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