Gonadotropin-releasing hormone type II antagonist induces apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells in vitro and in vivo

Abstract

Introduction: Triple-negative breast cancer does not express estrogen and progesterone receptors, and no overexpression/amplification of the HER2-neu gene occurs. Therefore, this subtype of breast cancer lacks the benefits of specific therapies that target these receptors. Today chemotherapy is the only systematic therapy for patients with triple-negative breast cancer. About 50% to 64% of human breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a target. New targeted therapies are warranted. Recently, we showed that antagonists of gonadotropin-releasing hormone type II (GnRH-II) induce apoptosis in human endometrial and ovarian cancer cells in vitro and in vivo. This was mediated through activation of stress-induced mitogen-activated protein kinases (MAPKs) p38 and c-Jun N-terminal kinase (JNK), followed by activation of proapoptotic protein Bax, loss of mitochondrial membrane potential, and activation of caspase-3. In the present study, we analyzed whether GnRH-II antagonists induce apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells that express GnRH receptors. In addition, we ascertained whether knockdown of GnRH-I receptor expression affects GnRH-II antagonist-induced apoptosis and apoptotic signaling.

Methods: Induction of apoptosis was analyzed by measurement of the loss of mitochondrial membrane potential. Apoptotic signaling was measured with quantification of activated MAPK p38 and caspase-3 by using the Western blot technique. GnRH-I receptor protein expression was inhibited by using the antisense knockdown technique. In vivo experiments were performed by using nude mice bearing xenografted human breast tumors.

Results: We showed that treatment of MCF-7 and triple-negative MDA-MB-231 human breast cancer cells with a GnRH-II antagonist results in apoptotic cell death in vitro via activation of stress-activated MAPK p38 and loss of mitochondrial membrane potential. In addition, we showed GnRH-II antagonist-induced activation of caspase-3 in MDA-MB-231 human breast cancer cells. After knockdown of GnRH-I receptor expression, GnRH-II antagonist-induced apoptosis and apoptotic signaling was only slightly reduced, indicating that an additional pathway mediating the effects of GnRH-II antagonists may exist. The GnRH-I receptor seems not to be the only target of GnRH-II antagonists. The antitumor effects of the GnRH-II antagonist could be confirmed in nude mice. The GnRH-II antagonist inhibited the growth of xenotransplants of human breast cancers in nude mice completely, without any apparent side effects.

Conclusions: GnRH-II antagonists seem to be suitable drugs for an efficacious and less-toxic endocrine therapy for breast cancers, including triple-negative breast cancers.

Figure 1

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.

Figure 2

Effects of GnRH-II antagonist treatment on apoptotic signaling in GnRH-I receptor-positive (wild-type; WT) and GnRH-I receptor-negative (GnRH-I receptor knockdown; KD) human breast cancer cells in vitro. (a) Amount of phosphorylated p38 after treatment without or with GnRH-II antagonist [Ac-D2Nal¹, D-4Cpa², D-3Pal^3,6, Leu⁸, D-Ala¹⁰]GnRH-II (10^-7 M) in nontransfected MCF-7 breast cancer cells and in MCF-7 breast cancer cells after knockdown of GnRH-I receptor expression. These are representative data obtained from three independent experiments in three different passages of the cell line. Experiments using MDA-MB-231 human breast cancer cells gave identical results. (b) Percentage of caspase-3 activity after 48 h of treatment of GnRH-I receptor-positive (wild-type; WT) and GnRH-I receptor-negative (GnRH-I receptor knockdown; KD) MDA-MB-231 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; (c) P < 0.05 versus control.

Figure 3

Tumor volume of MCF-7 (a) and triple-negative MDA-MB-231 (b) human breast cancers xenografted into nude mice. The mice were treated without (control 1), with 25 nmol of GnRH-I agonist Triptorelin (control 2) or with 25 nmol of GnRH-II antagonist [Ac-D2Nal¹, D-4Cpa², D-3Pal^3,6, Leu⁸, D-Ala¹⁰]GnRH-II. Treatment was repeated every 2 days. Tumor volumes were measured on days 7, 11, 16, and 21 of treatment (MCF-7; (a) or on days 4, 8, 12, and 16 of treatment (MDA-MB-231; (b)). The mice were killed after 21 (MCF-7 (a)) or 16 (MDA-MB-231 (b)) days. Each experimental group consisted of five animals. Vertical bars represent SEM. (a) P < 0.001 versus control; (b) P < 0.05 versus control.