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Introduction

Osteosarcoma (OS) is one of the most common malignant bone cancers, originating from mesenchymal tissue, with two high-incidence groups in adolescent and elderly individuals. The annual incidence rate of OS is approximately 4.4/1 million (1). In the early 1970s, with the advent of neoadjuvant chemotherapy combined with surgical resection, the 5-year survival rate of OS was increased from 20 to 70% (2-4). Unfortunately, the survival rate of OS has not improved in the past 30 years. Therefore, it is extremely urgent to study the pathogenesis of OS and explore new therapeutic targets.

Transforming growth factor-β (TGF-β) participates in the regulation of a variety of cellular biological processes through its TGF-β type I and type II receptors, including cell proliferation, differentiation, apoptosis, adhesion and migration, and acts on the downstream Smad pathway (classical) and non-Smad pathway (nonclassical) (5,6). Dysfunction of TGF-β leads to serious consequences, even tumorigenesis (7). Accumulating evidence has revealed that there is a close relationship between elevated TGF-β expression and the progression of tumors, such as lung, colorectal, prostate and gastric cancer. Additionally, the high expression of TGF-β in colorectal, prostate and breast cancer is positively correlated with tumor metastasis (8-10). Moreover, the serum TGF-β concentration in patients with OS lung metastasis is higher than that in patients without pulmonary metastasis (11,12). Therefore, it was hypothesized that the inhibition of the TGF-β signaling pathway may play a protective role in the occurrence and development of OS.

RepSox is a small molecule compound that acts as an inhibitor of TGF-βI receptor kinase (13,14). A recent study indicated that RepSox could inhibit osteoclast formation and bone resorption through the Smad3 and JNK/AP-1 pathway (15). Moreover, it has been reported that RepSox can induce mouse fibroblasts to reprogram into cardiomyocytes (16), improve the antitumor effects of antigen-presenting and immune-effector cells that are impaired in acute myeloid leukemia (17), and inhibit skin fibrosis (18). To date, no studies have been performed reporting the role of RepSox on tumors and its underlying mechanism. Therefore, the effects caused by RepSox on bone tumors remain unknown.

In the present study, the effects of RepSox on the proliferation, cell cycle, apoptosis and metastasis of OS cell lines in vitro and its effect on a tumor xenograft mouse model was therefore analyzed.

Materials and methods

Media and reagents

RepSox and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck KGaA. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco; Thermo Fisher Scientific, Inc. Cell Counting Kit-8 (CCK-8) reagent was obtained from Dojindo Molecular Technologies, Inc. Primary antibodies against JNK2 (product no. 9258S), phosphorylated (p)-JNK (product no. 4668S), ERK1/2 (product no. 5013S), p-ERK1/2 (product no. 4370S), p38 (product no. 8690S), p-p38 (product no. 4511S), Smad3 (product no. 9513S), p-Smad3 (product no. 9520S) and GAPDH (product no. 3683S) were purchased from Cell Signaling Technology, Inc. Primary antibodies against matrix metalloproteinase (MMP)-2 (cat. no. 10373-2-AP) and MMP-9 (cat. no. 10375-2-AP) were purchased from ProteinTech Group, Inc. Primary antibodies against Bax (product no. 5023S), Bcl-2 (product no. 3498S), E-cadherin (product no. 3195S), N-cadherin (product no. 4061S) and vimentin (product no. 5741S) were purchased from Cell Signaling Technology, Inc.

Cell culture

HOS and 143B cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin. Both cell lines were routinely stored at 37°C in a humidified incubator with 5% CO2.

Cell proliferation assays

To determine the effective inhibitory concentration of RepSox on cell viability, HOS and 143B cells were seeded at 3×103 cells/well into 96-well plates and incubated at 37°C overnight. Cells were then treated with increasing concentrations of RepSox ranging from 0 to 200 µM for 24, 48 or 96 h. The DMSO alone group was used as the negative control group. Subsequently, 10 µl of Cell Counting Kit-8 (CCK-8) buffer was added to each well containing 100 µl culture medium. The plate was incubated for another 2 h at 37°C, and the absorbance at a wavelength of 450 nm was measured using an ELX800 absorbance microplate reader (BioTek Instruments, Inc.). Cell viability was calculated as follows: Cell viability (%) = [A (RepSox)-A (blank)]/[A (Control)-A (blank)] ×100%; where A (RepSox) is the absorbance of wells with cells, CCK-8 solution and RepSox; A (blank) is the absorbance of wells with medium and CCK-8 solution but no cells; and A (Control) is the absorbance of wells with cells and CCK-8 solution but without RepSox. The cell growth rate was calculated by GraphPad Prism software (version 7.0; GraphPad Software, Inc.). Firstly, the drug concentration was converted into a logarithmic form as the x-axis, and then data fitting was performed through nonlinear regression, and finally the IC50 value was obtained. For cell colony formation, HOS and 143B cells were plated in 6-well plates at a cell density of 1,000 cells/well and then treated with different doses of RepSox (0, 50, 100 and 200 µM) supplemented with DMEM with 10% FBS for 2 weeks. The colonies were visualized under a light microscope (Carl Zeiss AG) and images were captured after staining with 0.5% crystal violet solution for 20 min at room temperature. Colonies containing at least 50 cells were recorded for statistical analysis.

