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Expression of Gonadotropin-Releasing Hormone II (GnRH-II) Receptor in Human Endometrial and Ovarian Cancer Cells and Effects of GnRH-II on Tumor Cell Proliferation
... The GnRH pathway is important in the hypothalamuspituitary-gonadal axis of reproduction [21]. Previous studies have demonstrated the direct effects of GnRH analogs in human endometrial cancer cells [22,23]. Furthermore, it has been demonstrated that GnRH-II has more potent effects than GnRH-I in extra-pituitary tissues, such as endometrial tumors, suggesting that GnRH-II could be considered as a possible therapeutic target for endometrial cancers [22]. ...
... Previous studies have demonstrated the direct effects of GnRH analogs in human endometrial cancer cells [22,23]. Furthermore, it has been demonstrated that GnRH-II has more potent effects than GnRH-I in extra-pituitary tissues, such as endometrial tumors, suggesting that GnRH-II could be considered as a possible therapeutic target for endometrial cancers [22]. Metastasis represents the main cause of death for patients with endometrial cancer, and the battle against this cancer would benefit greatly from the identification of factors involved in the metastatic process. ...
... These findings suggest that GnRH-II directly induces the cell migration and invasion of endometrial cancer cells and provide in vitro confirmation that GnRH-II induces cell motility in endometrial cancer. These findings confirmed the previous studies [22,23,[25][26][27][28][29] suggesting that GnRH-II may mediates the cell motility and anti-proliferation in gynecologic cancer cell lines. Therefore, differences in levels of GnRH-I receptor, GnRH-II receptor and signaling differentially affect the apoptotic and motile machinery within cell lines and contribute to the cell type-specific effects of GnRH analogues on cell growth and motility. ...
More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastases. Gonadotropin-releasing hormone (GnRH) plays an important role in reproduction. In mammals, expression of GnRH-II is higher than GnRH-I in reproductive tissues. Here, we examined the effect of a GnRH-II agonist on the motility of endometrial cancer cells and its mechanism of action in endometrial cancer therapy.
Immunoblotting and immunohistochemistry (IHC) were used to determine the expression of the GnRH-I receptor protein in human endometrial cancer. The activity of MMP-2 in the conditioned medium was determined by gelatin zymography. Cell motility was assessed by invasion and migration assay. GnRH-I receptor si-RNA was applied to knockdown GnRH-I receptor.
The GnRH-I receptor was expressed in the endometrial cancer cells. The GnRH-II agonist promoted cell motility in a dose-dependent manner. The GnRH-II agonist induced the phosphorylation of ERK1/2 and JNK, and the phosphorylation was abolished by ERK1/2 inhibitor (U0126) and the JNK inhibitor (SP600125). Cell motility promoted by GnRH-II agonist was suppressed in cells that were pretreated with U0126 and SP600125. Moreover, U0126 and SP600125 abolished the GnRH-II agonist-induced activation of MMP-2. The inhibition of MMP-2 with MMP-2 inhibitor (OA-Hy) suppressed the increase in cell motility in response to the GnRH-II agonist. Enhanced cell motility mediated by GnRH-II agonist was also suppressed by the knockdown of the endogenous GnRH-I receptor using siRNA.
Our study indicates that GnRH-II agonist promoted cell motility of endometrial cancer cells through the GnRH-I receptor via the phosphorylation of ERK1/2 and JNK, and the subsequent, MAPK-dependent activation of MMP-2. Our findings represent a new concept regarding the mechanism of GnRH-II-induced cell motility in endometrial cancer cells and suggest the possibility of exploring GnRH-II as a potential therapeutic target for the treatment of human endometrial cancer.
... Available studies have demonstrated that GnRH-R is expressed in a variety of tumor tissues, including non-reproductive mesenchymal tumors such as melanoma, glioblastoma, lung cancer and pancreatic cancer 63 . Application of GnRH agonists to activate GnRH-R may lead to significant antitumor effects such as proliferation, anti-metastasis, and anti-angiogenesis 64 , which may be related to intracellular signaling pathways coupled to GnRH-R in different tissues 65 . Transforming growth factor-β (TGF-β) is a family of structurally related proteins that are closely associated with a variety of cellular functions, including proliferation, apoptosis, differentiation, epithelialmesenchymal transition (EMT) and migration, regulate protein serine/threonine kinase activity and lead to phosphorylation of the intracellular effector SMAD protein, which promote tumor invasion and metastasis 66 . ...
Oxidative stress refers to the process of reactive oxide species (ROS) increase in human body due to various factors, which leads to oxidative damage in human tissues. Current studies have confirmed that sustained oxidative stress is one of the distinctive features throughout the development of tumors. Numerous reports have shown that lncRNAs can regulate the process of oxidative stress through multiple pathways. However, the relationship between glioma-associated oxidative stress and lncRNAs is not clearly investigated. RNA sequencing data of GBM (glioblastoma) and LGG (low grade glioma) and corresponding clinical data were retrieved from the TCGA database. Oxidative stress related lncRNAs (ORLs) were identified by Pearson correlation analysis. Prognostic models for 6-ORLs were structured in the training cohort by univariate Cox regression analysis, multivariate Cox regression analysis and LASSO regression analysis. We constructed the nomogram and verified its predictive efficacy by Calibration curves and DCA decision curves. The biological functions and pathways of 6-ORLs-related mRNAs were inferred by Gene Set Enrichment Analysis. Immune cell abundance and immune function associated with risk score (RS) were estimated by ssGSEA, CIBERSORT and MCPcounter synthetically. External validation of the signature was completed using the CGGA-325 and CGGA-693 datasets. 6-ORLs signature—AC083864.2, AC107294.1, AL035446.1, CRNDE, LINC02600, and SNAI3-AS1—were identified through our analysis as being predictive of glioma prognosis. Kaplan–Meier and ROC curves indicated that the signature has a dependable predictive efficacy in the TCGA training cohort, validation cohort and CGGA-325/CGGA-693 test cohort. The 6-ORLs signature were verified to be independent prognostic predictors by multivariate cox regression and stratified survival analysis. Nomogram built with risk scores had strong predictive efficacy for patients' overall survival (OS). The outcomes of the functional enrichment analysis revealing potential molecular regulatory mechanisms for the 6-ORLs. Patients in the high-risk subgroup presented a significant immune microenvironment of macrophage M0 and cancer-associated fibroblast infiltration which was associated with a poorer prognosis. Finally, the expression levels of 6-ORLs in U87/U251/T98/U138 and HA1800 cell lines were verified by RT-qPCR. The nomogram in this study has been made available as a web version for clinicians. This 6-ORLs risk signature has the capabilities to predict the prognosis of glioma patients, assist in evaluating immune infiltration, and assess the efficacy of various anti-tumor systemic therapy regimens.
... GnRH2 is produced in kidneys, bone marrow, lymph glands, heart, lungs, etc. [57,58]. GnRH2 has been proven capable of inhibiting proliferation of prostatic cancer [59,60], ovarian cancer, breast cancer and endometrial cancer [61][62][63][64]; it also exerts better effects than GnRH1 [65]. According to Ling Poon S et al., GnRH2 can inhibit ribosomal phosphoprotein of cancer cells and further inhibit protein translation and cell proliferation [66]. ...
