Effect of deguelin on mitochondrial permeability transition pore of human breast cancer cell line MDA-MB-231 / 第二军医大学学报

ABSTRACT

Objective: To investigate the effect of deguelin on mitochondrial permeability transition pore of human breast cancer cell line MDA-MB-231. Methods: The inhibitory effect of deguelin on cell proliferation was determined by MTT assay; cell apoptosis rate was analyzed by flow cytometry with AnnexinV FITC/PI double staining; MDA-MB-231 cells were stained by Rhodaminel23 to detect the changes of mitochondrial transmembrane potential by FCM; alteration of protein of Cyt c outside of mitochondria was detected by Western blotting analysis; caspase-3 activity was assessed by colorimetric assay; and MDA-MB-231 cells were stained by Fluo-3/AM to detect changes of intracellular Ca 2+ concentration by FCM. The expression of Bcl-2 and Bax was examined by RT-PCR and Western blotting analysis. Results: Deguelin significantly inhibited the growth of MDA-MB-231 cells in a time- and dose-dependent manner ( P < 0.05). After treatment with deguelin, mitochondrial transmembrane potential was decreased, the expression of Cyt c outside of mitochondria was increased, and caspase-3 activity was significantly increased compared with negative control group(P<0.01). FCM analysis showed that the apoptotic rate of MDA-MB-231 cells and intracellular Ca2+ concentration increased gradually with the increase of deguelin concentration. RT-PCR and Western blotting analysis showed that the expression of Bcl-2 mRNA and protein was down-regulated and that of Bax was up-regulated after deguelin treatment. Conclusion: Deguelin can inhibit proliferation and induce apoptosis of MDA-MB-231 cells, and the induction of apoptosis might be related to increased intracellular Ca2+ concentration and changes of mitochondrial permeability transition pore induced by altered Bcl-2, Bax expression.