTGGAC-3′.

There were at least three replicates for each reaction. β-actin was used as the internal control.

 Transfection

To silence EGFR, a set of synthesized small interfering (si)-RNAs against human EGFR (forward, 5′-CCUUAGCAGUCUUAUCUAATDT-3′ and reverse, 5′-TGGAAUCGUCAGAAUAGAUU-3′; Shanghai GeneChem Co., Ltd.) was used. HOS cells were transfected with pRS-siEGFR (forward, 5′-UUCUCCGAACGUGUCACGU-3′ and reverse, 5′-ACGUGACACGUUCGGAGAA-3′; Shanghai GeneChem Co., Ltd.), using Lipofectamine 2000® (Invitrogen; Thermo Fisher Scientific, Inc.) and then selected with puromycin (Sigma-Aldrich; Merck KGaA). Cells transfected with pRS-si-NC (negative control) and wild type tumour cell lines were used as the control groups. For EGFR overexpression, EGFR (NCBI accession no. NM_201283.1) was amplified using PCR with the following primers: Forward, 5′-GATCTGCAGGGGTTAGCTTGGGGACCTGAAC-3′ and reverse, 5′-GATCATATGAGAGTGACATACTGATGCCTAC-3′. Subsequently 2 µg EGFR was cloned into pcDNA3.1 vector then transfected into HOS cells using Lipofectamine 2000® (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. A total of 2 µg pcDNA3.1 vector (Shanghai GeneChem Co., Ltd.) was used as the control. All primers were purchased from Shanghai GeneChem Co., Ltd.. After transfection for 24 h, cells treated with DMSO or 20 µmol/l of ursolic acid and MTT, Transwell and western blot experiments were performed.

 Western blot analysis

HOS and MG63 cells (~1×105) were lysed with ice-cold lysis buffer containing NP40 buffer and a proteinase inhibitor cocktail (Invitrogen; Thermo Fisher Scientific, Inc.). The lysates were sonicated with an oscillation frequency of 15–25 KHz, at 4°C for 10 sec, followed by centrifugation at 12,000 × g for 10 min at 4°C and the supernatants were retained. Total protein concentration was quantified using a bicinchoninic acid protein assay kit (Applygen Technologies, Inc.), and western blot analysis was performed. A total of 30 µg protein/lane were resolved using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). Following blocking with 3% BSA (Beijing Solarbio Science & Technology Co., Ltd.) in TBS containing 0.1% Tween-20 for 3 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against EGFR (sc-80543; dilution 1:1,000), Akt1 (sc-135829; dilution 1:1,000), phosphorylated (p)-Akt1Ser473 (sc-293125; dilution 1:1,000), β-actin (sc-69879; dilution 1:1,000), cyclin D1 (sc-4074; dilution 1:1,000), CDK4 (sc-70831; dilution 1:1,000), CDK6 (sc-271364; dilution 1:1,000), caspase-3 (sc-7272; dilution 1:1,000), Bax (sc-70407; dilution 1:1,000), Bcl-2 (sc-23960; dilution 1:1,000), MMP-2 (sc-13595; dilution 1:1,000). After several washes, the membranes were incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, mouse IgG-HRP (sc-2748; dilution 1:5,000) and rat IgG-HRP (sc-2750; dilution 1:5,000) for 1 h at room temperature. All antibodies brought from Santa Cruz Biotechnology, Inc.. The protein bands were visualized using enhanced chemiluminescence kits (GE Healthcare), and the optical density of the protein bands was quantified using the ImageJ v1.8.0 software (National Institutes of Health), using β-actin as an internal control.

 Statistical analysis

All data were analysed using SPSS version 17.0 (SPSS, Inc.). Statistical significance between two groups of data was evaluated using two-tailed Student's t-test and one-way ANOVA. For multiple comparisons one-way ANOVA was used followed by LSD post hoc test. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated three times. Data are presented as the mean ± SD.

 Results <sec> <title>Ursolic acid inhibits the growth and invasion of HOS cells

The chemical structure of ursolic acid is shown in Fig. 1A. The MTT assay suggested that ursolic acid inhibited the proliferation of HOS cells in a concentration-dependent manner; through the calculation method of predecessors (14), the IC50 of ursolic acid was ~35 µmol/l (Fig. 1B and C). Ursolic acid affected the proliferation of cells following treatment for 24 h, at which time the cell proliferation of the 10, 20 and 30 µmol/l ursolic acid treatment groups was significantly different compared to that of control group, P<0.05; therefore, this time point was used for subsequent experiments. In addition, ursolic acid inhibited the proliferation of MG63 cells; the IC50 of ursolic acid was ~37 µmol/l (Fig. 1D and E). Cell cycle analysis was performed after HOS cells were exposed to ursolic acid for 24 h. 10, 20 and 30 µmol/l ursolic acid arrested HOS cells at the G1 phase of the cell cycle (Fig. 1F). Furthermore, Annexin-V-FITC staining demonstrated that 10, 20 and 30 µmol/l ursolic acid could induce cell apoptosis (Fig. 1G). Ursolic acid also significantly decreased the cell migration and invasion of HOS and MG63 cells (Fig. 1H-K).