Objective: For the identification of genes of prognostic significance related to tumor microenvironment (TME) in lung adenocarcinoma.
Methods and Materials: Transcriptome data and clinical data of lung adenocarcinoma originated from the Cancer Genome Atlas (TCGA) database. Immune scores and stromal scores were calculated by “Estimation of Stromal and Immune cells in Malignant Tumors using Expression data” algorithm. Based on these calculated scores, the samples were classified as high and low score groups. The average score of respective gene in different groups was calculated. A heat map was created to screen out genes exhibiting differential expressions. The interaction of up-regulated differentially expressed genes (DEGs) and down-regulated DEGs was harvested from a Venn diagram and then covered in the overlapping genes. The core genes influencing prognosis of lung adenocarcinoma were screened out by function enrichment analysis, protein-protein interaction network analysis and Kaplan-Meier (K-M) method on the overlapping genes.
Results: A total of 515 samples of lung adenocarcinoma were harvested from TCGA database. As revealed from the results, a high immune score was related to a high survival rate, while the matrix did not show significant relationships to survival rate. A total of 775 DEGs and 367 overlapping genes were harvested. The functions of these overlapping genes were tightly correlated with DEGs and immune response and were noticeably improved in cytokine-cytokine receptor interaction and chemokine signaling pathway. Eight genes, namely, CCR5, CCR2, CCL14, GNRH2, PKHD1L1, MS4A1, FCER2 and FDCSP, were correlated with prognosis of lung adenocarcinoma.
Conclusion: The genes and pathways affecting prognosis of lung adenocarcinoma were screened out, which offer ideas for subsequent study on lung adenocarcinoma.
... 2001), inhibition of gastric acid secretion (Chen et al. 2005), adhesion chemotaxis and homing in T cells (Chen et al. 2002), hCG release in the placenta ( Raga et al. 1998), steroidogenesis in ovarian cells (Kang et al. 2001, Pellicer et al. 1990Ramakrishnappa et al. 2003), sperm function and sperm oocyte interactions , growth inhibition in reproductive tumors ( Harrison et al. 2004;Finch et al. 2004;Franklin et al . 2003;Grundker et al. 2002Grundker et al. , 2003 and proliferation in melanoma cells (Limonta et al. 2003). ...
... A positive association between birthweight and BMI is well-documented in naturally conceived children (Oken and Gillman, 2003;Eriksson et al., 2008). It is postulated that the presence of GnRH receptors in cumulus –oocyte complexes and preimplantation embryos (Casan et al., 1999;Raga et al., 1999), as well as in the endometrium (Grundker et al., 2002) potentially provides a mechanism by which ovarian stimulation can directly impact offspring phenotype. An alternative or additional indirect mechanism is provided by the subsequent elevated estrogen concentrations that may act to advance the endometrium to impact phenotype (Savage et al., 2012). ...
STUDY QUESTION Are childhood measures of phenotype associated with peri-conception parental, IVF treatment and/or embryonic characteristics
of IVF children?
... The results of many studies on the application of GnRHII in tumor treatment have been published. GnRHII has a stronger anti-proliferative effect on oophoroma and endometrial carcinoma [8][9][10]. Endometriosis has similar characteristics of tumor implant, metastasis and recurrence. ...
To study the effect of gonadotropin-releasing hormone II (GnRHII) on the cell apoptosis of ectopic, eutopic and normal endometrial stromal cells cultured in vitro from endometriosis patients, and to provide theoretical basis for exploring new treatments for endometriosis (EMs).
Ectopic, eutopic and normal endometrial stromal cells were isolated, cultured and identified in vitro, then treated with different concentrations of GnRHII (0, 10(-10) M, 10(-8) M and 10(-6) M). Cell apoptosis was detected by Hoechst staining and flow cytometry.
GnRHII increased apoptosis in ectopic, eutopic and normal stromal cells in a dosage-dependent manner (P<0.05), and apoptosis of ectopic stroma cells was significantly higher than that of eutopic and normal cells (P<0.05); apoptosis in eutopic and normal cells had no different (P>0.05).
GnRHII can significantly induce apoptosis in ectopic, eutopic and normal endometrial stromal cells from patients with endometriosis, especially to the ectopic.
... GnRH and its receptor have been detected in most endometrial and ovarian cancer cell lines and in over 80% of biopsy specimens of these cancers (Imai et al., 1994;Emons et al., 2000). GnRH receptor expression in ovarian cancer cells directly mediates the antiproliferative effects of GnRH agonist (GnRHa) (Gründker et al., 2002b). Other studies have demonstrated the differential effects of GnRHa on the proliferation of human ovarian cancer cells. ...
Background and objective:
Gonadotropin-releasing hormone (GnRH) plays an important role in the regulation of ovarian function and ovarian cancer cell growth. In this study, we determined whether administration of the GnRH agonist (GnRHa), triporelin, prior to cisplatin treatment affects cisplatin and/or prevents cisplatin-induced ovarian damage.
Methods:
nu/nu mice were injected with ovarian cancer OVCAR-3 cells intraperitoneally. After two weeks, the mice were treated with saline (control), cisplatin, GnRHa, or cisplatin plus GnRHa for four weeks. At the end of the experimental protocol, blood, tumor, ovary, and uterine tissues were resected for hematoxylin and eosin (H&E) staining, immunohistochemical analyses of Ki67, nuclear factor-κB (NF-κB), and caspase-3, transmission electron microscopy of apoptosis, or enzyme-linked immunosorbent assay (ELISA) analyses of anti-Mullerian hormone (AMH).
Results:
Cisplatin treatment effectively inhibited tumor growth in mice treated with human ovarian cancer cells; however the treatment also induced considerable toxicity. Immunohistochemical analyses showed that Ki67 expression was reduced in cisplatin-treated mice compared to control (P<0.05), but there was no statistically significant differences between cisplatin-treated mice and cisplatin plus GnRHa-treated mice (P>0.05), while expressions of NF-κB and caspase-3 were reduced and induced, respectively, in cisplatin-treated mice and cisplatin plus GnRHa-treated mice. Apoptosis occurred in the GnRHa, cisplatin, and cisplatin plus GnRHa-treated mice, but not in control mice. Ovaries exposed to GnRHa in both GnRHa mice and cisplatin-treated mice (combination group) had significantly more primordial and growth follicles and serum levels of AMH than those in the control mice and cisplatin-treated mice (P<0.05).
Conclusions:
Administration of GnRHa to mice significantly decreased the extent of ovarian damage induced by cisplatin, but did not affect the anti-tumor activity of cisplatin.
