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.

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.

Gonadotropin-releasing hormone type II antagonists induce apoptotic cell death in human endometrial and ovarian cancer cells in vitro and in vivo.

In human endometrial and ovarian cancers, gonadotropin-releasing hormone type I (GnRH-I), GnRH-II, and their receptors are parts of a negative autocrine regulatory system of cell proliferation. Based on a tumor-specific signal transduction, GnRH-I and GnRH-II agonists inhibit the mitogenic signal transduction of growth factor receptors and related oncogene products associated with tyrosine kinase activity via activation of a phosphotyrosine phosphatase resulting in down-regulation of cancer cell proliferation. Induction of apoptosis is not involved. In this study, we show that treatment of human endometrial and ovarian cancer cells with GnRH-II antagonists results in apoptotic cell death via dose-dependent activation of caspase-3. The antitumor effects of the GnRH-II antagonists could be confirmed in nude mice. GnRH-II antagonists inhibited the growth of xenotransplants of human endometrial and ovarian cancers in nude mice significantly, without any apparent side effects. Thus, GnRH-II antagonists seem to be suitable drugs for an efficacious and less toxic endocrine therapy for endometrial and ovarian cancers.

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells.

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)­resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT­resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) I and II, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.

Antiproliferative effects of antiestrogens and inhibitors of growth factor receptor signaling on endometrial cancer cells.

AIM: In patients with advanced estrogen-dependent type I endometrial cancer (EC), pharmacological treatment with progestins or antiestrogens is recommended, but primary and secondary resistance are common. The aim of our study was to investigate single-agent and dual-agent therapeutic strategies in estrogen receptor-positive human EC cells. MATERIAL AND METHODS: Human EC cells Ishikawa and HEC1A were cultivated under estrogen-reduced conditions and exposed to 4-hydroxytamoxifen (OHT), fulvestrant, gefitinib, everolimus, and the AKT inhibitor perifosine. Effects of drugs were analyzed by proliferation and apoptosis assays. Additionally, we analyzed expression of aromatase, phosphatase and tensin homolog (PTEN), AKT and pAKT and G protein-coupled receptor 30 (GPR30). RESULTS: Neither OHT nor fulvestrant inhibited cell growth, nor did they induce apoptosis. Gefitinib, everolimus and perifosine inhibited proliferation in all cell lines. Only perifosine induced apoptosis. In PTEN-positive HEC1A cells, combined treatment of gefitinib plus OHT showed increased antiproliferative effects. In Ishikawa cells, combined treatment of everolimus plus gefitinib had synergistic antiproliferative effects. The most effective single-agent treatment and the only drug that induced apoptosis was perifosine. Activation of AKT had no predictive value for the effects perifosine. Due to mutation of PTEN, activated AKT was highly expressed in Ishikawa cells and scarcely detectable in HEC1A cells. CONCLUSION: Under estrogen-reduced conditions, growth of ER-positive EC cells can be reduced by inhibitors of AKT, mTOR and the erbB pathway, whereas antiestrogens have no effects. In PTEN-positive HEC1A cells, the absence of estradiol probably restores OHT-induced ER-mediated repression of nuclear co-activators and increases susceptibility to inhibitors of the erbB pathway. In PTEN-negative Ishikawa cells, OHT in combination with any drug had no effects, but inhibition of the PI3K/AKT/mTOR pathway by everolimus in combination with gefitinib showed synergistic effects.

GnRH-II antagonists induce apoptosis in human endometrial, ovarian, and breast cancer cells via activation of stress-induced MAPKs p38 and JNK and proapoptotic protein Bax.

Recently, we could show that gonadotropin-releasing hormone (GnRH)-II antagonists induce apoptosis in human endometrial, ovarian, and breast cancer cells in vitro and in vivo. In the present study, we have ascertained receptor binding and effects of GnRH-II antagonists on mitogenic signal transduction and on activation of proapoptotic protein Bax. The GnRH-II antagonists tested showed EC50 values for GnRH-I receptor binding in the range of 1 to 2 nmol/L. The GnRH-II agonist [D-Lys6]GnRH-II showed an EC50 value for GnRH-I receptor binding of approximately 1,000 nmol/L. Agonistic activity on GnRH-I receptor function with an EC50 of 13 nmol/L has been determined for [D-Lys6]GnRH-II. Antagonistic activities with EC50 values in the range of 1 nmol/L were determined for the GnRH-II antagonists. Treatment of human endometrial, ovarian, and breast cancer cells with GnRH-II antagonists resulted in time-dependent activation of stress-induced mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase. In addition, treatment with GnRH-II antagonists induced time-dependent activation of proapoptotic protein Bax. GnRH-II antagonists are not involved in activation of protein kinase B/Akt or extracellular signal-regulated kinase 1/2. The GnRH-II antagonists tested had similar binding affinities to the GnRH-I receptor comparable with that of GnRH-I antagonist Cetrorelix. Referring to the cyclic AMP response element reporter gene activation assay, the GnRH-II agonist [D-Lys6]GnRH-II has to be classified as an agonist at the GnRH-I receptor, whereas the GnRH-II antagonists tested are clear antagonists at the GnRH-I receptor. GnRH-II antagonists induce apoptotic cell death in human endometrial, ovarian, and breast cancer cells via activation of stress-induced mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase followed by activation of proapoptotic protein Bax.

