Neohesperidin has anti-oxidative and anti-inflammatory properties and exerts extensive therapeutic effects on various cancers. In this study, the osteosarcoma cell lines were exposed to different concentrations of neohesperidin. Cell proliferation and viability were assessed by CCK-8 and colony-formation assays. The role of neohesperidin in cell cycle progression and apoptosis were analyzed by flow cytometry and western blotting. To identify autophagosomes and autolysosomes, we used a tandem GFP-mRFP-LC3B lentiviral construct. In addition, autophagy was evaluated by examining autophagosome formation using transmission electron microscopy. Intracellular reactive oxygen species (ROS) production was detected by fluorescence microscopy and flow cytometry. Subsequently, the activation of the ROS/JNK signaling pathway was investigated. Neohesperidin could inhibit proliferation and induce apoptosis in SJSA and HOS cells. The formation of autophagosomes indicated that autophagy occurred in neohesperidin-treated cells and the apoptotic effect of neohesperidin was significantly increased after the use of autophagy inhibitors. Subsequently, we found that neohesperidin-induced apoptosis and autophagy were related to the increase in ROS generation and were significantly inhibited by GSH. Moreover, neohesperidin induced activation of the c-Jun N-terminal kinase (JNK) signaling pathway and inhibition of JNK with SP600125 attenuated neohesperidin-induced apoptosis and autophagy simultaneously. Our data indicated that neohesperidin caused G2/M phase arrest and induced apoptosis and autophagy by activating the ROS/JNK pathway in human osteosarcoma cells, suggesting that neohesperidin is a potential drug candidate for the treatment of osteosarcomas.

中文翻译：

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新橙皮苷通过人骨肉瘤细胞中的 ROS/JNK 信号通路诱导细胞周期停滞、凋亡和自噬

新橙皮苷具有抗氧化和抗炎特性，对各种癌症具有广泛的治疗作用。在这项研究中，骨肉瘤细胞系暴露于不同浓度的新橙皮苷。通过 CCK-8 和集落形成测定评估细胞增殖和活力。通过流式细胞术和蛋白质印迹分析新橙皮苷在细胞周期进程和细胞凋亡中的作用。为了识别自噬体和自溶酶体，我们使用了串联的 GFP-mRFP-LC3B 慢病毒构建体。此外，通过使用透射电子显微镜检查自噬体形成来评估自噬。通过荧光显微镜和流式细胞术检测细胞内活性氧 (ROS) 的产生。随后，研究了 ROS/JNK 信号通路的激活。Neohesperidin 可抑制 SJSA 和 HOS 细胞的增殖并诱导细胞凋亡。自噬体的形成表明新橙皮苷处理的细胞发生自噬，使用自噬抑制剂后新橙皮苷的凋亡作用显着增强。随后，我们发现新橙皮苷诱导的细胞凋亡和自噬与活性氧生成的增加有关，并被谷胱甘肽显着抑制。此外，新橙皮苷诱导 c-Jun N 末端激酶 (JNK) 信号通路的激活和用 SP600125 抑制 JNK 可同时减弱新橙皮苷诱导的细胞凋亡和自噬。我们的数据表明，新橙皮苷通过激活人骨肉瘤细胞中的 ROS/JNK 通路引起 G2/M 期阻滞并诱导细胞凋亡和自噬，