千里光菲灵碱诱导MEK/ERK1/2介导的宫颈癌细胞自噬效应

探讨千里光菲灵碱对人宫颈癌HeLa、Caski细胞增殖、自噬的影响及其可能的作用机制。MTT法检测千里光菲灵碱对HeLa、Caski细胞增殖的影响；免疫荧光法检测GFP-LC3/HeLa和GFP-LC3/Caski两种细胞内自噬体的形成情况；免疫印迹法检测千里光菲灵碱对自噬相关蛋白表达的影响；荧光共定位法检测自噬体与溶酶体的融合情况；建立荷宫颈癌HeLa、Caski细胞移植瘤裸鼠模型，检测千里光菲灵碱对移植瘤生长的抑制作用。结果显示，千里光菲灵碱呈时间和剂量依赖性抑制HeLa、Caski两种宫颈癌细胞的增殖；千里光菲灵碱可诱导HeLa、Caski细胞内自噬体的形成和LC3-II蛋白的增加及P62蛋白的减少，表明千里光菲灵碱可诱导HeLa、Caski细胞发生自噬；与单药千里光菲灵碱相比，千里光菲灵碱联合自噬下游抑制剂氯喹(CQ)显著增加了LC3-II及P62的蛋白表达，同时荧光共定位显示千里光菲灵碱诱导的自噬体可与溶酶体实现共定位，表明千里光菲灵碱可诱导完整自噬流；与单药千里光菲灵碱相比，千里光菲灵碱联合自噬上游抑制3-甲基腺嘌呤(3MA)剂显著增加了LC3-II的蛋白表达，显著降低了HeLa、Caski细胞的存活率，表明抑制自噬可增强千里光菲灵碱对宫颈癌细胞增殖的抑制作用；与单药千里光菲灵碱相比，千里光菲灵碱联合MEK抑制剂显著降低了P-ERK1/2的蛋白表达，减少了自噬体的形成，表明千里光菲灵碱诱导的HeLa、Caski细胞自噬依赖于MEK/ERK1/2途径；另外，千里光菲灵碱可显著抑制宫颈癌细胞裸鼠皮下移植瘤的生长。

Seneciphylline induced the autophagy of cervical cancer cells via MEK/ERK1/2 regulation

The purpose of this study was to explore the effects of seneciphylline on the proliferation and autophagy of cervical cancer HeLa and Caski cells and the possible mechanisms of autophagy. MTT assay was used to evaluate the effect of seneciphylline on the proliferation of cervical cancer cells. Immunofluorescence assay was applied to investigate the formation of autophagosomes in GFP-LC3/HeLa and GFP-LC3/Caski cells. The effect of seneciphylline on the expression of autophagy-related proteins was checked by Western blotting. In addition, fluorescence colocation assay was used to detect the fusion of autophagosomes and lysosomes. Human cervical cancer subcutaneous xenografts in nude mice were used to evaluate the effect of seneciphylline on the growth of the tumor in vivo. Results showed that HeLa cells proliferation was inhibited by seneciphylline in a dose- and time- dependent manner. Seneciphylline could induce formation of autophagosomes, increase the expression of LC3-II and decrease the expression of P62, suggesting that seneciphylline induced autophagy in HeLa and Caski cells. Compared with seneciphylline alone, seneciphylline combined with later-stage autophagy inhibitor chloroquine significantly increased the expression of LC3-II and P62. Moreover, and fluorescence colocation assay showed that autophagosomes induced by seneciphylline could colocate with lysosomes, indicating that seneciphylline could induce the complete autophagy flux. Compared with seneciphylline alone, seneciphylline combined with earlier-stage autophagy inhibitor 3MA significantly increased the expression of LC3-II and significantly decreased HeLa and Caski cells viability, suggesting that seneciphylline induced protective autophagy. Compared with seneciphylline alone, seneciphylline combined with MEK inhibitor significantly decreased the expression of P-ERK1/2 and formation of autophagosomes, suggesting that autophagy induced by seneciphylline activated MEK/ERK1/2 signal pathway. In addition, seneciphylline showed a significant inhibitory effect on growth of human cervical cancer cells subcutaneous xenografts.