 Ursolic acid suppresses EGFR signalling pathway in HOS cells

Western blot analysis suggested that ursolic acid could significantly inhibit the expression of EGFR and phosphorylated (p)-Akt (Ser473) without affecting the expression of Akt, an important protein in the EGFR signalling pathway (Fig. 2A). Therefore, we hypothesized that ursolic acid may regulate the EGFR signalling pathway. Compared with the DMSO group, there were no statistically significant differences in the mRNA level of EGFR in the 10 and 20 µmol/l ursolic acid groups; however, 30 µmol/l ursolic acid significantly reduced EGFR mRNA expression (Fig. 2B). The present results suggested that, at low concentrations, ursolic acid only affected the expression of EGFR protein and had little effect on mRNA expression, although higher doses may affect the synthesis of mRNA. When the concentration was increased to 30 µmol/l, ursolic acid inhibited EGFR expression at the protein and mRNA levels.

Since the biological function of osteosarcoma cells has been significantly affected by ursolic acid at 20 µmol/l for 24 h, this concentration and time point was used for the further experiments. To further verify the inhibitory effect of ursolic acid on EGFR, an EGFR overexpression plasmid was transfected into HOS cells (Fig. 2C and D), which were then treated with or without 20 µmol/l ursolic acid for 24 h (Fig. 2E). The present results suggested that ursolic acid decreased the level of EGFR and p-Akt (Ser473) in the vector and EGFR overexpression groups (Fig. 2E). The HOS/si-EGFR stable cell line was then used to detect the effect of ursolic acid on EGFR (Fig. 2F-H). Ursolic acid inhibited the p-Akt of EGFR overexpression group more strongly compared to that in the vector group (Fig. 2G). It also inhibited the p-Akt of NC group more strongly compared with that in the si-EGFR group (Fig. 2H). Ursolic acid treatment downregulated EGFR and p-Akt (Ser473) significantly in HOS/si-NC stable cell lines. The effect of ursolic acid was reduced after EGFR silencing in cells. Therefore, it was concluded that ursolic acid might inhibit proteins in the downstream signaling pathway by inhibiting EGFR.

 Ursolic acid inhibits the growth of HOS cells

Western blot analysis suggested that increasing concentrations of ursolic acid significantly decreased the protein expression level of cyclin D1, CDK4 and CDK6 (Fig. 3A). Furthermore, cyclin D1, CDK4 and CDK6 in HOS cells were also significantly downregulated at the mRNA level following treatment with increasing concentrations of ursolic acid (Fig. 3B). Apoptosis factors were investigated, and the present results suggested that Bcl-2 was significantly downregulated and caspase 3 and Bax were significantly upregulated at both the protein and mRNA levels (Fig. 3C and D).

The MTT assay showed that the proliferation of cells overexpressing EGFR was significantly inhibited by ursolic acid (Fig. 4A). Results from the HOS/si-EGFR cells suggested that there were significant decrease in the extent of proliferation inhibition between HOS/si-NC and HOS/si-EGFR cell lines (Fig. 4B).

Cell growth and apoptosis-related regulators were also detected in cells following overexpression or knockdown of EGFR, and after treatment with ursolic acid. Overexpression of EGFR induced the protein expression levels of cyclin D1, CDK4, CDK6 and Bcl-2 compared with that in the vector group. Moreover, the inhibitory effect of ursolic acid on cyclin D1, CDK4, CDK6 and Bcl-2 in EGFR overexpressed cells was greater compared with that of ursolic acid on the same proteins in the vector group (Fig. 4C and D). However, after knockdown of EGFR, the changes in these proteins were markedly reduced (Fig. 4E and F). By contrast, Bax and caspase 3 decreased following EGFR overexpression and decreased following EGFR knockdown.

 Ursolic acid represses the metastasis of HOS

Osteosarcoma is typically advanced due to its high invasiveness (15). MMP2 is an important protein regulating cell migration and a functional protein downstream of EGFR (16). Therefore, the regulatory effect of ursolic acid on MMP2 was investigated. The present results suggested that the expression of MMP2 was significantly decreased at increasing concentrations of ursolic acid (Fig. 5A and B). To investigate the involvement of EGFR in the ursolic acid-dependent inhibition of cell invasion and migration (Fig. 1H-K), a Transwell assay was performed (Fig. 5C-F). EGFR could promote the migration of HOS cells, whereas ursolic acid could significantly reduce the migratory ability of HOS cells. After the inhibition of EGFR, the effect of ursolic acid was reduced. In the HOS cell lines, the protein expression level of MMP2 decreased following treatment with ursolic acid (Fig. 5G and H).

