title: Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells.
abstract: Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables. The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated. Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Flow cytometry, fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors. Apigenin dose- and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines. The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3, PARP cleavage and Bax/Bcl-2 ratios. The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs) by flow cytometry. Furthermore, the results of the Western blot analysis revealed that the level of LC3-II, the processed form of LC3-I, was increased. Treatment with the autophagy inhibitor, 3-methyladenine (3-MA), significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the level of PARP cleavage. Similar results were also confirmed by flow cytometry and fluorescence microscopy. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in breast cancer cells. Autophagy plays a cyto-protective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.
authors.affiliation: National Key Laboratory of Breast Cancer Prevention and Treatment, Tianjin Medical University, Tianjin 300060, China ; Department of Breast Cancer Surgery of the Cancer Hospital, Tianjin Medical University, Tianjin 300060, China.
authors.forename: Xuchen
authors.initials: X
authors.lastname: Cao
authors.forename: Bowen
authors.initials: B
authors.lastname: Liu
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Cao
authors.forename: Weiran
authors.initials: W
authors.lastname: Zhang
authors.forename: Fei
authors.initials: F
authors.lastname: Zhang
authors.forename: Hongmeng
authors.initials: H
authors.lastname: Zhao
authors.forename: Ran
authors.initials: R
authors.lastname: Meng
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.forename: Ruifang
authors.initials: R
authors.lastname: Niu
authors.forename: Xishan
authors.initials: X
authors.lastname: Hao
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
keywords: 3-methyladenine; Apoptosis; apigenin; autophagy; breast cancer