Naringenin Induces ROS-Mediated ER Stress, Autophagy, and Apoptosis in Human Osteosarcoma Cell Lines

Osteosarcoma, a primary bone tumor, responds poorly to chemotherapy and radiation therapy in children and young adults; hence, as the basis for an alternative treatment, this study investigated the cytotoxic and antiproliferative effects of naringenin on osteosarcoma cell lines, HOS and U2OS, by using cell counting kit-8 and colony formation assays. DNA fragmentation and the increase in the G2/M phase in HOS and U2OS cells upon treatment with various naringenin con-centrations were determined by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and Annexin V/propidium iodide double staining, respectively. Flow cytom-etry was performed, and 2^′,7^′-dichlorodihydrofluorescein diacetate, JC-1, and Fluo-4 AM ester probes were examined for reactive oxygen species (ROS) generation, mitochondrial membrane potential, and intracellular calcium levels, respectively. Caspase activation, cell cycle, cytosolic and mitochon-drial, and autophagy-related proteins were determined using with western blotting. The results indicated that naringenin significantly inhibited the viability and proliferation of osteosarcoma cells in a dose-dependent manner. In addition, naringenin induced cell cycle arrest in osteosarcoma cells by inhibiting cyclin B1 and cyclin-dependent kinase 1 expression and upregulating p21 expression. Furthermore, naringenin significantly inhibited the growth of osteosarcoma cells by increasing the intracellular ROS level. Naringenin induced endoplasmic reticulum (ER) stress-mediated apoptosis through the upregulation of ER stress markers, GRP78 and GRP94. Naringenin caused acidic vesic-ular organelle formation and increased autophagolysosomes, microtubule-associated protein-light chain 3-II protein levels, and autophagy. The findings suggest that the induction of cell apoptosis, cell cycle arrest, and autophagy by naringenin through mitochondrial dysfunction, ROS produc-tion, and ER stress signaling pathways contribute to the antiproliferative effect of naringenin on osteosarcoma cells.