 Discussion

EGFR can regulate cellular processes via kinase-independent and -dependent signalling pathways. EGFR mutants with no kinase activity, such as EGFR (D813A) and EGFR (K721M), can induce activation of MAPK and activate survival signals by interacting with other important proteins, such as receptor tyrosine-protein kinase (17). In the EGFR kinase-dependent signalling pathway, the phosphorylation of EGFR induces the activation of a number of pathways, including MAPK/ERK, phosphoinositide phospholipase Cγ/protein kinase C/NF-κB, SRC proto-oncogene, non-receptor tyrosine kinase, PI3K and STAT3, which are associated with cell proliferation, apoptosis, cell cycle regulation and DNA damage repair (18). Due to the important role of EGFR, this receptor is one of the most promising therapeutic targets (6).

Ursolic acid has previously been used for the treatment of rheumatoid arthritis (19). In recent years, antitumour effects of ursolic acid have been found in synovial sarcoma, lung cancer, endometrial cancer cells and hepatocellular carcinoma (11,20–23).

In many types of cancer, including osteosarcoma, overexpression of EGFR is associated with aggressive invasiveness and resistance to therapy (24–26). Previous studies have indicated that EGFR may be an effective target for the treatment of human cancer (27,28). The US Food and Drug Administration has approved five anti-EGFR agents for cancer treatment including three small molecule inhibitors and two antibodies (29). The present study revealed that ursolic acid had inhibitory effects on the growth and metastatic ability of HOS cells in a concentration-dependent manner. Notably, ursolic acid could significantly inhibit the expression of EGFR. Thus, ursolic acid could be considered as a natural inhibitor of EGFR, and its antitumour effect may be partly due to the inhibition of EGFR, as EGFR can regulate various biological functions such as cell growth and metastasis (3). Therefore, it was identified that ursolic acid can inhibit EGFR, and the biological effect of ursolic acid was further investigated. The present study also suggested that ursolic acid could inhibit the cell cycle G1-to-S transition by inhibiting the expression of cyclin D1 and CDK4 and CDK6. This inhibition in cell cycle may be caused by the inhibitory effect of ursolic acid on EGFR. Furthermore, ursolic acid not only affected the proliferation of cells, but also inhibited the invasion and metastasis of cells; this inhibitory effect of ursolic acid on cell metastasis may involve the inhibition of MMP2.

In conclusion, the present study demonstrated that ursolic acid could inhibit the malignant phenotype of HOS cells by inhibiting EGFR expression and EGFR-associated signalling pathways. Although these results require further investigaton in vivo, the present findings support the hypothesis that ursolic acid may facilitate the development of a novel clinical treatment of osteosarcoma.

 Acknowledgements

Not applicable.

 Funding

No funding was received.

 Availability of data and materials

All data generated or analyzed during this study are included in this published article.

 Authors' contributions

YP contributed to the design of the study. YZ performed the experiments. KZ, GS and HW participated in supplementing experiments. KZ, GS drafted the manuscript and revised it critically for important intellectual content. YW drafted the manuscript and gave final approval of the version to be published. WW, EQ and XZ analyzed and interpreted the data.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

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Ursolic acid inhibits the growth and invasion of cells. (A) Chemical structure of ursolic acid. (B) Analysis of HOS cell proliferation using MTT assay following incubation with 10, 20 and 30 µmol/l ursolic acid for 12, 24, 36 and 48 h. Data are presented as the mean ± SD of three experiments performed in triplicate. (C) Inhibitory rate of HOS cells incubated with 30–40 µmol/l ursolic acid for 24 h was detected using MTT. (D) Analysis of MG63 cell proliferation using MTT assay following incubation with 10, 20 and 30 µmol/l concentrations of ursolic acid for 12, 24, 36 and 48 h. Data are presented as the mean ± SD of three experiments performed in triplicate. (E) Inhibitory rate of MG63 cells incubated with 30–40 µmol/l concentrations of ursolic acid for 24 h was detected using MTT. (F) Cell cycle analysis of HOS cells which were treated with DMSO, 10, 20 or 30 µmol/l ursolic acid was performed via a FACS Vantage flow cytometer. The experiments were repeated three times. Data represent the mean ± SD of three experiments. (G) Apoptosis of HOS cells which were treated with DMSO, 10, 20 or 30 µmol/l ursolic acid was analysed using flow cytometry. The experiment was repeated three independent times. Ursolic acid inhibits the growth and invasion of cells. (H-K) The (H) migration and (I) invasion of HOS and (J) migration and (K) invasion of MG63 cells were detected using a Transwell assay with or without Matrigel. Data represent the mean ± SD of three experiments. **P<0.01 vs. DMSO treated group. PI, propidium iodide; SD, standard deviation; OD, optical density.

