Monocyte chemotactic protein-1 promotes the proliferation and invasion of osteosarcoma cells and upregulates the expression of AKT

Monocyte chemotactic protein-1 (MCP-1/CCL2) is an important immune factor, which may be important in cancer progression by promoting proliferation, invasion, metastasis and the tumor microenvironment. Previous studies have demonstrated that CCL2 affects the proliferation of osteosarcoma cells via the RANKL signaling pathway. However, the underlying mechanisms remain to be elucidated. To investigate the role of CCL2 in osteosarcoma cells, MTT, spheroid forming, wound healing and transwell assays were performed to examine the proliferation and invasion abilities of the osteosarcoma cells. It was revealed that the high-grade osteosarcoma cells exhibited increased expression levels of CCL2 compared with the low-grade osteosarcoma cells (P<0.001). Furthermore, knockdown of CCL2 decreased the proliferation and invasion abilities of the osteosarcoma cells (P<0.01). These results suggested that the expression of CCL2 is high in high-grade osteosarcoma cells and promotes the proliferation and invasion of osteosarcoma cells.


Introduction
Chemokines are a superfamily of small, secreted molecules, which exert their activity by binding to G-protein-coupled receptors. Currently, ~50 ligands and 20 receptors have been identified in humans and mice (1). Chemokines are classified into four families, CXC, CC, CX3C and C, according to the presence of four cysteine residues in a conserved region (2). It has become clear that chemokines and receptors control the proliferation and metastasis of tumor cells (3). They are considered to have a critical and complicated role in a variety of cells, including tumor cells (4,5).
Osteosarcoma is the most common type of malignant bone tumor in childhood and adolescence, and represents 15% of all primary bone tumors and 0.2% of all malignant tumors in children (6,7). The prognosis of patients with primary metastatic disease is poor (8). Tumor cells, including osteosarcoma cells, and cells in the tumor microenvironment, including fiboclasts and endothelial cells, secret monocyte chemotactic protein-1 (MCP-1/CCL2) (9). As an inflammatory medium, CCL2 is the predominant chemotactic protein, which attracts macrophages into the tumor environment from the blood. It is hypothesized that by attracting macrophages into the microenvironment and inducing these cells to release a series of factors, CCL2 promotes tumor cell generation and angiogenesis, and increases the chance of metastasis (10)(11)(12)(13). The present study investigated the role of CCL2 in osteosarcoma cells.

Monocyte chemotactic protein-1 promotes the proliferation and invasion of osteosarcoma cells and upregulates the expression of AKT
MTT assay. The effects of CCL2 on the proliferation of untreated LM8 cells (mock) or those transfected with LM8-NC, LM8-sh1 or LM8-sh3 were assessed using an MTT assay. The cells were seeded into 96-well plates at a density of 2,000 cells/well in DMEM-h and were cultured for 1, 2, 3, 4 or 5 days in DMEM supplemented with 10% FBS. Following incubation, 20 µl MTT (5 mg/ml; Sigma-Aldrich) was added to each well and the cells were incubated for 4 h at 37˚C. The culture medium was removed and 150 µl dimethylsulfoxide (Sigma-Aldrich) was added, following which the optical densities (OD) were measured using an ELX800 Micro Plate Reader (Bio-Tec Instruments, Inc,. Winooski, VT, USA) at 490 nm. The cell viability was calculated using the following equation: Cell viability (%) = (OD 490nm treatment / OD 490nm control) x 100%.
Spheroid forming assay. The cells were seeded into 6-well plates at a density of 300 cells/well. Following incubation for 15 days, the colonies were washed three times with phosphate-buffered saline (PBS; Sangon Biotech Co., Ltd., Shanghai, China), fixed with methanol/acetic acid (3:1) and stained with 1% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China). The number of colonies (>10 cells) in four different field of views were quantified under a microscope (magnification, x50; DP72; Olympus Corporation, Tokyo, Japan) and the mean value was calculated.
Wound-healing assay. The cells (5x10 5 cells/well) were seeded into 24-well plates and cultured until 90% confluent, at 37˚C for ~24 h. The monolayer of cells was then scratched using a thin disposable tip to wound the layer, and the cells were incubated for 24 h at 37˚C. Images of cell migration were captured using an Olympus microscope (Olympus, Tokyo, Japan). The migratory activity of the cells was expressed as the percentage of the area covered by cells, which was measured using Olympus DP72 software (Olympus).
Transwell assay. The migration assay was performed using a Transwell (Corning Incorporated, Corning, NY, USA; pore size, 8 µm) in 24-well plates. Prior to performing the migration assay, the cells were pretreated for 24 h in FBS-free medium, with or without 30 µM AMD3100. The cells (~10x10 4 cells in 200 µl FBS-free DMEM-h) were placed in the upper chamber, and DMEM-h supplemented with 10% FBS was placed in the lower chamber. The plates were incubated for 48 h at 37˚C in 5% CO 2 , following which the culture medium was removed and the filters were washed with PBS twice. The cells on the upper side of filters were removed using cotton-tipped swabs, and the cells on the underside of the filters were fixed in 95% alcohol for 10 min and stained with 0.1% crystal violet for 15 min at room temperature. The cells, which had migrated from the upper to the lower side of the filter were counted under a light microscope by counting 10 random fields of view at magnification x100. The tumor cell migration assay was performed in triplicate. Statistical analysis. Statistical analyses were performed using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). The data were analyzed using an unpaired two-tailed Student's t-test and data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Results
Expression of CCL2 in the osteosarcoma cell lines. Since the importance of chemokines in the occurrence and development of various types of cancer is increasing, it is important to investigate the role of CCL2 in the biology and pathophysiology of osteosarcoma. The present study performed RT-qPCR and western blot analysis to analyze the expression of CCL2 in the osteosarcoma cell lines. It was observed that the expression of CCL2 was higher in the more malignant osteosarcoma cell lines (LM8 and K7M3; Fig. 1A and B) compared with the less malignant cell lines (Dunn, K7 and K12).    C in the LM8, NC, LM8-sh1 and LM8-sh3 cells was detected by RT-qPCR and western blot analysis. As shown in Fig. 2B and C, the LM8 cells transfected with the plasmid encoding shRNA-CCL2 (pGMLV-SC1 CCL2) exhibited reduced mRNA and protein expression levels of CCL2 compared with the control cells transfected with a negative control plasmid (pGMLV-SC1 NC) and the untransfected LM8 cells. In addition, an MTT assay and a clonogenic survival assay revealed that the CCL2-knockdown cells exhibited a reduced cell proliferation rate compared with the mock and NC cells (Fig. 3A-C).
Knockdown of CCL2 reduces the invasion of LM8 cells in vitro. The highly invasive LM8 osteosarcoma cell line exhibit significant migratory ability, and CCL2 has been found to induce the migration of bladder and prostate cancer cells (16,17). Therefore, it was hypothesized that CCL2 may be involved in LM8 cell migration. As shown in Fig. 4B and C, the wound healing and transwell assays revealed that the CCL2-knockdown cells exhibited a significantly slower rate of migration compared with the mock and NC cells ( Fig. 4A and D).

