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

Chiang Wen Lee, Cathy Chia Yu Huang, Miao Ching Chi, Kuan Han Lee, Kuo Ti Peng, Mei Ling Fang, Yao Chang Chiang, Ju Fang Liu

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15 Scopus citations


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.

Original languageEnglish
Article number373
Issue number2
StatePublished - 1 Jan 2022


  • Apoptosis
  • Autophagy
  • ER stress
  • Naringenin
  • Osteosarcoma
  • ROS


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