Bone cancer is one of the cancer-related diseases, and there are increased numbers of patients with bone cancer worldwide. Therefore the efficacy of treatment of bone cancer is considered extremely vital. Bufalin has been showed to have biological activities including anticancer activities in vitro and in vivo. However, the exact associated mechanisms for bufalin induced apoptosis in human bone cancer cells are still unclear. In the present study, we investigated the effect of bufalin on the cytotoxic effects in U-2 OS human osteosarcoma cells. For examining apoptotic cell deaths, we used flow cytometry assay, Annexin V/PI double staining, and TUNNEL assay. Reactive oxygen species (ROS), Ca²⁺, mitochondrial membrane potential (ΔΨ_m), and caspase-8, -9 and -3 activities were measured by flow cytometry assay. Furthermore, western blotting and a confocal laser microscopy examination were used for measuring the alterations of apoptotic associated protein expression and translocation, respectively. The results indicated that bufalin induced cell morphological changes, decreased the viable cell number, induced apoptotic cell death, and increased the apoptotic cell number, and affected apoptotic associated protein expression in U-2 OS cells. Bufalin increased apoptotic proteins such as Bak, and decreased anti-apoptotic proteins such as Bcl-2 and Bcl-x in U-2 OS cells. Furthermore, bufalin increased the protein levels of cytochrome c (Cyto c), AIF (Apoptosis inducing factor) and Endo G (Endonuclease G) in cytoplasm that were also confirmed by confocal microscopy examination. Based on those findings, bufalin induced apoptotic cell death in U-2 OS cells may be via endoplasmic reticulum (ER) stress, caspase-, and mitochondria-dependent pathways; thus, we may suggest that bufalin could be used as an anti-cancer agent for the treatment of osteosarcoma in the future, and further in vivo studies are needed. Full article

Osteosarcoma is the most common malignant primary bone tumor in children and young adults and lung metastasis is the main cause of death in those patients. Deguelin, a naturally occurring rotenoid, is known to be an Akt inhibitor and to exhibit cytotoxic effects, including antiproliferative and anticarcinogenic activities, in several cancers. In the present study, we determined if deguelin would inhibit migration and invasion in U-2 OS human osteosarcoma cells. Deguelin significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells which was associated with a reduction of activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). Furthermore, results from western blotting indicated that deguelin decreased the cell proliferation and cell growth-associated protein levels, such as SOS1, PKC, Ras, PI3K, p-AKT(Ser473), IRE-1α, MEKK3, iNOS, COX2, p-ERK1/2, p-JNK1/2, p-p38; the cell motility and focal adhesion-associated protein levels, such as Rho A, FAK, ROCK-1; the invasion-associated protein levels, such as TIMP1, uPA, MMP-2. MMP-9, MMP-13, MMP-1 and VEGF in U-2 OS cells. Confocal microscopy revealed that deguelin reduced NF-κB p65, Rho A and ROCK-1 protein levels in cytosol. MMP-7, MMP-9 and Rho A mRNA levels were suppressed by deguelin. These in vitro results provide evidence that deguelin may have potential as a novel anti-cancer agent for the treatment of osteosarcoma and provides the rationale for in vivo studies in animal models. Full article