Flow cytometry

HOS and 143B cells (5×104 cells/well) were seeded in 6-well plates and exposed to RepSox at concentrations of 0, 50, 100 and 200 µM for 48 h. An equal volume of DMSO was added to the negative control group. After 48 h, the cells were harvested with trypsin and washed twice with chilled PBS. Then, single-cell suspensions treated with different concentrations of RepSox were fixed overnight at 4°C with prechilled 70% ethanol. The following day, the cells were incubated with 500 µl PI/RNase solution for 30 min in the dark at room temperature. Subsequently, a flow cytometer (BD FACSCanto II; BD Biosciences) was used to analyze the samples and assess the cell cycle results. Similarly, to investigate the effect of RepSox on the viability of OS cells, the cells incubated with 0, 50, 100 and 200 µM RepSox were collected and washed with PBS. The cells were then stained with an Annexin V-fluorescein isothiocyanate (FITC)-PI cell apoptosis kit (cat. no. 556547; BD Biosciences) at room temperature for 15 min, according to the manufacturer's protocol, and apoptosis rates were analyzed by flow cytometry. Data were analyzed using FlowJo software 7.6.5 (FlowJo LLC).

Soft agar colony formation assay

The OS cells were trypsinized and harvested, and 5×103 cells were mixed with a 0.3% agar solution in DMEM containing 10% FBS. In addition, different doses of RepSox were added at concentrations of 0, 50, 100 and 200 µM. The suspensions were layered on top of a solidified 0.5% agarose layer in 6-well plates. The plates were then stored at 37°C for 2 weeks, and representative images of the cell colonies were obtained under a light microscope. Colonies containing at least 50 cells were recorded for statistical analysis.

Wound-healing assay

In brief, the cells (1×105 per well) were cultured in 6-well plates with DMEM containing 10% FBS overnight at 37°C until a confluence of 90% was reached. Then the cells were starved in serum-free medium for 24 h. A sterile pipette tip was used to scratch the cell layer to produce an artificial wound line. The cells were then rinsed with PBS to clear the detached cells. The drug was diluted to the specified concentration (0, 5, 10 and 20 µM) in serum-free DMEM medium and the cells were treated at 37°C for 24 h. An equal volume of DMSO was added to the negative control group. The cells were monitored and images were captured under a light microscope at 0 and 24 h.

Cell invasion and migration assays

A total of 24-well Transwell chambers with an 8-µm pore size polycarbonate membrane (Corning, Inc.) were used to evaluate the cell migration and invasion abilities. For the invasion assay, the Matrigel was pre-coated on the membranes of the upper chamber at 37°C for 1 h according to the manufacturer's protocol (BD Biosciences). For the migration and invasion assays, 100 µl suspension of cancer cells (5×104) was added to the upper compartment with serum-free DMEM, and the lower compartment was filled with 600 µl of DMEM supplemented with 10% FBS. A preset concentration of RepSox (0, 5, 10 and 20 µM), which was used as a source of chemoattractant, was concurrently added to the lower compartment of the chamber. After culturing at 37°C for 24 h, the non-invading/non-migrated cells were wiped off with a cotton swab. The invading/migrated cells that had invaded/migrated to the lower side were fixed at room temperature with 4% paraformaldehyde for 20 min and stained with 0.5% crystal violet solution at room temperature for 30 min. Finally, images of the invading/migrated cells were captured and cells were counted under an inverted light microscope.

