南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (1): 63-70.doi: 10.12122/j.issn.1673-4254.2022.01.07

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27-P-CAUA通过抑制HER2/PI3K/AKT信号通路诱导乳腺癌细胞凋亡和线粒体自噬

黄 超，王宏婷，王存琴   

  1. 皖南医学院药学院，安徽 芜湖 241002
  • 出版日期:2022-01-20 发布日期:2022-03-02

27-P-CAUA induces apoptosis and mitochondrial autophagy in breast cancer cells by inhibiting HER2/PI3K/AKT signaling pathway

HUANG Chao, WANG Hongting, WANG Cunqin   

  1. School of Pharmacy, Wannan Medical College, Wuhu 241002, China
  • Online:2022-01-20 Published:2022-03-02

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摘要： 目的 探讨大叶冬青叶中三萜类活性成分27-P-香豆酰基-乌索酸（27-P-CAUA）对乳腺癌细胞增殖抑制的作用机制。方法 27-P-CAUA处理HCC-1806细胞24、48、72 h后，CCK-8法检测HCC-1806细胞存活率；集落克隆法检测27-P-CAUA对细胞的增殖抑制作用；JC-1检测线粒体膜电位改变；流式细胞术检测细胞凋亡情况；细胞免疫荧光观察C-Caspase-3、P62表达；氯喹预处理后，Western blot观察27-P-CAUA对LC3I/II、P62以及HER2信号通路蛋白的影响。结果 CCK-8和集落克隆结果显示，随着药物浓度增加和时间延长，27-P-CAUA对HCC-1806乳腺癌细胞的抑制作用逐渐增强（P<0.01），24、48、72 h的IC50值分别为83.671、49.479、19.578 μmol/L；JC-1检测结果显示，线粒体膜电位改变明显（P<0.01）；流式细胞术结果显示，27-P-CAUA可诱导细胞发生凋亡，细胞早期调亡比率增加 3.34%；免疫荧光结果显示，C-Caspase-3表达增强，促进细胞凋亡，40 μmol /L 27-P-CAUA能显著诱导细胞凋亡（P<0.01）。Western blot和细胞免疫荧光结果显示，27-P-CAUA降低LC3II表达，导致P62降解，诱导自噬发生。经氯喹预处理后，27-P-CAUA诱导自噬可被逆转。此外，27-P-CAUA可抑制细胞内Her2及AKT蛋白的磷酸化，细胞内的Her2以及磷酸化AKT蛋白表达逐渐减少（P<0.01）。结论 27-P-CAUA可通过抑制HER2/PI3K/AKT信号通路，抑制HCC-1806细胞增殖，诱导细胞发生线粒体自噬和凋亡。

关键词: HCC-1806细胞；27-P-CAUA；HER2；细胞凋亡；线粒体自噬

Abstract: Objective To investigate the inhibitory effect of 27-P-coumayl-ursolic acid (27-P-CAUA), the active ingredient in triterpenoids from the leaves of Ilex latifolia Thunb, against breast cancer cells and explore the underlying mechanism. Methods CCK-8 assay was used to assess the changes in viability of breast cancer HCC-1806 cells after 27-P-CAUA treatment for 24, 48, or 72 h. The inhibitory effect of 27-P-CAUA on proliferation of the cells was determined by clonogenic assay. JC-1 was used to detect the changes in mitochondrial membrane potential and flow cytometry was performed for analyzing cell apoptosis following 27-P-CAUA treatment. Immunofluorescence assay was used to observe the expression of cl-caspase-3 and P62 in the treated cells. Western blotting was performed to observe the effect of 27-P-CAUA and chloroquine pretreatment on the expressions of LC3I/II,P62 and HER2 signaling pathway proteins in the cells. Results The results of CCK-8 and clonogenic assays showed that 27-P-CAUA treatment significantly inhibited the proliferation of HCC-1806 cells (P<0.01) with IC50 values of 81.473, 48.392 and 18.467 μmol/L at 24, 48, and 72 h, respectively. 27-P-CAUA treatment also caused obvious changes in mitochondrial membrane potential (P<0.01) and induced cell apoptosis in HCC-1806 cells with a 3.34% increase of the early apoptosis rate. Immunofluorescence assay revealed a significant increase of cl-caspase3 expression in 27-P-CAUA-treated HCC-1806 cells, and treatment with 40 μmol/L 27-P-CAUA resulted in significant cell apoptosis (P<0.01). 27-P-CAUA obviously reduced the expression of LC3II, caused P62 degradation and induced autophagy in HCC-1806 cells. Chloroquine pretreatment obviously blocked the autophagy-inducing effect of 27-P-CAUA. 27-P-CAUA treatment also inhibited the phosphorylation of HER2 and AKT proteins and progressively lowered the expressions of HER2 and phosphorylated AKT protein in HCC-1806 cells (P<0.01). Conclusion 27-P-CAUA can inhibit the proliferation and induce mitochondrial autophagy and apoptosis of HCC-1806 cells by inhibiting the HER2/PI3K/AKT signaling pathway.

Key words: HCC-1806 cells; 27-P-coumayl-ursolic acid; HER2; mitochondrial autophagy; apoptosis