Effects of GnRH-II antagonist treatment on induction of apoptosis in GnRH-I receptor-positive (wild-type; WT) and GnRH-I receptor-negative (GnRH-I receptor knockdown; KD) MCF-7 human breast cancer cells in vitro. (a) Percentage of mitochondrial membrane potential (ΔΨ) after 72 h of treatment of GnRH-I receptor-positive (wild-type; WT) and GnRH-I receptor-negative (GnRH-I receptor knockdown; KD) MCF-7 breast cancer cells without (control = 100%) or with cytotoxic agent doxorubicin (10-9 M; positive control) or with GnRH-II antagonist [Ac-D2Nal1, D-4Cpa2, D-3Pal3, D-Lys6, D-Ala10]GnRH-II (10-7 M and 10-9 M). Columns represent mean ± SEM of data obtained from three independent experiments in three different passages of the cell line. (a) P < 0.001 versus control; (b) P < 0.01 versus control. Experiments using MDA-MB-231 human breast cancer cells gave identical results. (b-d) Immune histochemical detection of GnRH-I receptor protein by using a monoclonal mouse anti-human GnRH-I receptor antibody. (b) Control performed by omission of the primary antibody. (c) Nontransfected cells. (d) Cells transfected with pGnRH-I-R antisense expression vector.