Western blot analysis

After treatment with the indicated concentrations of RepSox, the cells were rinsed with precooled PBS and collected with a cell scraper. Total protein was obtained by lysing cells with RIPA lysis buffer (Dalian Meilun Biology Technology Co., Ltd.) containing phosphatase inhibitor and protease inhibitor for 20 min at 4°C, and centrifugation (12,000 × g for 15 min at 4°C). Protein concentration determination was measured by BCA kit. Equal amounts (10 µg/lane) of protein samples were separated by 8% or 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels by electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore). Then, the membrane was blocked with 5% skimmed milk or 5% bovine serum albumin (BSA) (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and incubated overnight at 4°C with the appropriate primary antibodies (dilution 1:1,000) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution 1:3,000; cat. nos. FDM007 and FDR007; Hangzhou Fude Biological Technology Co., Ltd.) for 1 h at room temperature. Subsequently, an enhanced chemiluminescence kit (FD8030; Hangzhou Fude Biological Technology Co., Ltd.) was used for the analysis of the protein interactions after exposure in an imaging system, and the protein band images were quantified with ImageJ (version 1.48; National Institutes of Health).

RNA extraction and reverse transcription-quantitative (RT-q) PCR

The MMP-2 and MMP-9 gene expression levels in HOS and 143B cells were detected by qPCR. Total RNA was isolated from OS cell lines and tumor tissues by TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) after treatment with different doses of RepSox. cDNA templates were synthesized by reverse transcription using a PrimeScript™ RT reagent Kit (Takara Bio, Inc.) according to the manufacturer's instructions. Then, the cDNAs were quantified by a LightCycler® 480II PCR instrument (Roche Diagnostics) to determine the expression of related genes by using UltraSYBR Mixture (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 15 sec, and 60°C for 1 min. The sequences of the primers used were as follows: GAPDH forward, 5′-GGAAGGTGAAGGTCGGAG TCA-3′ and reverse, 5′-GTCATTGATGGCAACAATATCC ACT-3′; MMP-9 forward, 5′-TGTACCGCTATGGTTACAC TCG-3′ and reverse, 5′-GGCAGGGACAGTTGCTTCT-3′; MMP-2 forward, 5′-TTGATGGCATCGCTCAGATC-3′ and reverse, 5′-TTGTCACGTGGCGTCACAGT-3′; TGF-β forward, 5′-GGCCAGATCCTGTCCAAGC-3′ and reverse, 5′-GTGGGT TTCCACCATTAGCAC-3′; cyclin E1 forward, 5′-AAGGAGC GGGACACCATGA-3′ and reverse, 5′-ACGGTCACGTTTG CCTTCC-3′; cyclin-dependent kinase (CDK)2 forward, 5′-CCAGGAGTTACTTCTATGCCTGA-3′ and reverse, 5′-TTC ATCCAGGGGAGGTACAAC-3′; cyclin A2 forward, 5′-CGCTGGCGGTACTGAAGTC-3′ and reverse, 5′-GAGGAACGG TGACATGCTCAT-3′; p21 forward, 5′-TGTCCGTCAGAAC CCATGC-3′ and reverse, 5′-AAAGTCGAAGTTCCATCG CTC-3′. Relative mRNA expression was calculated as 2−ΔΔCq (19).

Xenograft tumor model

All animal experiments were approved (approval no. 20190712) by the Animal Care Ethics Committee of Sir Run Run Shaw Hospital (Hangzhou, China). All animal studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were kept under standard laboratory conditions (temperature 22±2°C; relative humidity 50±10%; 12-h light/dark cycle) with access to food and water ad libitum. A xenograft model of 6-week-old male nude mice with an average weight of 18.05 g was established by subcutaneous injection of 143B cells on one side of the back. In our in vivo experiments, 3×106 143B cells were made into a suspension with sterile PBS and inoculated subcutaneously into one side of the 6-week-old male nude mice (Shanghai Institutes for Biological Sciences Shanghai Laboratory Animal Center) to establish tumor formation models. One week later, when tumors grew to be visible to the naked eye, a total of 15 nude mice were randomly assigned to three groups. RepSox treatment was initiated until the tumor volume reached approximately 50 mm3. Treatment (5 and 20 mg/kg) groups were administered RepSox at 5 and 20 mg/kg every other day, respectively, while the control group was injected with DMSO. The tumor volume (V) was monitored and calculated every week based on the formula V (volume) = π x L (length) x W (width)2/6. After continuous observation for 3 weeks, all the mice were euthanized, and the tumors were resected and weighed for further study. Animal euthanasia was performed by cervical dislocation under 2% isoflurane anesthesia.

TUNEL assay

The TUNEL assay was performed according to the manufacturer's instructions of a One-Step TUNEL Apoptosis Assay kit (Beyotime Institute of Biotechnology), which was used to analyze the level of apoptosis in cell and tissue specimens. The pretreated cells were seeded in a 24-well plate at a density of 1×104 cells/well, and then fixed with 4% formaldehyde for 30 min, blocked with 5% bovine serum albumin (BSA) and incubated with 0.1% Triton X-100 at room temperature for 5 min. The slides were washed with PBS, and 50 µl of TUNEL reaction mixture was added to the cells, followed by incubation at 37°C in the dark for 1 h. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) at 37°C for 10 min. Images of the sections were captured under a fluorescence microscope, and the positive cells were counted. The nucleus stained with DAPI was blue. The cells exhibiting green fluorescence were considered apoptotic cells. Analysis of TUNEL-positive cells was carried out on five fields of view on each of the four slides in three independent experiments.

