Furthermore, the normal osteoblast cell line hFOB1.19 was employed to explore whether salidroside induced osteoblast apoptosis with the same concentrations. reported that salidroside has anti-fatigue, anti-aging, anti-oxidant, anti-inflammatory, neuroprotective and cardiovascular protective effects (9-12). A literature review revealed that salidroside exhibits antitumor effects in various tumors, including fibrosarcoma (13), bladder carcinoma (14), lung carcinoma (15), breast carcinoma (16) and renal cell carcinoma (17) and the underlying molecular mechanism. Materials and methods Cell culture and treatment Human osteosarcoma cell lines MG63 and U2OS (ZQXZBIO, Shanghai, China), were selected to assess the antitumor effects of salidroside. Cells were cultured in Dulbecco’s modified Eagle’s medium combined with high-glucose medium (DMEM-HG) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and were maintained in a 37C humidified incubator with 5% CO2. Cells were harvested with a 0.25% trypsin-0.02% EDTA solution and passaged when the cells attained ~80% confluence. Salidroside (Fig. 1A; purity 99%, MedChem Express, Monmouth Junction, NJ, USA) was dissolved in PBS at room temperature and filtered through a 0.22-(24) reported that salidroside combined with antitumor agents exerted excellent antitumor effects in colorectal cancer. Qi (25) revealed that salidroside had a direct inhibitory effect on the proliferation, migration and invasion of gastric cancer cells. In the TMB present study, we first assessed the antitumor effects of salidroside in the treatment of osteosarcoma. We demonstrated that salidroside induced the growth and invasion TMB of osteosarcoma cells, which indicated its therapeutic potential. The pharmacological mechanism of salidroside may be related to the JAK2/STAT3 signaling pathway (Fig. 7). Open in a separate window Figure 7 Diagram of the mechanism of salidroside-induced apoptosis, cell cycle arrest and suppressed invasion of osteosarcoma cells via inhibition of the JAK2/STAT3 signaling pathway. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; JAK, Janus kinase; MMP, matrix metalloproteinase; STAT3, signal transducer and activator of transcription 3. HDAC-A Cell proliferation is an important marker for tumor development. Therefore, inhibiting tumor growth (by promoting tumor cell apoptosis) is the most important objective in preventing tumor progression (26). The MTT assay is widely used in bioactive factor activity assays, large-scale antitumor drug screening and cytotoxicity assays (27). In the present study, the results of the MTT assay revealed that salidroside significantly inhibited the viability of osteosarcoma cells in a time- and concentration-dependent manner. The results of cell morphological observations and flow cytometric apoptosis detection further indicated that the decrease in cell viability induced by salidroside was associated with cell apoptosis. We investigated whether the expression of apoptotic-related proteins via western blot analysis, and the expression of the Bcl-2 and caspase families, critical apoptosis-related proteins, were regulated by salidroside. The Bcl-2 and caspase families are specific regulatory proteins of the mitochondrial apoptosis pathway, which is one of the main pathways of apoptosis (28). Our results indicated that TMB the mitochondrial apoptosis pathway is involved in salidroside-mediated apoptosis of osteosarcoma cells. In addition, dysregulated cell cycle distribution is another feature of tumor development, and the induction of cell apoptosis is accompanied with cell cycle arrest (29). Flow cytometric cell cycle analysis is widely used for evaluating changes in cell cycle distribution (30). We reported that salidroside triggered G0/G1 phase arrest in osteosarcoma cells, which was consistent with previous reports (16,31). Then, the present study investigated the expression of cell cycle-related proteins using western blot analysis; the expression of cyclin D1 and p21 were revealed to be regulated TMB by salidroside. Therefore, we concluded that salidroside induced the apoptosis of osteosarcoma cells by inducing G0/G1 phase arrest. We suggested that salidroside may function as an agonist to induce the apoptosis and G0/G1 phase arrest of osteosarcoma cells, and may represent as an alternative therapeutic strategy for the treatment of osteosarcoma. Invasion, which generally leads to metastasis, can be used to predict tumor malignancy (32). Previous research reported that early metastasis (particularly pulmonary metastasis) remains as the main cause of mortality in ~40% of patients with osteosarcoma (33). The results of Transwell assays demonstrated that salidroside significantly.