枞酸在顺铂耐药鼻咽癌细胞增殖、侵袭、迁移中的作用及分子机制

The role and the molecular mechanism of abietic acid in the proliferation,invasion and migration of cisplatin-resistant nasopharyngeal carcinoma cells

目的探讨枞酸在顺铂耐药鼻咽癌细胞增殖、侵袭、迁移的作用及分子机制。方法①药物浓度递增法构建顺铂耐药鼻咽癌C666/DDP细胞株。②枞酸对C666/DDP细胞增殖、侵袭及迁移的影响研究:CCK-8法、活性氧(ROS)及线粒体膜电位(MMP)水平检测及裸鼠皮下成瘤实验检测枞酸对C666/DDP细胞体外、体内增殖及凋亡能力的影响;Transwell法检测细胞侵袭和迁移能力。③Western blot及IHC法检测PI3K/AKT/mTOR通路相关蛋白的表达情况。结果①顺铂对C666-1细胞毒性IC50约为25μmol/L。RI=25μmol/L/4μmol/L=6.25,获得耐药性,C666-1-DDP耐药株构建成功。②枞酸可促进C666/DDP细胞凋亡,抑制其增殖,且表现为G2/M期阻滞;Transwell显示枞酸抑制C666/DDP细胞迁移及侵袭能力,提高C666/DDP细胞ROS水平并使MMP降低。③动物实验显示枞酸在体内呈浓度梯度抑制顺铂耐药鼻咽癌的增殖,抑制PI3K/AKT/mTOR信号通路相关蛋白的表达。结论枞酸可抑制顺铂耐药鼻咽癌细胞增殖、侵袭及迁移,其机制与抑制PI3K/AKT/mTOR信号通路有关。

Objective To investigate the effects and molecular mechanisms of abietic acid in the cell proliferation,invasion and migration of cisplatin-resistant nasopharyngeal carcinoma cells.Methods①Cisplatin-resistant C666/DDP cell line was constructed by increasing drug concentration method.②The effects of abietic acid on proliferation,invasion and migration of C666/DDP cells were investigated by CCK-8 method,reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)level assay and subcutaneous tumorigenesis assay in nude mice to detect the effects of abietic acid on proliferation and apoptosis of C666/DDP cells in vitro and in vivo.The effect of abietic acid on the proliferation and apoptosis of C666/DDP cells in vitro and in vivo was measured by Transwell assay.③Western blot and IHC method to detect the expression of PI3K/AKT/mTOR pathway related proteins.Results①The IC50 of cisplatin cytotoxicity to C666-1 was about 25μmol/L.RI=25μmol/L/4μmol/L=6.25,resistance was obtained,and the C666-1-DDP resistant strain was successfully constructed.②Abietic acid promoted apoptosis and inhibited proliferation of C666/DDP cells,and showed G2/M phase block;transwell showed that abietic acid inhibited C666/DDP cell migration and invasion,increased ROS level of C666/DDP cells and decreased MMP.Transwell showed that abietic acid inhibited the migration and invasion ability of C666/DDP cells,increased the ROS level of C666/DDP cells and decreased MMP.③Animal experiments showed that abietic acid inhibited the proliferation of cisplatin-resistant nasopharyngeal carcinoma in vivo in a concentration gradient and suppressed the expression of PI3K/AKT/mTOR signaling pathway-related proteins.Conclusion Abietic acid inhibits proliferation,invasion and migration of cisplatin-resistant nasopharyngeal carcinoma cells by a mechanism related to inhibition of PI3K/AKT/mTOR signaling pathway.