Histological and immunohistochemical analyses

Tissue blocks from excised tumors were fixed with 10% formalin for 48 h at room temperature and embedded in paraffin. Then, 4-µm sections were stained with hematoxylin for 5 min and eosin for 2 min at room temperature for histological examinations and morphometric analysis. For immunohistochemical staining, the paraffin sections were first deparaffinized, and then the sections were heated in citrate buffer for antigen retrieval. Sections were then incubated with 3% hydrogen peroxide in phosphate-buffered saline (PBS) at 37°C for 5 min, and blocked with 3% bovine serum albumin (BSA) in PBS at 37°C for 1 h. Slides were incubated overnight at 4°C with primary antibodies against Bax (dilution 1:100), Bcl-2 (dilution 1:400; product no. 15071; Cell Signaling Technology, Inc.), E-cadherin (dilution 1:400), N-cadherin (dilution 1:125; product no. 13116; Cell Signaling Technology, Inc.), vimentin (dilution 1:100), MMP-2 (dilution 1:100), MMP-9 (dilution 1:100) and Ki67 (dilution 1:500; product no. 9449; Cell Signaling Technology, Inc.), followed by incubation with biotinylated secondary antibodies (dilution 1:50; product nos. 8114 and 8125; Cell Signaling Technology, Inc.) for 1 h at room temperature.

Statistical analysis

All the data presented in this study were obtained from at least three independent experiments and were analyzed using GraphPad Prism software (version 7.0; GraphPad Software, Inc.). Values are presented as the mean ± SD of 3 independent experiments. Statistical analyses were performed using unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

Results

RepSox inhibits cell proliferation and colony formation and induces S-phase arrest

The chemical structure of RepSox is revealed in Fig. 1A. Firstly, to evaluate the inhibitory effect of RepSox on the viability of human OS cells, RepSox with concentration gradients ranging from 0 to 200 µM was used to treat HOS and 143B cells, and a CCK-8 assay was used to detect cell viability at 24, 48 and 96 h (Fig. 1B). As revealed in Fig. 1B, RepSox significantly inhibited the proliferation of HOS and 143B cells in a time- and concentration-dependent manner. In the following colony formation assays, it was also revealed that the colony size and number of HOS and 143B cells treated with RepSox were significantly inhibited. The results revealed a concentration dependence with statistically significant differences in the number and size of colonies (Fig. 1C and D). In addition, a soft agar colony formation assay was further performed, which is a three-dimensional environment that simulates the growth of OS in vivo. It was revealed that as the drug concentration increased, the inhibitory effect on the number and size of colonies tended to become markedly more pronounced (Fig. 1E and F).

Figure 1 RepSox inhibits cell proliferation and colony formation and induces S-phase arrest. (A) Chemical structure of RepSox. (B) HOS and 143B cells were exposed to different concentrations of RepSox, and Cell Counting Kit-8 assays were used to assess cell viability at 24, 48 and 96 h. The calculated IC50 values of RepSox in HOS and 143B cells at 96 h were 140 and 149.3 µM, respectively. (C and D) HOS and 143B cells were induced by different concentrations of RepSox for 14 days and then stained with crystal violet dye. Scale bars, 500 µm. (E and F) The proliferation ability of HOS and 143B cells was evaluated by soft agar colony formation assay, and the number of colonies was counted. Scale bars, 100 µm. (G and H) HOS and 143B cells were exposed to different concentrations of RepSox for 48 h, and then cell cycle assays were performed by flow cytometry, and the cell cycle distribution after RepSox treatment was analyzed. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. IC50, half maximal inhibitory concentration.

To investigate whether the inhibitory effect of RepSox on OS proliferation is related to the cell cycle, the cell cycle distribution of HOS and 143B cells induced by RepSox was further verified using PI/RNase staining. As demonstrated in Fig. 1G and H, RepSox led to the reduction of the number of cells in the G0/G1 phase and a corresponding accumulation in the S phase in both HOS and 143B cells. Based on the aforementioned results, it could be concluded that RepSox can inhibit the proliferation of OS cells by inducing S-phase arrest. To further elucidate the mechanism of the S-phase arrest of OS cells induced by RepSox, the mRNA levels of cycle-related genes were examined. As revealed in Fig. S1, the mRNA levels of cyclin E1 and p21 were upregulated after RepSox treatment, while the mRNA levels of CDK2 and cyclin A2 were decreased. The aforementioned results indicated that RepSox could inhibit cell proliferation and induce S-phase arrest of OS cells by upregulating the expression of cyclin E1 and p21 mRNA, and downregulating the expression of CDK2 and cyclin A2 mRNA.

