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.
Subject(s)
Female , Humans , Apoptosis , Autophagy , Breast Neoplasms , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Proliferation , Liver Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
27-O-(E)-p-coumaric acyl ursolic acid( DY-17) from Ilex latifolia is a compound of the monomer. To investigate the DY-17 inducing apoptosis in the human breast cancer cell line, the MDA-MB-231 cells were used as research object in this experiment. The proliferation activity of the MDA-MB-231 cells stimulated with the different concentrations of DY-17 (20, 40 µmol · L(-1)) was detected at different time( 12, 24, 36, 48, 60,72 h) . We surveyed the DY-17 inducing apoptosis of the MDA-MB-231 cells with the fluorescent staining technology. The rate of MDA-MB-231 cells apoptosis and necrosis was determined by flow cell cytometry (FCC). Moreover, expression of JNK, phosphorylated JNK, Bax, PARP shear and caspase-3 shear related to JNK/SAPK pathways were investigated in every group ( control group, EGF group, EGF + DY-17 40 µmol · L(1) group and EGF + SP600125 group) with Western blot. The MTT results showed that, in the presence of DY-17, the proliferation activity of MDA-MB-231 cells decreased in a dose-dependent and time-dependent manner. The apoptosis and necrosis rates of MDA-MB-231 cells with DY-17(20, 40 µmol · L(-1)) groups was respectively 31.86%, 49.91% by flow cytometry and significantly increased compared with control group under Fluores- cence microscopy. Up-regulation of the JNK phosphorylation protein expression was observed in EGF group compared with control group. In addition, markedly decreased the expression of JNK phosphorylation protein were also surveyed in EGF + DY-17 40 µmol · L(-1) group compared with EGF group. The expression of Bax, shear PARP and shear caspase-3 protein in EGF + DY-17 40 µmol · L(-1) group were significantly increased in comparison with EGF group. The results showed DY-17 induced apoptosis of human breast cancer MDA-MB-231 cell line related to down-regulating JNK/SAPK signal pathways.
Subject(s)
Female , Humans , Apoptosis , Breast Neoplasms , Drug Therapy , Genetics , Cell Line, Tumor , Cell Proliferation , Drugs, Chinese Herbal , Pharmacology , MAP Kinase Kinase 4 , Genetics , Metabolism , Mitogen-Activated Protein Kinase 8 , Genetics , Metabolism , Signal Transduction , Triterpenes , Chemistry , PharmacologyABSTRACT
Nine compounds were isolated from the leaves of Ilex latifolia. Their structures were respectively identified as 5-hydroxy-6, 7, 8, 4'-tetramethoxyflavone (1), tangeretin (2), nobiletin (3), 5-hydroxy-6, 7, 8, 3', 4'-pentamethoxyflavone (4), 5, 6, 7, 8, 4'-pentamethoxyflavonol (5), 5, 6, 7, 8, 3', 4'-hexamethoxy-flavonol (6), 5-hydroxy-3', 4', 7-trimethoxyflavanone (7), soyacerebroside I (8), and soyacerebroside II (9) by their physicochemical properties and spectroscopic data Compounds 1-9 were isolated from this plant for the first time.