MMPs contribute to this metastatic process by degrading basement membrane. In addition, MMPs can, due to their proteolytic activities, promote tumor growth by increasing the bioavailabilities of growth factors in the ECM [11]. Furthermore, it is becoming

increasingly clear that MMPs play a central role in ECM degradation [13]. Among MMPs, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), are present in large quantities in cancer tissues [14, 15], and accumulating evidence indicates that MMP-2 and MMP-9 play critical role during tumor invasion and metastasis [14, 16–20]. Furthermore, Matrix metalloproteinases (MMPs) and their endogenous inhibitors participate in the invasive process of human osteosarcoma [21]. Bisphosphonates (BPs) are stable analogues of pyrophosphonate, GSK1210151A supplier and are potent inhibitors of osteoclast-mediated bone resorption. They are widely used to treat metabolic bone diseases, such as, Paget’s disease [22] and hypercalcemia [23] and to treat postmenopausal osteoporosis [24]. Recently, it was reported that BPs may significantly help control the pain associated with bone tumors [25]. Preclinical evidence suggest that BPs have direct antitumor effects on a variety of human cancer cells [26], and it is known that they

decrease cell proliferation in human osteosarcoma cell line panels, disturb the cell cycle, and PND-1186 induce the apoptosis of SaOS-2 cells [27, 28]. These findings suggest that BPs could play a beneficial Ribonucleotide reductase adjuvant role in the treatment of osteosarcoma. However, the inhibitory effects of BPs on osteosarcoma cell have not been Tanespimycin comprehensively studied, and therefore, in the present study, we examined the effects of the third-generation BPs, risedronate, on osteosarcoma cell invasion. Methods Reagents Risedronate [1-hydroxy-2-(3-pyridinyl)ethylidene]bis [phosphonic acid] was purchased from (Sanofi-Aventis, Korea). A stock solution of risedronate was prepared in phosphate-buffer saline (PBS). All other chemicals and reagents

used were of analytical grade. Cell Culture SaOS-2 and U2OS were purchased from the Korean Cell Line Bank (KCLB). Cells were cultivated in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL, Grand Island, NY). Cultures were maintained at 37°C in a 5% CO2/95% air atmosphere. The medium was changed every 2–3 days, and cells were passaged twice a week. Risedronate treatment of SaOS-2 and U2OS cells SaOS-2 and U2OS cells were seeded in 6-well plates at a density of 2 × 105cells/well in DMEM/10% FBS overnight. The cells were then washed and treated with different concentrations of risedronate (0, 0.1, 1, 10 μM) for 48-h at 37°C in 5% CO2. Conditioned media were then collected and cells were harvested. MTT cell viability assay SaOS-2 and U2OS cells were seeded onto a 96-well culture plate at a density of 1 × 104 cells/well in 100 μl of complete DMEM.