RepSox promotes OS cell apoptosis

To clarify whether apoptosis is responsible for the inhibition of proliferation in OS cells following RepSox treatment, flow cytometry and TUNEL staining were used to detect the apoptosis levels. To quantify apoptosis, FITC-Annexin V/PI staining was performed to examine the proapoptotic effect of RepSox. In both HOS and 143B cell lines, the proportion of apoptosis in the treatment group exposed to RepSox for 48 h was significantly higher than that in the control group, regardless of early or late apoptotic status, and the apoptotic rate in early apoptotic cells was affected in a dose-dependent manner (Fig. 2A and B). TUNEL staining assays were used to detect the effect of RepSox on DNA damage in apoptotic OS cells. Fig. 2C and D revealed that RepSox-induced TUNEL-positive cells could be clearly observed, whereas the proportion of TUNEL-positive cells was negligible in the control group. Furthermore, western blotting was used to detect apoptosis-related proteins, and the results revealed that the expression of pro-apoptotic Bax protein increased with increasing drug concentration, while the expression of anti-apoptotic Bcl-2 protein was decreased (Fig. 2E and F). Collectively, these results indicated that RepSox induced typical apoptosis in human OS cells.

Figure 2 RepSox promotes osteosarcoma cell apoptosis. (A) HOS and 143B cells were treated with different concentrations of RepSox for 48 h, detected with an Annexin V-FITC/PI cell apoptosis kit, and finally analyzed by flow cytometry. (B) The proportion of apoptotic cells was quantified. (C) HOS and 143B cells were treated with 200 µM RepSox, apoptotic cells exhibited green fluorescence, and nuclei were stained with DAPI (blue). Scale bars, 50 µm. (D) The TUNEL-positive cells were quantified. (E) After exposure to different concentrations of RepSox, apoptosis-related proteins in HOS and 143B cells were assessed by western blotting. (F) The densitometric levels of Bax and Bcl-2 were quantified and normalized to that of GAPDH. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. FITC/PI, fluorescein isothiocyanate/propidine iodide; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

RepSox inhibits migration in OS cells

To further evaluate the effect of RepSox on OS cell migratory behavior, a wound-healing assay was performed. Here, the drug concentration was reduced below the toxicity level (from 50-200 µM to 5-20 µM) in order to rule out the bias caused by cell proliferation inhibition. The migration distance was obviously shorter in cells subjected to RepSox treatment for 24 h than in untreated cells. The effect of inhibition was dose-dependent (Fig. 3A and B). Concurrently, longitudinal migration experiments were carried out by using a Transwell chamber. Representative images of cell migration and invasion are presented in Fig. 3C, and it was revealed that the number of transmembrane cells in the RepSox treatment group was significantly less than that in the control group. Furthermore, the number of transmembrane cells decreased with higher concentrations of RepSox (Fig. 3D). The Transwell chamber was then coated with a layer of Matrigel to detect the invasion ability of OS cells, and the obtained results were consistent with the migration experiment (Fig. 3C and D). In addition, the results of RT-q PCR (Fig. 3E) and western blotting (Fig. 3F) revealed that RepSox significantly inhibited the expression of MMP-2 and MMP-9 in HOS and 143B cells. Next, the expression of EMT-related proteins was further examined and it was revealed that in cells pretreated with RepSox the protein levels of mesenchymal markers (N-cadherin and vimentin) were significantly downregulated, but the levels of the epithelial cell marker E-cadherin were significantly upregulated (Fig. 3G and H). All these results indicated that RepSox could inhibit migration in OS cells.

