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

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.

Keywords: 27-P-coumayl-ursolic acid; HCC-1806 cells; HER2; apoptosis; mitochondrial autophagy.

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27-P-CAUA对HCC-1806细胞的增殖抑制作用 Inhibitory effects of 27-P-CAUA on HCC-1806 cells. A: CCK-8 assay; B: Clonogenic assay; 1: Control; 2: 27-P-CAUA (20 µmol/L); 3: 27-P-CAUA (40 µmol/L); 4: 27-P-CAUA (60 µmol/L); C: Morphological changes of HCC1806 cells. ^*P < 0.01 vs control.

2

27-P-CAUA诱导HCC-1806细胞发生凋亡 27-P-CAUA induces apoptosis in HCC1806 cells. A: Effect of 27-P-CAUA on mitochondrial membrane potential of the cells. B: Bar graph of changes in mitochondrial membrane potential. C: Flow cytometry of the cells with different treatments. D: C-caspase-3 immunofluorescence graph and bar graph.

3

抑制自噬增强HCC-1806细胞对27-P-CAUA的敏感性 Inhibition of autophagy enhances the sensitivity of HCC-1806 cells to 27-P-CAUA. A: CCK-8 assay of the cells incubated with 27-P-CAUA (40 and 60 µmol/L) for 24 h with or without pretreatment with CQ for 4 h. B: Western blot analysis. C: LC3A/B and P62 protein expression bar graph. D: P62 protein immunofluorescence graph and bar graph.

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27-P-CAUA降低HCC-1806细胞内Her2和AKT蛋白的磷酸化水平 27-P-CAUA reduces phosphorylation of Her2 and AKT proteins in HCC-1806 cells.

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27-P-CAUA诱导HCC-1806发生凋亡和线粒体自噬信号通路图 Signaling pathway of 27-P-CAUA-induced apoptosis and mitophagy in HCC-1806 cells.