... The transferrin-conjugated nanoparticles demonstrated greater cellular uptake and reduced exocytosis, yielding greater antiproliferative activity and more sustained effects compared to the free drug or unconjugated nanoparticles. Luteinizing hormone-releasing hormone (LHRH) is another targeting moiety; the LHRH receptor is barely present on the surfaces of most healthy human cells, but is over-expressed in ovarian and some other cancer cells [54,55]. Dharap et al. [55] recently developed the LHRH-PEG-camptothecin targeted anticancer drug delivery system, wherein LHRH targets the corresponding receptors in cancer cells: PEG is used as a carrier to prolong the circulation time in blood, and camptothecin functions as the anticancer drug. ...
Nanomedicine, an offshoot of nanotechnology, refers to highly specific, molecular-scale medical intervention for treating disease or repairing damaged tissues. In recent years, polymer-based nanomedicine, a field that includes the use of polymer–DNA complexes (polyplexes), polymer–drug conjugates, and polymer micelles bearing hydrophobic drugs, has received increasing attention for its ability to improve the efficacy of cancer therapeutics. Owing to their small size and excellent biocompatibility, nanosized polymer therapeutic agents can circulate in the bloodstream for long periods of time, allowing them to reach the target site. In addition, chemical modification of polymer therapeutic agents with ligands capable of specifically binding receptors that are over-expressed in cancer cells can markedly augment therapeutic efficiency. This review highlights the characteristics of cancer that provide nanodrug targeting opportunities and discusses rational approaches for future development of polymeric nanomedicines.
... GnRH-II has been less well studied than GnRH-I. However, GnRH-II and its receptor are expressed in human ovarian cancer cell lines and native GnRH-II dose- and time-dependently reduces proliferation in those cancer cells (Gründker et al., 2002). The observed effect is significantly more potent than the anti-proliferative ability of GnRH-I. ...
Vertebrate gonads are the sites of synthesis and binding of many peptides that were initially classified as neuropeptides. These gonadal neuropeptide systems are neither well understood in isolation, nor in their interactions with other neuropeptide systems. Further, our knowledge of the control of these gonadal neuropeptides by peripheral hormones that bind to the gonads, and which themselves are under regulation by true neuropeptide systems from the hypothalamus, is relatively meager. This review discusses the existence of a variety of neuropeptides and their receptors which have been discovered in vertebrate gonads, and the possible way in which such systems could have evolved. We then focus on two key neuropeptides for regulation of the hypothalamo-pituitary-gonadal axis: gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH). Comparative studies have provided us with a degree of understanding as to how a gonadal GnRH system might have evolved, and they have been responsible for the discovery of GnIH and its gonadal counterpart. We attempt to highlight what is known about these two key gonadal neuropeptides, how their actions differ from their hypothalamic counterparts, and how we might learn from comparative studies of them and other gonadal neuropeptides in terms of pharmacology, reproductive physiology and evolutionary biology.
... 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]. ...
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.
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.
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.
GnRH-II antagonists seem to be suitable drugs for an efficacious and less-toxic endocrine therapy for breast cancers, including triple-negative breast cancers.
... Although GnRH II is capable of mimicking the biological actions of GnRH I in extrapituitary tissues, evidence shows differential effects of these two hormones. For instance, GnRH II exhibited more potent anti-proliferative effects than an equimolar dose of GnRH I in human endometrial and ovarian cancer cells [32]. In human placenta, GnRH I was more effective than GnRH II on hCG synthesis and secretion [33], while GnRH II appeared to be more potent than GnRH I in stimulating leptin secretion [34]. ...
Matrix metalloproteinase-26 (MMP-26), one of the main mediators of extracellular matrix (ECM) degradation, has been shown to exist in trophoblasts of human placenta and to play a role in trophoblast cell invasion. However, little is known about the regulation of MMP-26 expression in human trophoblasts. Recently, gonadotropin-releasing hormone I (GnRH I) and GnRH II have been shown to regulate the expression of MMP-2, MMP-9/tissue inhibitor of metalloproteinases 1 (TIMP-1), and urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) in human trophoblasts, suggesting that these two hormones may work as paracrine and/or autocrine regulators in modulating the activities of various protease systems at the feto-maternal interface. In this study, we determined the regulatory effects of GnRH I and GnRH II on the expression of MMP-26 in human immortalized cytotrophoblast-like cell line, B6Tert-1.
Real-time PCR was used to quantify mRNA levels of MMP-26 in human trophoblast-like cell line, B6Tert-1 and primary cultured cytotrophoblasts. Western blotting was used to characterize the expression of MMP-26 and the phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) in B6Tert-1 cells after treatment with GnRH I and GnRH II.
We found that GnRH I increased MMP-26 expression in B6Tert-1 cells after 12 h of treatment at both the mRNA and protein level, while GnRH II increased MMP-26 expression beginning at 3 h of treatment. Treatment of GnRH I at 1 nM resulted in maximal increase of MMP-26 mRNA and protein levels, whereas GnRH II treatment at a concentration of 100 nM was required to induce maximal increase in MMP-26 expression. In addition, we demonstrated that the activation of JNK, but not ERK1/2, was required for GnRH I and II-stimulated MMP-26 production in B6Tert-1 cells and primary cytotrophoblasts.
These novel findings indicated that GnRH I and II could up-regulate MMP-26 expression through the JNK signaling pathway in human trophoblast-like/trophoblast cells.
... T he two GnRHs (GnRH-I and GnRH-II) and the GnRH receptor (GnRHR) have been detected in human ovarian surface epithelial cells and ovarian cancer cell lines (1), and these GnRH subtypes regulate the growth (2)(3)(4) and metastatic activity (5, 6) of ovarian cancer cells. Despite advances in our knowledge of the functional role of GnRH-II in ovarian cancer, the endocrine regulation of GnRH-II expression in ovarian cancer cells is poorly understood. ...
GnRH-II modulates ovarian cancer cells invasion and is expressed in normal ovary and ovarian epithelial cancer cells; however, the upstream regulator(s) of GnRH-II expression in these cells remains unclear. We now demonstrate that epidermal growth factor (EGF) increases GnRH-II mRNA levels in several human ovarian carcinoma cell lines and up-regulates GnRH-II promoter activity in OVCAR-3 cells in a dose-dependent manner, whereas an EGF receptor inhibitor (AG148) abolishes EGF-induced increases in GnRH-II promoter activity and GnRH-II mRNA levels. EGF increases the phosphorylation of cAMP-responsive element-binding protein (p-CREB) and its association with the coregulator, CCAAT/enhancer binding protein beta, whereas blocking the EGF-induced ERK1/2 phosphorylation with MAPK inhibitors (PD98059/U0126) markedly reduced these effects. Moreover, depletion of CREB using small interfering RNA attenuated EGF-induced GnRH-II promoter activity. Chromatin immunoprecipitation assays demonstrated that EGF induces p-CREB binding to a cAMP responsive-element within the GnRH-II promoter, likely in association with CCAAT/enhancer binding protein beta, and mutagenesis of this cAMP responsive-element prevented EGF-induced GnRH-II promoter activity in OVCAR-3 cells. Importantly, GnRH-II acts additively with EGF to promote invasion of OVCAR-3 and CaOV-3 cells, but not SKOV-3 cells that express low levels of GnRH receptor (GnRHR). Treatment with GnRHR small interfering RNA also partially inhibited the EGF-induced invasion of OVCAR-3 and CaOV-3 cells. Furthermore, EGF treatment transiently increases GnRHR levels in OVCAR-3 and CaOV-3, which likely accentuates the effects of increase GnRH-II production on cell invasion. These results provide evidence that EGF is an upstream regulator of the autocrine actions of GnRH-II on the invasive properties of ovarian cancer cells.