Increase of doxorubicin-induced apoptosis after knock-down of gonadotropin-releasing hormone receptor expression in human endometrial, ovarian and breast cancer cells.

The majority of human endometrial, ovarian and breast cancers express receptors for gonadotropin-releasing hormone (GnRH). Their proliferation is time- and dose-dependently reduced by GnRH and its agonistic analogs. GnRH agonists inhibit the mitogenic signal transduction of growth factor receptors via activation of a phosphotyrosine phosphatase, resulting in downregulation of cancer cell proliferation. Induction of apoptosis is not involved. Recently we showed that the GnRH agonist triptorelin induces activation of nuclear factor-kappaB (NFkappaB) and thus reduces the apoptosis induced by the cytotoxic agent doxorubicin in human endometrial and ovarian cancer cells. The triptorelin-induced reduction of doxorubicin-induced apoptosis was blocked by inhibition of NFkappaB translocation into the nucleus. The present study was conducted to investigate whether knock-down of GnRH receptor expression reduces GnRH agonist-induced anti-apoptotic action. We show that knock-down of GnRH receptor expression results in an increase of doxorubicin-induced apoptosis in human endometrial and ovarian cancers and in the human breast cancer cell line MCF-7. These data further demonstrate that GnRH agonists suppress chemotherapeutic drug-induced apoptosis via activation of the GnRH receptor in these cancers. The situation is different with T-47-D breast cancer cells. After knock-down of GnRH receptor expression doxorubicin-induced apoptosis was decreased, indicating that GnRH agonists do not suppress chemotherapeutic drug-induced apoptosis in T-47-D breast cancer cells.

No supra-additive effects of goserelin and radiotherapy on clonogenic survival of prostate carcinoma cells in vitro.

BACKGROUND: Oncological results of radiotherapy for locally advanced prostate cancer (PC) are significantly improved by simultaneous application of LHRH analoga (e.g. goserelin). As 85% of PC express LHRH receptors, we investigated the interaction of goserelin incubation with radiotherapy under androgen-deprived conditions in vitro. METHODS: LNCaP and PC-3 cells were stained for LHRH receptors. Downstream the LHRH receptor, changes in protein expression of c-fos, phosphorylated p38 and phosphorylated ERK1/2 were analyzed by means of Western blotting after incubation with goserelin and irradiation with 4 Gy. Both cell lines were incubated with different concentrations of goserelin in hormone-free medium. 12 h later cells were irradiated (0 - 4 Gy) and after 12 h goserelin was withdrawn. Endpoints were clonogenic survival and cell viability (12 h, 36 h and 60 h after irradiation). RESULTS: Both tested cell lines expressed LHRH-receptors. Changes in protein expression demonstrated the functional activity of goserelin in the tested cell lines. Neither in LNCaP nor in PC-3 any significant effects of additional goserelin incubation on clonogenic survival or cell viability for all tested concentrations in comparison to radiation alone were seen. CONCLUSION: The clinically observed increase in tumor control after combination of goserelin with radiotherapy in PC cannot be attributed to an increase in radiosensitivity of PC cells by goserelin in vitro.

GnRH analogs reduce invasiveness of human breast cancer cells.

OBJECTIVE: Bone, besides lung and liver, is one of the most preferential metastatic target sites for breast cancers. Although the precise molecular mechanisms underlying this preference need to be elucidated, it appears that bone microenvironments possess unique biological features that enable circulating cancer cells to home, survive and proliferate, and destroy bone. The majority of human breast cancers and in addition most breast cancer cell lines express GnRH receptors. Their proliferation is time- and dose-dependently reduced by GnRH-I and GnRH-II agonists by counteracting of the mitogenic signal transduction. METHODS: We have established a coculture system of different breast cancer cell lines stable transfected with red fluorescence (DS-Red) and human primary osteoblasts (hOB) or MG63 human osteosarcoma cells to analyze tumor cell invasion to bone. RESULTS: We could show that breast cancer cell invasion was increased when cocultured with hOB or MG63. Treatment with GnRH-I and GnRH-II analogs reduced the ability to invade a reconstituted basement membrane (Matrigel) and to migrate in response to the cellular stimulus. Searching for the molecular mechanisms we found that GnRH treatment reduces expression of the osteoblast derived chemokine SDF-1 by hOB or MG63 cells cocultured with breast cancer cells. CONCLUSION: These data represent the first report that the activation of tumor GnRH receptors reduces the metastatic potential of breast cancer cells. The crosstalk between metastatic breast cancer cells and bone is critical to the development and progression of bone metastases. Disruption of this interaction will allow us to design mechanism-based effective and specific therapeutic interventions for bone metastases.