CCL2 promotes the growth of osteosarcoma tumors in mice.
The cells from each of the treatment groups were implanted into mice to investigate the extent to which CCL2 affects the growth or invasion of cell-derived osteosarcoma tumors. The results indicated that CCL2-shRNA LM8 cells formed smaller nodes compared with the CCL2-NC and mock LM8 cells ( Fig. 5A and B). The necrotic degree of lung metastasis was detected by hematoxylin-eosin staining. Microtumors were not observed in the lungs of the of mice models and they may have been associated with the injection of tumor cells into the muscle.
Expression of p-AKT is downregulated in the CCL2-shRNA LM8 cells. The significance of the AKT pathway in cancer cell proliferation and metastasis has been evaluated in several studies (18,19). The expression of p-AKT was detected in the CCL2-shRNA, CCL2-NC and mock LM8 cells by western blot analysis, which revealed that the expression of p-AKT was downregulated in the CCL2-shRNA LM8 cells (Fig. 5C) compared with the mock and NC cells.

Discussion
Osteosarcoma is the most common malignant bone tumor and exhibits a high propensity for metastatic spread, predominantly to the lung and bones (20). As a CCL2 receptor, the expression of CCR2 was detected in 100% of the 27 osteosarcoma samples (21). The expression of CCL2 correlated with a poor prognosis and metastatic disease in patients with several types of cancer, including breast and prostate cancer (22,23). CCL2 is involved in other cancer types, including bladder and prostate, by promoting the migration and invasion of cancer cells (16,17). Previous studies demonstrated that CCL2 is synthsized by breast tumors and the stroma recruitment metastasis-associated macrophages, and this promoted metastasis in vivo and shortened the survival rate of tumor-bearing mice (13).
The role of CCL2 in osteaosarcoma cells remains to be elucidated. The present study demonstrated that CCL2 promoted the proliferation and invasion of osteosarcoma cells. The mRNA and protein expression levels of CCL2 were upregulated in high-grade osteosarcoma cells. It was also suggested that CCL2 may be important in osteosarcoma cells. A pGMLV-SC1 RNAi vector, which inhibited the expression of CCL2 was transfected into LM8 cells to investigate the importance of CCL2 in vivo and in vitro. It was demonstrated that inhibition of the expression of CCL2 inhibited LM8 cell proliferation, decreased clonogenic formation and invasion in vitro. In murine models, inhibition of CCL2 decreased the weight of the cancer nodes. To further examine the molecular mechanism underlying growth promotion by CCL2, the protein expression of p-AKT was demonstrated to decrease in LM8 cells with CCL2-inhibition. The activation of AKT signaling is a common hallmark in various human cancers, including osteosarcoma (24,25). In high-grade osteosarcoma cells, the inhibition of AKT signaling may be a potential target for cancer therapy (26).
Previous studies have demonstrated that Carlumab (CNTO 888), which is a human IgG 1 κ monoclonal antibody with high affinity and specificity for human CCL2, does not inhibit the CCL2/CCR2 axis or demonstrate antitumor activity as a single agent in metastatic castration-resistant prostate cancer (27). However, whether carlumab is enriched in the tumor microenvironment remains to be elucidated. In conclusion, the present study provided a novel insight into how CCL2 affected tumor progression. Whether activation of CCL2 affected the expression of AKT will be determined in a future investigation.