... Consequently, it undergoes desensitization and internalization. PCR and immunocytochemistry also showed that the GnRHR-II is expressed ubiquitously in human tissues (Grundker et al. 2002), including brain, pituitary and reproductive tissues (Millar et al. 2001, Neill et al. 2001, couples to G q/11 proteins (White et al. 1998) and stimulates the production of IP 3 when activated by its specific agonist GnRH-II (Millar et al. 2001). So far, there is no evidence for the presence of this receptor subtype in rats and the stimulatory action of GnRH-II on IP 3 production and gonadotropin release appears to be mediated by GnRHR-I (Okada et al. 2003, Mongiat et al. 2004). ...
Two forms of gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are commonly present in mammals. The main hormone controlling reproduction is GnRH-I acting through its receptor (GnRHR-I), whereas the function of GnRH-II is unknown. In primates, it has been suggested that GnRH-II is a specific agonist for the structurally distinct GnRHR-II. Here we compared effects of GnRH-I and GnRH-II on intracellular calcium and gonadotropin hormone release in neonatal rat gonadotrophs in vitro and the dependence of agonist actions on cyclic nucleotide levels. Both agonists elevated intracellular calcium and stimulated gonadotropin secretion in a concentration-dependent manner, with comparable peak amplitudes, but GnRH-I was three times more potent than GnRH-II. Antide, a specific GnRHR-I antagonist, completely blocked the action of both agonists on gonadotropin release. Inhibition of adenylyl cyclase activity by melatonin and MDL significantly attenuated GnRH-I- and GnRH-II-induced calcium signaling and gonadotropin release, whereas inhibition of soluble guanylyl cyclase activity was ineffective. GnRH-II also generated calcium oscillations in a fraction of gonadotrophs not expressing melatonin receptors. These results indicate that GnRH-I and GnRH-II act on the same GnRHR to stimulate gonadotropin release through intracellular calcium and cyclic nucleotide signaling, and that GnRH-II is less potent agonist for this receptor in neonatal rat gonadotrophs.
Cancer is a prime healthcare problem that is significantly responsible for universal mortality. Despite distinguished advancements in medical field, chemotherapy is still the mainstay for the treatment of cancers. During chemotherapy, approximately 90% of the administered dose goes to normal tissues, with mere 2–5% precisely reaching the cancerous tissues. Subsequently, the resultant side effects and associated complications lead to dose reduction or even discontinuance of the therapy. Tumor directed therapy therefore, represents a fascinating approach to augment the therapeutic potential of anticancer bioactives as well as overcomes its side effects. The selective overexpression of LHRH receptors on human tumors compared to normal tissues makes them a suitable marker for diagnostics, molecular probes and targeted therapeutics. These understanding enabled the rational to conjugate LHRH with various cytotoxic drugs (doxorubicin, DOX; camptothecin etc.), cytotoxic genes [small interfering RNA (siRNA), micro RNA (miRNA)], as well as therapeutic nanocarriers (nanoparticles, liposomes or dendrimers) to facilitate their tumor specific delivery. LHRH conjugation enhances their delivery via LHRH receptor mediated endocytosis. Numerous cytotoxic analogs of LHRH were developed over the past two decades to target various types of cancers. The potency of LHRH compound were reported to be as high as 5,00–10,00 folds compared to parent molecules. The objective of this review article is to discuss reports on various LHRH analogs with special emphasis on their prospective application in the medical field. The article also focuses on the attributes that must be taken into account while designing a LHRH therapeutics with special account to the biochemistry and applications of these conjugates. The record on various cytotoxic analogs of LHRH are also discussed. It is anticipated that the knowledge of therapeutic and toxicological aspects of LHRH compounds will facilitate the development of a more systematic approach to the targeted delivery of cytotoxic agents using peptides.
In addition to its well-documented role in the reproductive hormone cascade, GnRH may play a role as an autocrine/paracrine regulator in the inhibition of cell proliferation and/or induction of apoptosis in normal and neoplastic OSE cells. The use of GnRH and its analogs is being considered for pre-clinical trials as a possible therapeutic agent in gynecological tumors such as ovarian and endometrial cancers. The exact mechanisms of GnRH action in the inhibition of cell growth need to be delineated using in-vitro and in-vivo models for ovarian cancer. The recent discovery of a second form of GnRH (GnRH-II) is posing a new challenge in understanding the role of the GnRH/GnRH-R system in normal OSE and its neoplastic counterpart. It appears that GnRH-II may have a more potent effect in the inhibition of cell proliferation when compared to that of GnRH-I in ovarian cancer cells.
On the other hand, E and ER may play a role in ovarian carcinogenesis as indicated by epidemiological and experimental observations. Further studies are needed to elucidate the influence of E and other hormones such as P4, gonadotropins and growth factors in normal and neoplastic OSE. Interestingly, it appears that E may be involved in the regulation of GnRH and GnRH-R genes in normal OSE and ovarian cancer cells. This potential cross-talk between the E and GnRH systems in the ovary may be important in the multi-faceted regulation of cell proliferation, apoptosis and/or differentiation of normal OSE and its neoplastic counterparts.
Introduction:
Suppression of sex-steroid secretion is required in a variety of gynecological conditions. This can be achieved using gonadotropin releasing hormone (GnRH) agonists that bind pituitary gonadotropin receptors and antagonize the link-receptor of endogenous GnRH, inhibiting the mechanism of GnRH pulsatility. On the other hand, GnRH antagonists immediately reduce gonadal steroid levels, avoiding the initial stimulatory phase of the agonists. Potential benefits of GnRH antagonists over GnRH agonists include a rapid onset and reversibility of action. Older GnRH antagonists are synthetic peptides, obtained by modifications of certain amino acids in the native GnRH sequence. They require subcutaneous injections, implantation of long-acting depots. The peptide structure is responsible for histamine-related adverse events and the tendency to elicit hypersensitivity reactions. Areas Covered: Research has worked towards the development of non-peptidic molecules exerting antagonist action on GnRH. They are available for oral administration and may have a more beneficial safety profile in comparison with peptide GnRH antagonists. This article focuses on the data of the literature about elagolix, a novel non-peptidic GnRHantagonist, in the treatment of endometriosis. Expert Opinion: Elagolix demonstrated efficacy in the management of endometriosis-associated pain and had an acceptable safety and tolerability profile. However, further studies are necessary to evaluate its non-inferiority in comparison with other endometriosis's treatments.
