articleGonadotropin-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

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 gonadotropinreleasing 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 antagonistinduced 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. * Correspondence: grundker@med.uni-goettingen.de Department of Gynecology and Obstetrics, Georg-August-University, RobertKoch-Street 40, 37075 Göttingen, Germany Full list of author information is available at the end of the article © 2010 Gründker et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gründker et al. Breast Cancer Research 2010, 12:R49 http://breast-cancer-research.com/content/12/4/R49 Page 2 of 9 Introduction Breast cancer is the most frequent malignant disease in women, with more than 1,000,000 new cases and 370,000 deaths yearly worldwide [1]. About 75$ to 80% of breast cancers are hormone-receptor positive and express estrogen and progesterone receptors [2,3]. Approximately 15% to 20% of breast cancers overexpress/amplify the HER2neu gene, with about half of these co-expressing steroidhormone receptors. For patients with hormone-receptorpositive or HER2-neu-positive tumors, effective targeted therapies have been developed. About 10% to 15% of breast cancers do not express either estrogen and progesterone receptor and also do not overexpress/amplify the HER2-neu gene [4-6]. These so-called triple-negative breast cancers lack the benefits of specific therapies that target these receptors. Triple-negative breast cancer is relatively chemosensitive to conventional cytotoxic agents such as cisplatin, but the effectiveness is for only a short duration. Therefore, the development of new therapies is of great interest. The expression of gonadotropin-releasing hormone (GnRH-I) and its receptor as a part of a negative autocrine/paracrine regulatory mechanism of cell proliferation has been demonstrated in a number of malignant tumors, including cancers of endometrium, ovary, and breast [7]. In these cancers, the in vitro proliferation can be inhibited by agonistic analogues of GnRH-I in a doseand time-dependent manner [7-11]. GnRH-I antagonists also have marked antiproliferative activity in most endometrial, ovarian, and breast cancer cell lines tested in vitro, indicating that the dichotomy of GnRH agonists and antagonists might not apply to the GnRH system in cancer cells [7-11]. Besides GnRH-I, a second structural variant of GnRH exists in mammals. GnRH-II is totally conserved in structure from fish to mammals. It differs from GnRH-I in three amino acids. GnRH-II receptor was found in different species, including nonhuman primates. Its existence in the human is controversial. Several lines of evidence, however, exist for a functional GnRH-II receptor [12]. GnRH-II has antiproliferative effects on human endometrial, ovarian, and breast cancer cells that are significantly greater than those of the superactive GnRH-I agonist triptorelin [13]. Induction of apoptosis is not involved in the downregulation of cancer cell proliferation induced by agonists of GnRH-I or GnRH-II [7]. GnRH-I and GnRH-II agonists rather inhibit mitogenic signal transduction of growth-factor receptors via activation of a phosphotyrosine phosphatase, resulting in downregulation of cancer cell proliferation [14-16]. Recently, we showed that antagonistic analogues of GnRH-II induced apoptotic cell death in human endometrial and ovarian cancer cells in vitro, via dose-dependent loss of mitochondrial membrane potential and activation of caspase-3 [17]. These antitumor effects could be confirmed in nude mice. GnRH-II antagonists significantly inhibited the growth of xenotransplants of human endometrial and ovarian cancers in nude mice, without any apparent side effects [17]. Apoptosis induced by GnRH-II antagonists is mediated through the intrinsic apoptotic pathway via stress-induced MAPKs p38and JNKinduced activation of the pro-apoptotic protein Bax, loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 [17,18]. In addition, we demonstrated that GnRH-II antagonists couple to the GnRH-I receptor and have binding affinities to the GnRH-I receptor similar to those of the GnRH-I antagonist cetrorelix [18]. Furthermore, we showed that [D-Lys6 ]-GnRH-II is an agonist at the GnRH-I receptor, whereas GnRH-II antagonists are clear antagonists at the GnRH-I receptor [18]. About 50% to 64% of human breast cancers express GnRH-I receptors [19-22]. Because GnRH-II antagonists appear to be suitable drugs for efficacious and less-toxic endocrine therapy for endometrial and ovarian cancers, the question arises whether GnRH-II antagonist treatment could also be a new therapeutic option for breast cancers. Especially for triple-negative breast cancers, defined by the lack of both estrogen and progesterone receptors, as well as a lack of overexpression/amplification of the HER2-neu gene, the therapeutic options today are very limited. In the present study, we analyzed whether a GnRH-II antagonist induces apoptosis in estrogen receptor/progesterone receptor-positive MCF-7 human breast cancer cells, which have a normal expression of HER2-neu and triple-negative MDA-MB-231 human breast cancer cells in vitro and in vivo. In addition, we ascertained whether knockdown of GnRH-I receptor expression affects GnRH-II antagonist-induced apoptosis, measured by loss of mitochondrial membrane potential and GnRH-II antagonist-induced apoptotic signaling, analyzed by activation of stress-activated MAPK p38 and caspase-3. Materials and methods Cell lines and culture conditions The human breast cancer cell lines MCF-7 and MDAMB-231 (triple-negative) were obtained from American Type Culture Collection (ATCC, Manassas, Virginia). To guarantee the identity of the cell lines over the years, the cells were expanded after purchase, and aliquots were stored in liquid nitrogen. Every half year, a new frozen stock was opened and expanded to carry out the experiments. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air, as previously described [810]. Gründker et al. Breast Cancer Research 2010, 12:R49 http://breast-cancer-research.com/content/12/4/R49 Page 3 of 9