Figure 3 RepSox inhibits migration in OS cells. (A) When the HOS and 143B cells in 6-well plates were confluent, 200 µl pipette tips were used to scratch wounds, after which the medium was replaced with different concentrations of RepSox prepared in serum-free medium, and the cells were cultured for 24 h. Scale bars, 200 µm. (B) The migration ability was quantified. (C) Cells induced by different concentrations of RepSox were seeded into Transwell chambers for the cell migration assay for 24 h. In contrast to the cell migration assay, the cell invasion assay used Matrigel-coated Transwell chambers for 24 h. Scale bars, 100 µm. (D) Cells that migrated and invaded were counted. (E) Reverse transcription-quantitative PCR was used to detect the gene expression levels of MMP-2 and MMP-9 in HOS and 143B cells after RepSox induction for 24 h. (F) Western blotting was used to detect the protein expression levels of MMP-2 and MMP-9 in HOS and 143B cells after RepSox induction for 24 h. The densitometric levels of MMP-2 and MMP-9 were quantified and normalized to GAPDH. (G) Western blotting was conducted to assess the expression of EMT-related proteins. (H) The protein bands of EMT-related proteins were quantified and normalized to GAPDH. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. MMP, matrix metalloproteinase; EMT, epithelial-mesenchymal transition; ns, not significance.

RepSox exerts anti-OS effects via the JNK/Smad3 pathway

Previous studies indicated that MAPK and Smad signaling pathways have great importance for tumor progression (20-25). Firstly, analysis of gene expression revealed that treatment with 200 µM RepSox significantly inhibited the expression of TGF-β mRNA (Fig. 4A). Next, it was verified whether MAPKs and Smad play a crucial role in the inhibitory effects of RepSox in OS cells. The results revealed that the phosphorylation of JNK and Smad3 was significantly inhibited after RepSox treatment (Fig. 4B and C), while the phosphorylation of p38 and ERK was not significantly altered (Fig. 4D and E). All this data indicated that RepSox exerted an anti-OS effect via the JNK/Smad3 pathway.

Figure 4 RepSox exerts an anti-osteosarcoma effect via the JNK/Smad3 pathway. (A) Reverse transcription-quantitative PCR was used to detect the gene expression level of transforming growth factor-β in HOS and 143B cells after RepSox induction for 24 h. (B) After treatment with RepSox at different concentrations, western blotting was performed to compare the expression levels of JNK, p-JNK, Smad3 and p-Smad3 proteins. (C) Western blot analysis revealed the p-JNK and p-Smad3 protein levels and then normalized them to total JNK and Smad3. (D) HOS and 143B cells were treated as indicated in B, and western blotting was performed to compare the expression levels of p38, p-p38, ERK and p-ERK proteins. (E) Western blot analysis revealed the p-p38 and p-ERK protein levels and then normalized to total p38 and ERK. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. JNK, c-Jun amino-terminal kinase; TGF-β, transforming growth factor-β; p-, phosphorylated; ERK, extracellular signal-regulated kinase; ns, no significance.

RepSox inhibits OS growth in vivo

To clarify the anticancer effect of RepSox in vivo, a xenograft model was established and randomly divided into three groups, receiving intraperitoneal administration of RepSox (5 mg/kg or 20 mg/kg every other day) or DMSO. In terms of drug concentration selection for the in vivo experiment, on the one hand, the CCK-8 data of our in vitro experiment were considered, and on the other hand, some published literature (26-28) on drug concentrations of other drugs in OS xenograft models was consulted. After three weeks, the tumors were removed from each nude mouse and measured (Fig. 5A and B). The RepSox-treated group exhibited a significant decrease in both tumor volume and weight (Fig. 5C and D). However, there was no significant difference in body weight between the control group and RepSox-treated groups (Fig. 5E). The results of immunohistochemistry revealed an obvious decrease in Bcl-2, N-cadherin, vimentin, MMP-2 and MMP-9 expression, along with a reduction in the proliferation marker Ki67. Conversely, the expression of E-cadherin and Bax was upregulated (Fig. 5F). A TUNEL assay was utilized to confirm the proapoptotic effect of RepSox on tumors in vivo. It was revealed that as the RepSox concentration increased, TUNEL-positive cells also increased in tumor tissue compared with the control group (Fig. 5G and H). The hearts, livers, spleens, lungs and kidneys of the mice were removed for further histological observation. H&E-stained sections revealed intact tissue structure in all three groups, no OS metastatic lesions and no cytotoxic effects (Fig. 5I). In conclusion, these results revealed that RepSox efficiently suppressed the growth of OS and had low toxicity in nude mice.

Figure 5 RepSox inhibits osteosarcoma growth in vivo. A xenograft model in 6-week-old male nude mice was established by subcutaneous injection of 143B cells on both sides of the back. After one week of modeling, mice randomly divided into three groups began to receive intraperitoneal administration of RepSox (5 mg/kg or 20 mg/kg every other day) or DMSO (control group). (A) Tumors in vivo are presented. (B) Tumors were removed from the nude mice. (C) The tumors were weighed and analyzed. (D and E) The tumor volume and body weight of mice were measured weekly. (F) Representative images of Bax, Bcl-2, MMP-2, MMP-9, E-cadherin, N-cadherin, vimentin and Ki67 immunostaining obtained by a light microscope. Scale bars, 50 µm. (G) Tumor tissue from the three groups was stained with a TUNEL kit. Scale bars, 50 µm. (H) TUNEL-positive tissue was quantified. (I) Hematoxylin and eosin staining of major organs is presented. Scale bars, 50 µm. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. MMP, matrix metalloproteinase; TUNEL, terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.