Hormone based drug targeting is a promising tool for selective tumor therapy. In this study, synthesis and systematic comparative biological evaluation of novel drug containing analogs of gonadotropin-releasing hormone GnRH-I and GnRH-II is reported demonstrating their suitability for tumor targeting. The cytotoxic conjugates were prepared by the attachment of the chemotherapeutical agent daunorubicin (Dau) to GnRH analogs directly or through an enzyme-labile spacer with oxime linkage. All conjugates were found to be proteolytically stable under circumstances applied in biological assays. Both GnRH-I and GnRH-II were able to bind similarly to high-affinity GnRH-I receptors on human pituitary and human prostate cancer cells. The in vitro long-term cytotoxic effect of the conjugates was comparable with that of the free drug in human breast and colon cancer cell lines. Furthermore, a concentration-dependent cellular uptake profile was observed. The in vitro apoptotic effect of the compounds was evaluated by flow cytometry analysis using annexin-V. Our results show that both the GnRH-I and the GnRH-II based analogs might be applied for targeted tumor therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
In this study we aimed to address the poor drug-like properties of Gonadotropin-Releasing Hormone (GnRH) peptide through modification with lipids and carbohydrates.
GnRH peptide was conjugated to 2-amino-D,L-octanoic acid (C8) and 2-amino-D,L-dodecanoic acid (C12) in monomer and dimer, along with (6-9) or without (2-5 and 11) a glucose moiety. Peptides were tested for their biological activity using different tumour cell lines. The toxicity of the constructs was evaluated in peripheral blood mononuclear cells (PBMC).
All (glyco)lipopeptides showed improved metabolic stability in Caco-2 cell homogenates. Those with single lipid moiety (2, 4 and 8) exhibited prodrug-like properties. Permeability across Caco-2 cell monolayers was enhanced in the dimer C8-modified (glyco)lipopeptide (3) and the lipopeptide with C12 inserted mid-sequence (11). Most of the constructs showed moderate-to-high antiproliferative activity against GnRH-receptor positive DU145 and OVCAR-3 cells (up to 60%). Compound 11 was the most effective with IC50 = 26.4 ± 1.07 μg.ml(-1), which was comparable to triptorelin (25.1 ± 1.14 μg.mL(-1)). The sensitivity of OVCAR-3 cells to the effect of all analogues except for 11 decreased significantly in estrogen-reconstituted media. Only compounds 2, 4, 5 and 8 showed a steroid-dependent effect in DU145 cells. No compounds exhibited significant toxicity on PBMCs.
These results indicated lipidation and glycosylation improves the druggability of GnRH and could lead to an increased direct antitumour activity in some hormone dependent and independent reproductive cancers.
The lack of efficient and non-toxic gene delivery, preferably with non-viral DNA vectors, is generally re-garded as a major limitation for gene therapy. In this study, a wheat histone H4 gene was cloned from Triticum aesti-vum, sequenced, modified and expressed in E. coli. The wheat histone H4 gene and reconstructed H4TL gene en-coded wheat histone H4 and a recombinant protein of 141 amino acids with an approximate molecular weight of 15500. Gel electrophoresis mobility shift assays demonstrated that the purified protein had high affinity for DNA. Most significantly, the complex of plasmid pEGFP/C1 with H4TL was transfected with increased efficiency into MCF-7, HO8910, LNCap, A549 and HeLa cells in vitro. These results demonstrate that the targeting of non-viral vectors to tumor-specific receptors provides a cheap, simple and highly efficient tool for gene delivery.
Das Ovarialkarzinom wird zumeist in fortgeschrittenem Stadium mit entsprechend schlechter Prognose diagnostiziert. Prävention und Screening könnten diesen fatalen Verlauf beeinflussen. Charakterisierung und Identifikation der Risikogruppen, bereits verfügbare präventive Maßnahmen fördern und mögliche in Studien evaluieren, Verbesserung der Diagnostik und Evaluation möglicher Screeningmethoden – dies sind einige Aspekte des Problemfeldes. Betrachtet werden hormonelle Kontrazeption, weitere endokrine und chemopräventive Optionen sowie chirurgische Aspekte. Die Bedeutung von nichthereditären Risiken, welche die Inzidenz des Ovarialkarzinoms erhöhen, wird erörtert. Bereits heute bestehen Interventions- und Vermeidungsmöglichkeiten zur Prävention des Ovarialkarzinoms, die mit diagnostischen Maßnahmen verbunden werden können. Sekundär- und Tertiärprävention werden diskutiert.
Our objective was to synthesize LHRH-conjugated amphiphilic copolymer for micellar delivery of CBDIV17, a novel antiandrogen for treating prostate cancer.
LHRH-PEG-b-p(CB-co-LA) was synthesized by opening polymerization of carbonate (CB), lactide (LA), and HOOC-PEG-OH followed by conjugation with LHRH analogue. Bicalutamide analogue CBIDV17 loaded micelles were formulated by film hydration method, and characterized for critical micelle concentration (CMC), drug loading and in vitro drug release. Formulations were tested on LNCaP and C4-2 cells for cellular uptake, induction of apoptosis, viability and dowregulation of androgen receptor (AR). In vivo studies were performed in ectopic tumor bearing athymic nude mice after tail vein injection at a dose of 10 mg/kg. Tumor volume and body weight were measured for 25 days followed by immunohistochemistry (IHC) of tumor samples for Ki-67, caspase-3, and prostate specific antigen (PSA).
HOOC-PEG-b-p(CB-co-LA) and LHRH-PEG-b-p(CB-co-LA) were characterized by (1)HNMR and used for preparing micelles, which had a mean particle size of 75.60 ± 2.25 and 72.64 ± 1.15 nm, respectively and CBDIV17 loading of 4.6% w/w. LHRH conjugated micelles showed higher cellular uptake, cytotoxicity, and apoptosis in LNCaP and C4-2 cells compared to non-targeted micelles. CBDIV17 loaded LHRH micelles more efficiently inhibited the proliferation and induced apoptosis of tumor cells according to Ki-67, caspase-3, and PSA expression. There was significant inhibition of tumor growth with the treatment of CBDIV17 loaded LHRH-conjugated micelles.
These results demonstrated that LHRH-b-PEG-p(CB-co-LA) micelles have the potential for targeted delivery of CBDIV17 to treat advanced prostate cancer.