Ursolic acid suppresses the EGFR signalling pathway in HOS cells. The expression of EGFR in HOS cells with 10, 20 and 30 µmol/l ursolic acid was detected using (A) western blot analysis and (B) RT-qPCR. Data represent the mean ± SD of three experiments performed in triplicate. The ratio p-Akt/total Akt was down regulated by ursolic acid. The expression of EGFR in HOS cells was detected using (C) western blot analysis and (D) RT-qPCR. Data represents the mean ± SD of three experiments performed in triplicate. (E) HOS cells were transfected with either Flag-vector or Flag-EGFR and subsequently treated with or without 20 µmol/l ursolic acid. EGFR, p-Akt (Ser473), Akt and β-actin were detected in the indicated groups of HOS cells. Experiments were repeated three times. The expression of EGFR in HOS cells was detected using (F) western blot analysis and (G) RT-qPCR results. The ratio p-Akt/total Akt was up regulated by EGFR. Data represents the mean ± SD of three experiments performed in triplicate. (H) HOS cells transfected with either si-NC or si-EGFR and subsequently treated with or without 20 µmol/l ursolic acid. EGFR, p-Akt (Ser473), Akt and β-actin were detected in the indicated groups of HOS cells. The ratio p-Akt/total Akt was down regulated by si-EGFR. Experiments were repeated independently three times. *P<0.05, **P<0.01 vs. DMSO-treated group; ##P<0.01 vs. vec group; ^^P<0.01 vs. si-NC group. EGFR, epidermal growth factor receptor; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Vec, vector; Urs, ursolic acid; si, small interfering; p, phosphorylated.

Ursolic acid inhibits the growth of HOS cells. Following treatment with 10, 20 and 30 µmol/l ursolic acid, the (A) protein and (B) mRNA levels of cyclin D1, CDK4, CDK6, and (C) protein and (D) mRNA levels of caspase 3, Bax, Bcl-2 were detected in HOS cells. Data represent the mean ± SD of three experiments performed in triplicate. β-actin was used as control. **P<0.01 vs. DMSO-treated group.

Ursolic acid inhibits the growth of HOS cells. MTT assay was performed in HOS cells transfected with (A) Flag-vector or Flag-EGFR and (B) si-EGFR or si-NC cells, following incubation with 20 µmol/l ursolic acid for 0, 12, 24, 36 and 48 h. Data represent the mean ± standard deviation of three experiments done in triplicate. ##P<0.01 vector group treated with DMSO vs. EGFR group treated with DMSO, ^^P<0.01 si-NC group treated with DMSO vs. si-EGFR group treated with DMSO. **P<0.01 vector group treated with 20 µmol/l ursolic acid group vs. EGFR group treated with 20 µmol/l ursolic acid. *P<0.05 si-NC group treated with 20 µmol/l ursolic acid group vs. si-EGFR group treated with 20 µmol/l ursolic acid. Protein levels of (C) cyclin D1, CDK4 and CDK6, and (D) caspase 3, Bax and Bcl-2 were detected using western blot analysis in HOS cells expressing Flag-vector or Flag-EGFR and treated with or without ursolic acid. Experiments were repeated independently three times. The protein levels of (E) cyclin D1, CDK4 and CDK6, and (F) caspase 3, Bax and Bcl-2 were detected in HOS/si-EGFR and HOS/si-NC cells using western blot analysis. Experiments were repeated independently three times. *P<0.05, **P<0.01. EGFR, epidermal growth factor receptor; si, small interfering; NC, negative control; URS, ursolic acid, Vec, vector; OD, optical density.

Ursolic acid represses the metastasis of HOS cells. Following treatment with 10, 20 and 30 µmol/l ursolic acid, the MMP2 (A) protein and (B) mRNA levels were detected in HOS cells. Data are presented as the mean ± SD of three experiments performed in triplicate. (C-F) The (C and E) migration and (D and F) invasion of the indicated cells were detected using a Transwell assay with or without Matrigel. The experiments were repeated independently three times. (G) MMP2 protein expression was detected in the HOS cells transfected with either Flag-vector or Flag-EGFR and treated with or without 20 µmol/l ursolic acid. Experiments were repeated independently three times. (H) MMP2 protein expression was detected in the HOS cells transfected with either si-NC or si-EGFR and treated with or without ursolic acid. Experiments were repeated independently three times. ##P<0.01 vs. DMSO-treated group; *P<0.05, **P<0.01. MMP, matrix metalloproteinase; EGFR, epidermal growth factor receptor; si, small interfering; NC, negative control; Vec, vector; URS, ursolic acid.