Discussion

OS is a malignant primary bone tumor that mainly occurs in children and adolescents (1). Its prognosis is poor, especially in patients with metastases at diagnosis and patients who are resistant to chemotherapy (29, 30). In this context, it is urgent to study the mechanism of OS development and find new targets for OS treatment. Previous studies revealed that TGF-β plays an important role in tumor progression and can promote the metastasis of numerous solid tumors, such as breast, colon and prostate cancer (8,31-33). Interestingly, TGF-β exhibits dual effects on tumor progression, which is, inhibiting tumor progression in premalignant tumors and promoting tumor development in advanced tumors (34-36). However, some studies have indicated that compared with its role in malignant epithelial neoplasms, TGF-β in sarcoma mainly exerts a tumor-promoting effect, especially in OS (12,37). In patients with OS, the expression of TGF-β1 and TGF-β2 in serum is significantly higher than that in healthy donors (11,12). Previous studies have reported that high levels of TGF-β1 mRNA expression in tumor cells were associated with high-grade OS and frequent metastasis to the lung or other organs (38,39). According to the literature, SD-208, a TGF-βRI inhibitor, can significantly reduce the incidence of lung metastasis in OS, which is similar to the results of our study (12). The present study explored the inhibitory effect of RepSox on OS cells. In brief, RepSox inhibited the proliferation and metastasis of OS cells and induced apoptosis in vitro through the JNK/Smad3 signaling pathway, and its inhibitory effect on tumors was also confirmed in vivo.

In our experiments, a small molecule TGF-βRI/ALK5 inhibitor (RepSox) was selected to evaluate its effect on OS cells. It was revealed that RepSox significantly inhibited the proliferation of OS cells in vitro. Mechanistically, it was revealed that this result was mainly caused by cell cycle arrest and increased apoptosis. Therefore, a literature search was conducted and it was revealed that TGF-β can increase the expression of CDK inhibitors 1A (CDKN1A or p21), 2B (CDKN2B or p15) and 1C (CNKN1C or p57) and apoptosis inducer death-associated protein kinase (DAPK) (40-42), thereby inducing cell cycle arrest and promoting apoptosis. Cell cycle arrest is recognized as an important cause of proliferation inhibition. This process is closely monitored by some checkpoints and will be induced to pause when necessary (43,44). Cyclins and CDKs are key regulators of the cell cycle (45). CDK2 is activated by binding to cyclin E, while cyclin E, in complex with CDK2, drives the transition from the late G1 phase to the S phase (46). In addition, the association of CDK2 and cyclin A are also essential for G1/S progression (47). Concurrently, as an important protein inhibitor in the cell cycle, p21 can also bind to CDK, resulting in a block in cell cycle progression (48). In the present study, RepSox promoted the transition of cells to the S phase by upregulating cyclin E1 and p21, and reduced cyclin A2 and CDK2 to arrest the cell cycle in the S phase. In addition, c-Myc, another key transcriptional activator of cell proliferation, has been revealed to be inhibited (49).

As aforementioned, TGF-β plays the role of a tumor suppressor, which is exactly the opposite of our experimental results. A possible explanation is that cancer cells bypass these cytostatic effects by inactivating key molecules in the TGF-β signaling pathway and crosstalk with other oncogenic pathways (49). Malignant tumor cells may retain the TGF-β/Smad pathway, but they can promote tumor progression by acquiring noncanonical PI3K/AKT, RAS/MAPK, or p53 pathway carcinogenic mutations (50). In our research, it was revealed that the JNK/Smad3 signaling pathway was inhibited (Fig. 6), which may explain the aforementioned phenomenon, but the specific mechanism still requires further study.

Figure 6 Schematic representation of how RepSox induces cell cycle arrest and apoptosis and inhibits epithelial-mesenchymal transition in osteosarcoma cells. EMT, epithelial-mesenchymal transition; TGF-β, transforming growth factor-β.

In the subsequent wound-healing, cell migration and invasion assays, the drug concentration was changed in order to exclude the biased results caused by cell proliferation. The results revealed that RepSox could also significantly inhibit the migration and invasion of tumor cells below the cytotoxic concentration, which fully indicated that RepSox had a quite evident inhibitory effect on the metastasis of OS cells.