Prostate cancer is androgen-dependent in its early stages and androgen deprivation therapy represents the most effective first-line therapeutic approach. However, after an initial remission, prostate cancer progresses towards the castration resistant prostate cancer (CRPC) stage, with increased malignancy and resistance to conventional chemotherapy. Pituitary gonadotropin-releasing hormone receptors (GnRH-Rs) represent the most effective molecular target for the treatment of steroid-dependent prostate cancer. GnRH agonists (through GnRH-Rs desensitization) suppress the pituitary-testicular axis and, therefore, represent the treatment of choice for prostate cancer patients. GnRH-Rs are also expressed in prostate cancer, even when the tumor has reached the CRPC stage, and are endowed with antitumor activity, supporting the notion that they might represent a molecular target for GnRH analog-based therapeutic strategies. In addition to GnRH agonists and antagonists, GnRH-based bioconjugates (cytotoxic GnRH bioconjugates, GnRH-conjugated lytic peptides and GnRH-toxin bioconjugates) have been developed and are now undergoing intensive investigations; some of them (i.e., AN-152, Dox-[d-Lys(6)]-GnRH) have entered clinical trials. The advantage of these treatments is the specific delivery of cytotoxic agents to cancer cells. Interestingly, other isoforms of the peptide have been identified. One of them is GnRH-III, which was isolated from sea lamprey. GnRH-III specifically binds to GnRH-Rs in cancer cells and exerts antiproliferative effects; on the other hand, its endocrine effects at pituitary level are insignificant, supporting its selective antitumor activity. Based on these observations, different cytotoxic GnRH-III bioconjugates have recently been synthesized; preliminary in vitro studies suggest that these compounds might represent a new promising treatment strategy for prostate cancer.
Gonadotrophin-releasing hormone (GnRH) significantly inhibits proliferation of a proportion of cancer cell lines by activating GnRH receptor-G protein signaling. Therefore, manipulation of GnRH receptor signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GnRH receptor activation in sensitive cells (HEK293-GnRHR, SCL60) in in vitro and in vivo settings, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic SCL60 response to the GnRH super-agonist Triptorelin. Early and mid-phase changes (0.5-1.0 h) comprised mainly transcription factors. Later changes (8-24 h) included a GnRH target gene, CGA, and up or down-regulation of transcripts encoding signaling and cell division machinery. Pathway analysis exposed identified altered mitogen-activated protein kinase and cell cycle pathways, consistent with occurrence of G2/M arrest and apoptosis. NFκB pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenografts in vivo during Triptorelin anti-proliferation. Increased phosphorylated NFκB (p65) occurred in SCL60 in vitro, and p-NFκB and IκBε were higher in treated xenografts than controls after 4 days Triptorelin. NFκB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GnRH receptor signaling, identifies potential anti-proliferative target genes and implicates the NFκB survival pathway as a node for enhancing GnRH agonist-induced anti-proliferation.
Objective:
Increased glycolysis for energy production is necessary for survival of tumor cells and thus represents a selective therapeutic target. We have analyzed in vitro whether inhibition of glycolysis can reduce the viability of human endometrial and ovarian cancer cells and whether it can enhance the antitumor efficacy of GnRH receptor-targeted therapies.
Materials and methods:
Cell viability of ovarian and endometrial cancer cells treated without or with glycolysis inhibitor 2-Deoxy-D-Glucose (2DG) alone or in combination with GnRH-II antagonist [Ac-D2Nal(1), D-4Cpa(2), D-3Pal(3,6)(8),Leu, D-Ala(10)]GnRH-II or with cytotoxic GnRH-I agonist AEZS-108 (AN-152) was measured using alamar blue assay. Induction of apoptosis was analyzed using TUNEL assay and quantified by measurement of loss of mitochondrial membrane potential. Apoptotic signaling was measured by quantification of activated caspase-3 by using the Western blot technique.
Results:
Treatment of endometrial and ovarian cancer cells with glycolysis inhibitor 2DG resulted in a significant decrease of cell viability and a significant increase of apoptosis. Treatment with 2DG in combination with the GnRH-II antagonist or with AEZS-108 resulted in a significant reduced viability compared with single-agent treatments. The observed reduction in viability was due to induction of apoptosis. Also for apoptosis induction, a significant stronger effect in the case of cotreatments compared with single-agent treatments could be observed. These additive effects could be correlated to increased activation of caspase-3.
Conclusions:
The glycolytic phenotype of human endometrial and ovarian cancer cells can be targeted for therapeutic intervention. In addition, cotreatment of a glycolysis inhibitor with GnRH receptor-targeted therapies might be a suitable therapy for GnRH receptor-positive human endometrial and ovarian cancers.
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. OHT‑resistant 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.
The sections in this article are
Introduction
Cancer
Conventional Approaches to Cancer/Metastases Detection
Current Chemotherapeutic Approaches and their Disadvantages in Cancer Treatments
Multidrug Resistance
Drug Delivery to Tumors
Nanoparticles as Vehicles for Drug Delivery and Diagnosis
Targeting Tumor Cells
Passive Targeting
Active Targeting
Detection of Tumors and Metastases using Nanoparticles
Nanoparticles for Magnetic Resonance Imaging
Targeted Delivery of Nanoparticles to Increase Cellular Uptake for Higher MRI Resolution
LHRH and its Receptors
The Ligand Luteinizing Hormone Releasing Hormone – LHRH
Analogs of LHRH
Receptors for LHRH
Function–Signal Transduction Pathways
LHRH Receptor‐mediated Uptake
LHRH Receptor Type II
LHRH ‐bound Magnetic Nanoparticles
Synthesis and Characterization
Treatment using Hyperthermia
Treatment using Lytic Peptides
Destruction of Metastases through LHRH ‐ SPION ‐Hecate
Detection of Tumors and Metastases
Targeted Delivery of SPION Contrast Agents for MRI
In Vitro Studies on Receptor‐targeted LHRH ‐ SPION Uptake
In Vivo Studies on Receptor‐targeted LHRH ‐ SPION Uptake
Future Outlook
Acknowledgments
Chemotherapy together with debulking surgery is a major treatment for cancer. There are, however, major limitations of conventional
cytotoxic drugs that result from their nonspecific toxicity (e.g., the lack of selectivity) in the body and the intrinsic
or acquired multidrug resistance (MDR) of cancer cells. To this end, polymeric drug carriers have been developed to address
this nonspecificity and MDR [1]. It is believed that these drug carriers alter the biodistribution and increase the bioavailability
of incorporated anticancer agents to the target cells [2].
Gonadotropin-releasing hormone is a neuropeptide that acts via Gq coupled G-protein coupled receptors in the pituitary that mediate central control of reproduction. GnRH receptors (GnRHR) and GnRH ligands are also found in extra-pituitary sites including the CNS as well as reproductive tissues and cancer cells derived from such tissues. Much of the interest in the extra-pituitary receptors stems from the fact that they mediate anti-proliferative and/or pro-apoptotic effects and may therefore be directly targeted for cancer therapy. Type I mammalian GnRHR are atypical in that they do not bind to (or signal via) arrestins. In spite of this restriction on their signaling repertoire, there is good evidence for existence of multiple active GnRHR conformations and for activation of multiple upstream effectors (heterotrimeric and monomeric G-proteins). In this review GnRHR signaling is described, with emphasis on the relevance of functional selectivity for pharmacological characterization of GnRHR ligands, as well as its possible contribution to contextdependent GnRHR signaling and relevance for GnRHR-mediated effects on cell fate as well as GnRHR trafficking.
