Necroptosis of osteoblasts was induced by breast cancer cells in vitro

BACKGROUND: Bone metastasis of breast cancer could lead to serious osteolysis and severe pain. This study is aimed to investigate the existence of necroptosis, a new type of programmed cell necrosis pathway, in breast cancer-induced osteoblast cell death. METHODS: In this study, conditioned medium (CM) of breast cancer cells was prepared to simulate the micro-environment of bone metastasis in breast cancer in vitro and co-cultured with osteoblast. Then the percentage of cell survival and death was detected via cell viability and flow cytometry. Western blot and PCR were taken to measure protein and mRNA expression level of RIPK 3, MLKL and caspase 3 respectively. RESULTS: CM could induce osteoblasts death, including apoptosis and necroptosis and necrostatin-1 plus Z-IETD-FMK could decrease the percentage of death cells significantly in the flow cytometry detection. Moreover, CM could increase cleaved caspase 3, RIPK 3 and p-MLKL significantly, while RIPK 3 and p-MLKL was reduced statistically when osteoblasts were treated with Necrostatin-1 (Nec-1). In addition, the mRNA level of three proteins was not consistent with the change of their corresponding protein level. CONCLUSIONS: In conclusion, the necroptosis pathway exists in osteoblast cell death pathway induced by breast cancer cells and could be inhibited by Necrostatin-1 (Nec-1).