As aforementioned, TGF-β has a dual effect in tumors, and ultimately, the function of TGF-β is related to its ability to induce epithelial-mesenchymal transition (EMT) programs (51). Sarcoma has a mesenchymal origin, and its mesenchymal phenotype is maintained by the functions of EMT transcription factors (EMT-TFs), including TWIST1, NAIL, SLUG, ZEB1 and ZEB2, and associated with more aggressive behaviors (52). A number of studies indicated that overexpression of EMT-related transcription factors is implicated in the OS pathogenesis, which may be a significant reason for EMT (53-56). In addition, several in vitro studies have confirmed that the ability of TGF-βs to promote EMT programs in various OS cell lines may be linked to the promigratory effect of TGF-βI (57-60). In addition, Smad and MAP-kinase signaling are essential for the transcriptional induction of numerous extracellular matrix (ECM) proteins, such as MMPs (e.g., MMP-2 and MMP-9) (61,62). Multiple previous studies (63-66) demonstrated that inhibition of the phosphorylation of JNK can reduce the expression of MMP-2/MMP-9 in OS cells. Furthermore, a study by Lamora et al (67) revealed that halofuginone decreased the expression and activity of MMP-2 by inhibiting the TGF-β/Smad3 cascade in OS. In our study, RepSox inhibited the expression of MMP-2 and MMP-9 in a concentration-dependent manner, reducing transcription-induced stimulation of ECM-related proteins. In parallel, hallmarks of mesenchymal phenotypes include the expression of vimentin-based intermediate filaments, the exchange of E-cadherin-based with N-cadherin-based junctions, and the extensive synthesis of ECM proteins and matrix remodeling enzymes (metalloproteases) that facilitate the migration and invasiveness of mesenchymal and inflammatory cells (68). Our data revealed that the expression of E-cadherin was increased under the effect of RepSox, while the expression of N-cadherin and vimentin was inhibited, indicating that RepSox enhanced the adhesion between tumor cells and reduced the migration and invasion of tumor cells.

In in vivo experiments, several tumor xenograft models in literature have been used, and it was revealed that they all adopted the 143B cell line as the source of tumor cells. In addition, in our in vitro experiments, it was observed that 143B cells proliferated faster than HOS cells. It was hypothesized that this may render the experimental results more intuitive, therefore the 143B cell line was selected. Meanwhile, in addition to the 143B cell line, other OS cell lines should also be used for in vivo xenograft studies. However, due to constraints on research funds and time, only the 143B cell line was selected for in vivo experiments in the present study, which is also a limitation of the present study.

With regard to the tumors in the in vivo experiments that appear to have a more pronounced inhibitory effect than in the in vitro experiments, it was hypothesized that this may be due to the effect of migration inhibitory factor (MIF) on the tumor microenvironment. Several studies have revealed that treatment with anti-MIF antibodies significantly inhibited the growth of murine colon cancer, and concurrently suppressed its angiogenesis (69,70). Moreover, a series of in vitro and in vivo experimental results indicated that MIF deficiency attenuated tumor-polarized macrophage alternative activation, immunosuppression, neoangiogenesis, and melanoma tumor outgrowth (71). The aforementioned results revealed the effect of MIF and its inhibitors on the tumor microenvironment and that is also our future direction of the present study. In addition, in vivo pharmacokinetic and bioavailability studies on RepSox are required.

In addition, in recent years, nanomedicine is rapidly advancing as an emerging field. Nanomaterials are gradually used as an effective carrier for the diagnosis and treatment of diseases (72-76). In the chemotherapy of OS, conventional small-molecule therapeutics exhibit low efficacies and severe side effects, while the drug-delivery platforms based on nanotechnology can significantly improve the antitumor efficacy and diminish the side effects (74). It has broad application prospects in OS chemotherapy. In addition, the development and innovation of biocomposites also provide novel insights for the treatment of OS (77).

In summary, our study revealed that RepSox could induce cycle arrest and apoptosis of OS cells and inhibit EMT by inhibiting the JNK/Smad3 pathway, which was also further demonstrated in in vivo experiments. Therefore, RepSox may be potentially valuable as an alternate therapeutic agent for OS treatment.

Supplementary Data

Availability of data and materials

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

DH and SF conceived and designed the experiments. DH, JG, LZ, SL, LY and HL performed the experiments. DH, BP, WP and ZC conducted the statistical analyses. CL helped to perform the analyses with constructive suggestions. DH wrote the paper and SF revised the paper. DH and JG confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The animal experiments were approved by the Animal Care Ethics Committee of Sir Run Run Shaw Hospital (Hangzhou, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

References