Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways

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), Ca(2+), 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.