Fig. 1. Different ER-α and caspase-3 status and genistein responses in MCF-7 and MDA-MB-231 cells. (A) MDA-MB-231 cells are more sensitive to genistein than MCF-7 cells. Cells from each line were inoculated into 96-well plates at 1000 cells/well. Genistein was added 24 h later for a 6-day treatment. Cell survival fractions were measured using MTT assay as detailed in Materials and methods. (B) Expression of ER-α and caspase-3 in MCF-7 and MDA-MB-231 cells detected with Western blot.

Fig. 2. Caspase-3 reconstitution sensitizes MCF-7 cells to genistein. (A) Caspase-3 status in control (MCF-7/PV) and caspase-3 reconstituted (MCF-7/C3) MCF-7 cells detected with Western blot. (B) Genistein-mediated growth inhibition in MCF-7/PV and MCF-7/C3 cells. Survival fractions of genistein treated cells based on 8 parallel samples were determined using MTT assay. *p < 0.01 as compared to MCF-7/PV cells under the same conditions. (C) PARP cleavage in genistein-treated MCF-7/PV and MCF-7/C3 cells. Each cell line was treated with genistein at indicated concentrations for 48 h, followed by Western blot detection of PARP. (D) Genistein-induced apoptosis in control (MCF-7/PV) and MCF-7/C3 cells. Cells from each line were inoculated into 24 well plates at 1 × 10⁵/well 24 h, followed by genistein treatment for 30 h. Apoptosis in the treated cells was detected using an apoptosis ELSIA kit from Roche.

Fig. 3. Caspase-3 knockdown in MDA-MB-231 cells renders the cells resistant to genistein. (A) Caspase-3 levels in control and caspase-3 shRNA transfected cells. MDA-MB-231 cells were transfected with control or caspase-3 specific shRNA as described in Materials and methods. Protein lysate were collected 48 h, followed by caspase-3 detection using Western blot. (B) Genistein-mediated growth inhibition in control and caspase-3 shRNA transfected cells. MDA-MB-231 cells transfected with control or caspase-3 specific shRNA were treated with genistein 16 h post transfection. The survival fractions of the treated (a 4-day treatment) cells based on three parallel samples were determined by MTT assay. *p < 0.01 as compared to MDA-MB-231 cells transfected with control siRNA under the same conditions. (C) PARP cleavage in genistein treated MDA-MB-231 cells transfected with control or caspase-3 specific shRNA. shRNA transfected cell were treated with genistein at indicated concentrations for 48 h, followed by Western blot detection of PARP. (D) Effect of caspase-3 knockdown on genistein-induced apoptosis in MDA-MB-231 cells. The cells were inoculated in a 24-well plate at 8 × 10⁴/well 16 h, followed by transfection with control (−) or caspase-3 specific shRNA (+) and genistein treatment at 25 μM for 30 h. Apoptosis in the treated cells was detected using an apoptosis ELSIA kit from Roche. *p < 0.01 as compared to genistein treated MDA-MB-231 cells transfected with control siRNA.

Fig. 4. Expression of caspase-3 in 18 breast cancer cell lines and 9 primary breast cancer tissues. Protein lysates were prepared from non-synchronized cells of the indicated cell lines (A and B) or primary breast cancer tissues (C). Caspase-3 and actin were probed with corresponding specific antibodies using Western blot.

Fig. 5. Differential expression of more apoptotic factors between MFC-7 and MDA-MB-231 cells. (A) mRNA levels of caspases-3, -5, -8, -9, and Apaf-1 (panel A1); and caspases 2, 4, 6, 7, and 10 (panel A2) in genistein treated MCF-7 and MDA-MB-231 cells. Total RNA was extracted from control (−) and genistein (50 μM for 20 h) treated (+) cells. Three μg of total RNA were used for cDNA synthesis. One tenth of the RT product was used for amplification. mRNA levels of the selected caspases were amplified by PCR for 30 cycles using a multi-complex PCR kit (BioSource) containing specific primers of corresponding caspases according to the manufacturer's protocol. P.C., positive control from the kits. (B) Protein levels of caspases-4 and -10 in MCF-7 and MDA-MB-231 cells detected with Western blot.

Fig. 6. Genistein-induced apoptosis is not enhanced by caspase-10 overexpression in MCF-7 cells. (A) Caspase-10 status in control (MCF-7/PV) and caspase-10 reconstituted (MCF-7/C10) MCF-7 cells detected with Western blot. (B) Genistein-mediated growth inhibition in control (MCF-7/PV) and MCF-7/C10 cells. After genistein treatment at indicated concentrations for 6 days, the survival fractions of the treated cells based on 8 parallel samples were determined using MTT assay. (C) Genistein-induced apoptosis in control (MCF-7/PV) and MCF-7/C10 cells. Cells from each line were inoculated into 24 well plates at 1 × 10⁵/well 24 h, followed by genistein treatment for 30 h. Apoptosis in the treated cells was detected using an apoptosis ELSIA kit from Roche.