BACKGROUND The impact of gonadotrophin-releasing hormone (GnRH) antagonists used in IVF protocols on endometrial tissue remodeling,
embryo implantation and the programming of early pregnancy is still unclear. Pregnancy and infant outcomes after treatment
with GnRH antagonist for IVF are particular causes of concern. The purpose of this study was to investigate the mechanisms
of GnRH antagonist-induced apoptosis of human decidual stromal cells and the effects of GnRH antagonist on the activation
of ERK1/2, JNK and GADD45α signaling.
The debate exists whether or not gonadotropin-releasing hormone (GnRH) analogs used in controlled ovarian hyperstimulation (COH) impair endometrial receptivity. Homeobox A11 (Hoxa11), Meis homeobox 1 (Meis1), cadherin 1 (Cdh1), and catenin beta 1 (Ctnnb1) are well known to be involved in successful implantation. In this study, the endometrial expression of Hoxa11, Meis1, Cdh1, and Ctnnb1 during the peri-implantation period was investigated in an in vitro fertilization (IVF) mouse model by real-time RT-PCR and Western blot to evaluate the relationship between Hoxa11, Meis1, Cdh1, and Ctnnb1 expression and the impact of the COH on endometrial receptivity. The mimic COH protocols included GnRH agonist plus human menopausal gonadotropin (HMG) (GnRH agonist group), GnRH antagonist plus HMG (GnRH antagonist group), and HMG alone (HMG group). The expression levels of Hoxa11, Meis1, Cdh1, and Ctnnb1 mRNA and protein were decreased in all of the COH groups. The expression levels of Hoxa11 and Ctnnb1 were the lowest in the GnRH agonist group, and those of Meis1 and Cdh1 were lower in the GnRH analog groups than the HMG group. There were positive correlations between the expression of Hoxa11 and Ctnnb1, as well as the expression of Meis1 and Cdh1 among all the groups. In conclusion, the COH protocols, particularly with GnRH analogs, suppressed Hoxa11, Meis1, Ctnnb1 and Cdh1 expression, in mouse endometrium during the peri-implantation period. Our data reveal a novel molecular mechanism by which the COH protocols might impair endometrial receptivity.
Endometrial cancer is the most common malignant tumor of the female genital tract in the developed world. Increasing evidence suggests that the majority of cases can be divided into two different types ofendometrial cancer based on clinico-pathological and molecular characteristics. Type I is associated with an endocrine milieu of estrogen predominance. These tumors are ofendometroid histology and develop from endometrial hyperplasia. They have good prognosis and are sensitive to endocrine treatment. Type II endometrial cancers are not associated with a history of unopposed estrogens and develop from the atrophic endometrium of elderly women. Mainly, they are of serous papillary or clear cell morphology, have a poor prognosis and do not react to endocrine treatment. Both types of endometrial cancer probably differ markedly with regard to the molecular mechanisms of transformation. The transition from normal endometrium to a malignant tumor is thought to involve a stepwise accumulation of alterations in cellular mechanisms leading to dysfunctional cell growth. This chapter reviews the current knowledge of the molecular mechanisms commonly associated with development of type I and type II endometrial cancer.
The objective of this study was to elucidate the effects of GnRH antagonist Cetrorelix on proliferation and apoptosis in human leiomyoma cells cultured in vitro. Isolated leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions in the presence or absence of graded concentrations of Cetrorelix (10(-5) to 10(-8) mol/liter) for 6 d. Cultured leiomyoma cells were used for semiquantitative RT-PCR, immunocytochemistry, Western blot analysis, and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay. RT-PCR analysis revealed the presence of mRNAs encoding for GnRH receptor and epidermal growth factor (EGF) in cultured leiomyoma cells. The number of viable cultured leiomyoma cells was significantly (P < 0.01) decreased by treatment with Cetrorelix compared with untreated control cultures. Immunocytochemical examination demonstrated that treatment with Cetrorelix attenuated the expression of proliferating cell nuclear antigen (PCNA) and EGF in cultured leiomyoma cells. Western blot analysis revealed that treatment with 10(-5) mol/liter Cetrorelix significantly (P < 0.01) decreased PCNA expression. In addition, treatment with 10(-5) mol/liter Cetrorelix remarkably increased the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling-positive rate and poly(ADP-ribose) polymerase expression at 24 h of treatment compared with untreated control cultures (P < 0.01). Furthermore, treatment with 10(-5) mol/liter Cetrorelix decreased immunoreactive EGF protein and EGF mRNA expression in cultured leiomyoma cells at 4 d of treatment. GnRH antagonist Cetrorelix may directly inhibit leiomyoma cell growth by down-regulating proliferation in association with a decrease in EGF mRNA expression and by up-regulating apoptosis in those cells.
We recently demonstrated the effect of gonadotrophin-releasing hormone agonist (GnRHa) on tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma. Here, we investigated expression of GnRH receptors (GnRHRs) and effect of GnRHa on the proliferation of cells derived from endometria and pathological lesions of women with these reproductive diseases.
Biopsy specimens were collected from lesions and corresponding endometria of 35 women with pelvic endometriosis, 45 women with ovarian endometrioma, 35 women with adenomyosis and 56 women with uterine myoma during laparoscopy or laparotomy. The gene and protein expressions of GnRHR in eutopic/ectopic cells and tissues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The immunoreactivity of GnRHR in tissue was analysed by quantitative-histogram (Q-H) scores. The exogenous effect of GnRHa on cell proliferation was examined by 5-bromo-2-deoxyuridine incorporation assay. The Ki-67-immunoreactive cell proliferation index was analysed in biopsy specimens derived from GnRHa-treated and -non-treated women.
Types I and II GnRHRs mRNA and proteins were expressed in eutopic endometria and pathological lesions derived from women with endometriosis, adenomyosis and uterine myoma. GnRHR expression was the highest in the menstrual phase when compared with other phases of the menstrual cycle. Higher Q-H scores of GnRHR immunoreaction were found in blood-filled opaque red lesions than in other peritoneal lesions. Exogenous treatment with GnRHa significantly suppressed the proliferation of cells derived from respective endometria and pathological lesions when compared with GnRHa-non-treated cells.
Local tissue expression of GnRHR was detected in endometriosis, adenomyosis and uterine myoma. In addition to a hypo-estrogenic effect, a direct anti-proliferative effect of GnRHa may be involved in the regression of these reproductive diseases with consequent remission of clinical symptoms.
To investigate the expression and function of GnRH and GnRH receptor (GnRHR) subtypes at the maternal-fetal interface.
In vitro experiments using freshly isolated human trophoblast cells, decidual stromal cells (DSCs), and immortalized cell lines.
University teaching hospital.
Placenta-fetal membranes from term deliveries.
Human trophoblast and DSCs were isolated, purified, and cultured.
Expression of GnRH-I, GnRH-II, and GnRHR-I mRNA and protein in human trophoblast cell lines and tissues were evaluated by reverse-transcription polymerase chain reaction and Western blot. The effect of GnRH-I and -II on the production of select cytokines (hCG, interleukin [IL] 8, IL-6, matrix metalloproteinase 3, monocyte chemoattractant protein 1, vascular endothelial growth factor, soluble Fms-like tyrosine kinase 1, urokinase-type plasminogen activator, and plasminogen activator inhibitor 1) were measured by ELISA and normalized for protein content.
