Abstract
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 concentrations were determined by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and Annexin V/propidium iodide double staining, respectively. Flow cytometry 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 mitochondrial, and autophagy-related proteins were determined using western blotting. The results indicated that naringenin significantly inhibited 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 vesicular 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 production, and ER stress signaling pathways contribute to the antiproliferative effect of naringenin on osteosarcoma cells.
Highlights
Cancer is a public health problem, with high mortality and disability rates worldwide [1]
To determine the effect of naringenin on human osteosarcoma, HOS and U2OS cells were treated with varying concentrations of naringenin for 24 h
The results indicated that naringenin reduced the viability of osteosarcoma cells but not normal hFOB 1.19 cells in a concentration-dependent manner (Figure 1B–D), indicating that naringenin selectively inhibited the growth of osteosarcoma cells but exhibited less cytotoxicity in normal human bone cells
Summary
Cancer is a public health problem, with high mortality and disability rates worldwide [1]. Song et al indicated that naringenin (200 μM; 24 h) caused colon cancer apoptosis through p38-dependent ATF3 activation [17]. Autophagy, which refers to the intracellular degradation of cytoplasmic materials caused by vacuoles or lysosomes in eukaryotic cells, eliminates and recycles damaged proteins to prolong the lifespan of cells [19]. It is a crucial homeostasis and cell survival mechanism that responds to environmental stresses such as starvation or pathogen infection [20]. Many anticancer drugs which lead to apoptosis can induce autophagy-related cell death in cancer cell lines [23]. The findings of this study can provide the proof-of-concept for evaluating naringenin as an antiosteosarcoma agent
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