Yan Bin, Liu Qingbai, Liu Gang, Huang Xiaoyi, Zhu Guangming, Gao Luoluo, Xu Yaozeng
Department of Orthopaedics, The First Affiliated Hospital of Soochow University 899 Pinghai Road Suzhou Jiangsu 215006 China
Department of Orthopaedics, Taixing People's Hospital Taixing Jiangsu 225400 China.
RSC Adv. 2020 Jan 30;10(9):5032-5039. doi: 10.1039/c9ra07332a. eCollection 2020 Jan 29.
Osteosarcoma is the most common type of bone tumor, which severely threatens the health of adolescents and young adults. Tumor-infiltrating macrophages have been shown to mediate cancer progression extracellular vesicles. However, their potential mechanisms in osteosarcoma progression and in drug-resistance are still not yet known. The macrophage cell line THP1 was stimulated by phorbol myristate acetate (PMA) to secrete exosomes. The exosomes isolated from THP1 were characterized transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and by a western blot. Cell proliferation was determined using CCK-8. A transwell assay and flow cytometry were conducted to detect cell migration and apoptosis, respectively. The expression levels of AKT and its phosphorylation status were determined using a western blot. PMA-treated activated THP1 cells secreted an abundance of exosomes with the characteristics of being less than 200 nm in diameter, and showing the robust expression of exosome markers CD63 and CD81. The THP1-derived exosomes promoted cell proliferation, migration and drug-resistance to the chemical drug docetaxel in both osteosarcoma cell lines MG63 and 143B. The inhibition of the generation of exosomes by the knockdown of ALIX clearly suppressed the cell proliferation, migration and drug-resistance. Mechanistically, the THP1-derived exosomes activated AKT signaling by inducing the increased expression of the phosphorylated AKT at serine 473 (p-AKT). The AKT inhibitor MK2206 significantly abolished exosome-mediated cell proliferation and drug-resistance in osteosarcoma cells. In summary, our data demonstrated that macrophage-derived exosomes promoted osteosarcoma progression and drug-resistance by activating AKT signaling that could be used as a potential molecular target for osteosarcoma treatment.
骨肉瘤是最常见的骨肿瘤类型，严重威胁青少年和年轻成年人的健康。肿瘤浸润巨噬细胞已被证明可通过细胞外囊泡介导癌症进展。然而，它们在骨肉瘤进展和耐药性中的潜在机制仍不清楚。用佛波酯（PMA）刺激巨噬细胞系THP1分泌外泌体。从THP1分离的外泌体通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）和蛋白质免疫印迹进行表征。使用CCK-8测定细胞增殖。分别进行Transwell实验和流式细胞术检测细胞迁移和凋亡。使用蛋白质免疫印迹测定AKT的表达水平及其磷酸化状态。PMA处理激活的THP1细胞分泌大量外泌体，其直径小于200nm，且外泌体标志物CD63和CD81表达强烈。THP1来源的外泌体促进骨肉瘤细胞系MG63和143B的细胞增殖、迁移以及对化学药物多西他赛的耐药性。通过敲低ALIX抑制外泌体生成明显抑制了细胞增殖、迁移和耐药性。机制上，THP1来源的外泌体通过诱导丝氨酸473磷酸化AKT（p-AKT）表达增加来激活AKT信号通路。AKT抑制剂MK2206显著消除了外泌体介导骨肉瘤细胞的增殖和耐药性。总之，我们的数据表明巨噬细胞来源的外泌体通过激活AKT信号通路促进骨肉瘤进展和耐药性，这可能成为骨肉瘤治疗的潜在分子靶点。
