Brazilin induces FOXO3A-dependent autophagic cell death by disturbing calcium homeostasis in osteosarcoma cells.

Abstract

Osteosarcoma is a common primary malignant bone tumour, and its cure rate has stagnated over the past 25-30years. Brazilin, a purified natural product from sappan wood (Caesalpinia sappan L.), has been proved to possess potent anti-cancer effects. In this study, we investigated the anti-cancer effect of brazilin on human osteosarcoma and elucidated the underlying mechanisms. We exposed MG-63 cells to different concentrations of brazilin (5, 10 and 20µM) for 24h. Western blotting, immunocytofluorescence, luciferase reporter assays, and RT-PCR were used to evaluate whether brazilin activates FOXO family-dependent autophagy. Brazilin increased autophagic flux in the human osteosarcoma cell line MG-63, as evidenced by the upregulation of LC3-II and the downregulation of P62/SQSTM1. Moreover, the pharmacological or genetic blockade of autophagy decreased brazilin-induced cell death, indicating that brazilin triggered autophagic cell death in MG-63 cells. Specifically, brazilin induced FOXO3A(Ser7) phosphorylation, activated FOXO3A nuclear translocation and increased FOXO3A reporter activity, which contributed to the expression of autophagy-related genes and subsequently initiated autophagic cell death in MG-63 cells. Importantly, the increased expression and nuclear translocation of FOXO3A were tightly related to the disturbance of calcium homeostasis, which could be prevented by chelating intracellular calcium. Taken together, these data demonstrate that brazilin induces osteosarcoma cell death by inducing excessive autophagy, which is mediated through the Ca2+-FOXO3A pathway. Our study provides a new anti-tumour mechanism for brazilin treatment in osteosarcoma patients.