Figure 4
Effect of buthionine sulfoximine (BSO) and 17β-estradiol (E2) on Bcl-2 family protein expression and mitochondrial function in MCF-7 and MCF-7:2A cells. (a) Western blot analysis for pBcl-2, Bcl-2, Bcl-x L , and Bax protein expression in parental MCF-7 cells and MCF-7:2A cells following 48 h of treatment with ethanol vehicle (Control), 1 nM E2, 100 μM BSO, or E2 + BSO. Equal loading was confirmed by reprobing with an antibody against β-actin. (b) Small interfering RNA (siRNA) knockdown of Bcl-2 partially sensitizes MCF-7:2A cells to E2-induced apoptosis. Cells were transfected with 100 nM siRNA-Bcl-2 or siRNA-Con (control) and expression levels of Bcl-2 was determined by immunoblot analysis (top). Annexin V staining (bottom) showing the effects of siRNA-con and siRNA-Bcl-2 on apoptosis induced by estradiol treatment in MCF-7:2A cells. *, p < 0.001. (c) Loss of mitochondrial potential in MCF-7:2A cells was determined by rhodamine 123 (Rh123) retention assay. The percentage of cells retaining Rh123 in each treatment group was compared with untreated control. (d) Cytochrome c release from the mitochondria to the cytosol after treatment with E2 alone or BSO and E2 for 48 h was determined as described in Materials and methods. Anti-Cox IV antibody was used as a control to demonstrate that mitochondrial protein fractionation was successfully achieved. (e) Cleavage of caspase 7 and poly(ADP-ribose) polymerase (PARP) (72 h) was assessed by western blot using specific antibodies. The upper band of caspase 7 represents the full-length protein and the lower band (p20, arrow) represents the cleaved activated product; NS, nonspecific. Full length PARP is approximately 116 kDa; cleaved (active) PARP is 85 kDa (arrow). The results are representative of three independent experiments.