BPA and E2 protect T47D cells from cisplatin cytotoxicity. (A) Cells were treated with 1 nM BPA or E2 for 24 hrs, followed by incubation with increasing concentrations of cisplatin (0–800 ng/ml) for 72 hrs. (B) T47D cells were treated with increasing concentrations of BPA (0–10nM) for 24 hrs prior treatment with 800 ng/ml cisplatin for 24 hrs. (C) T47D cells were treated with increasing concentrations of E2 (0–10nM) for 24 hrs before treatment with 800 ng/ml cisplatin for 24 hrs. In all cases, cell viability was determined by the MTT assay. Each value is a mean±SEM of six replicates. In (A) * significant (p< 0.05) vs cisplatin alone. In (B&C) * significant vs control, and ** significant vs cisplatin.

BPA and E2 protect T47D cells from cisplatin-induced decreases in cell proliferation and increases in apoptosis. (A) T47D cells were treated with 10 nM BPA or E2 for 24 hrs followed by cisplatin (800 ng/ml) for 72 hrs. Cells were fixed and incubated with a BrdU antibody for 8 hrs. After adding substrate, the product was quantified by measuring absorbance. Each value is a mean±SEM of four replicates. * significant (p< 0.05) vs control. ** significant vs cisplatin. (B) T47D cells were treated as in (A), stained with Annexin V and propidium iodide (PI), and analyzed by flow cytommetry. The table depicts the percentage of cells in each treatment group that are alive (no stain), in early apoptosis (Annexin V-positive), late apoptosis/necrosis (Annexin V+PI-positive) or dead (PI- positive). Shown is a representative experiment repeated 3 times

BPA and E2 mediate their protective effects independent of classical estrogen receptors. (A) T47D cells were treated with 100 nM ICI or PHTPP for 1 hr before incubation with 10 nM BPA or E2 for 24 hrs. Cells were then exposed to 800 ng/ml cisplatin for 24 hrs and cell viability was determined by the MTT assay. Each value is a mean±SEM of six replicates. * significant (p <0.05) vs control, and ** significant vs cisplatin. (B) MDA-MB-468 cells were treated with 10 nM of BPA or E2 for 24 hrs followed by increasing doses of cisplatin for 24 hrs. Cytotoxicity was determined by the MTT assay. Each value is a mean±SEM of six replicates. * significant vs corresponding doses of cisplatin alone.

BPA and E2 antagonize cisplatin in ERβ-knockdown T47D cells. (A) Cells were transfected with siRNA targeted towards ERβ or with scrambled siRNA. On days (d) 1, 4 and 8 post-transfection, ERβ expression was determined by RT-PCR; GAPDH was used as a control. (B) ERβ knockdown cells were incubated with 10 nM BPA or E2 for 24 hrs, followed by 800 ng/ml cisplatin for 24 hrs. Cytotoxicity was determined by the MTT assay. Each value is a mean±SEM of six replicates. * significant (p <0.05) vs control and ** significant vs cisplatin.

BPA and E2 may confer chemoresistance by increasing Bcl-2 expression. (A) T47D cells were treated with 10 nM BPA or E2 for 24 hrs, followed by 800 ng/ml cisplatin for 24 hrs. DNA was isolated and platinum content was analyzed by inductively coupled plasma mass spectroscopy. (B) Cells were treated as in (A). Western blots were probed for Bcl-2, Bcl-xL and Bax; β-actin serves as a loading control. Shown are representative blots, repeated at least three times. (C) Cells were pre-treated for 1 hr with 10 µM HA14-1 and then with 10 nM BPA or E2 for 24 hrs. This was followed by exposure to 800 ng/ml cisplatin for 24 hrs. Cytotoxicity was determined by the MTT assay. Each value is a mean±SEM of six replicates. * significant (p < 0.05) vs control, and ** significant vs cisplatin.