GnRH-I, GnRH-II, and GnRHR-I mRNA and protein were identified in trophoblasts and decidua. GnRH-I and -II stimulated hCG production by trophoblast and trophoblast-derived cell lines in a dose-dependent fashion (e.g., 2.8-fold, from 2.5 ± 0.5 to 7.0 ± 0.4 ng/mg protein per 24 h, for 1,000 nmol/L GnRH-I and 2.4-fold, from 2.5 ± 0.5 to 6.1 ± 0.6 ng/mg protein per 24 h, for 1,000 nmol/L GnRH-II) without affecting the production of other cytokines.
Trophoblasts and decidua express GnRH-I, GnRH-II, and GnRHR-I mRNA and protein. GnRH-I and -II selectively stimulate hCG production by trophoblast cells without altering the production of select cytokines by trophoblasts or decidua. The role of GnRH-GnRHR signaling at the maternal-fetal interface therefore appears to be limited to the regulation of trophoblast hCG production.
Recent studies propose the role of gonadotropins in the development and growth of endometrial carcinoma. The present research was undertaken to establish the expression of human chorionic gonadotropin (hCG), gonadotropin-releasing hormones 1 (GnRH1 and GnRH2, respectively) and their receptors in endometrial hyperplasias and carcinoma.
The expression of analyzed genes in endometrial carcinoma and hyperplasia with and without atypia was evaluated using reverse transcriptase polymerase chain reaction and immunohistochemistry.
The results of the experiments demonstrated the presence of hCG and GnRH1 at both messenger RNA and protein levels in endometrial carcinoma and atypical hyperplasia. Noncancerous tissue and hyperplasia without atypia demonstrated the lack of these gene coexpressions. The expression of GnRH2, LH/hCGR, and GnRHRs was heterogeneous, and the study molecules were found only in part of the analyzed tissues. The presence of hCG and GnRH1 and their receptors in cancer tissue and atypical hyperplasia suggests autocrine/paracrine action of hormones regulating the endometrial carcinoma cell proliferation.
The interaction between the hCG and LH/hCGR in endometrial tissue might stimulate cell growth and promote neoangiogenesis, whereas GnRHs, by binding to their receptors, could be responsible for the antiproliferative effect and stimulation of apoptosis. The identification of differences in the expression profile of the analyzed genes could be relevant for better understanding of the development of endometrial carcinomas and could be useful in clinical diagnostics.
The somatic mutation in the FOXL2 gene c.402C>G (p.Cys134Trp) has recently been identified in the vast majority of adult ovarian granulosa cell tumors (OGCTs) studied. In addition, this mutation seems to be specific to adult OGCTs and is likely to be a driver of malignant transformation. However, its pathogenic mechanisms remain elusive.
We have sequenced the FOXL2 open reading frame in a panel of tumor cell lines (NCI-60, colorectal carcinoma cell lines, JEG-3, and KGN cells). We found the FOXL2 c.402C>G mutation in the adult OGCT-derived KGN cell line. All other cell lines analyzed were negative for the mutation. In order to gain insights into the pathogenic mechanism of the p.Cys134Trp mutation, the subcellular localization and mobility of the mutant protein were studied and found to be no different from those of the wild type (WT). Furthermore, its transactivation ability was in most cases similar to that of the WT protein, including in conditions of oxidative stress. A notable exception was an artificial promoter known to be coregulated by FOXL2 and Smad3, suggesting a potential modification of their interaction. We generated a 3D structural model of the p.Cys134Trp variant and our analysis suggests that homodimer formation might also be disturbed by the mutation.
Here, we confirm the specificity of the FOXL2 c.402C>G mutation in adult OGCTs and begin the exploration of its molecular significance. This is the first study demonstrating that the p.Cys134Trp mutant does not have a strong impact on FOXL2 localization, solubility, and transactivation abilities on a panel of proven target promoters, behaving neither as a dominant-negative nor as a loss-of-function mutation. Further studies are required to understand the specific molecular effects of this outstanding FOXL2 mutation.
To investigate the expression of the second form of GnRH (GnRH-II) in tumor tissue and peripheral blood mononuclear cells (PBMCs) in malignant and benign ovarian tumors in humans.
Sixty-six women were studied: 24 with epithelial ovarian carcinomas, 22 with benign ovarian tumors and 20 in the control group undergoing surgery. Malignant, benign and normal ovarian tissue and PBMCs were obtained for measurement of GnRH-II mRNA levels using quantitative real-time RT-PCR.
The expression of GnRH-II was found to be 1.5 times higher in malignant ovarian tumors compared with benign ovarian tumors and the control group in post-menopausal patients (P<0.01). In the post-menopausal patient group with malignant ovarian tumors, there were significant positive correlations between serum FSH level and ovarian tissue GnRH-II mRNA expression (r=0.68; P=0.03), and serum LH level and ovarian tissue GnRH-II mRNA expression (r=0.71; P=0.02). Controls, benign and malignant groups were similar in terms of GnRH-II expression in PBMCs in the pre- and post-menopausal periods. There was no significant correlation between ovarian tissue GnRH-II mRNA expression vs. PBMC GnRH-II mRNA expression in patient and control groups.
We have shown increased GnRH-II expression in human ovarian cancer tissue in post-menopausal women in vivo. Expression of GnRH-II in PBMCs did not reflect the local GnRH-II expression levels in ovarian tissue. These preliminary data suggest that local GnRH-II may participate in the regulation of ovarian tumor growth in post-menopausal women.
Tumor targeting with peptides is based on the discovery that receptors for many regulatory peptides are overexpressed in tumor cells, compared with their expression in normal tissues. Consequently, these peptides and their analogues can be used as carriers/targeting moieties for the preparation of diagnostic and therapeutic agents that have increased selectivity and decreased peripheral toxicity.
Here an overview is given of the most relevant gonadotropin-releasing hormone (GnRH) and somatostatin derivatives, as well as of their applications in cancer diagnosis and therapy. For this purpose, recently published data in these areas (mostly articles published from 2000 to 2009) were reviewed.
In contrast to other regulatory peptides that stimulate the tumor growth, GnRH and somatostatin derivatives have inhibitory effect; therefore, they were used primarily for the preparation of various conjugates to be used in targeted chemotherapy, targeted radiotherapy, photodynamic therapy, boron neutron capture therapy and cancer diagnosis. Some of these conjugates have already found clinical applications, whereas others are now in preclinical and clinical trials.
Tumor targeting with hormone peptides provides a basis for the development of new diagnostic and therapeutic approaches for cancer.
Researchers have launched a new area of febrile investigations on the autophagy-related gene Beclin 1. Our aim is to investigate whether Beclin 1 expression is altered in eutopic endometrium of women with adenomyosis and its association with clinical characteristics.
We collected tissue samples from the eutopic endometria of 30 women with adenomyosis and 32 healthy women undergoing surgery for benign indications. We cultured the stromal cells of the eutopic endometria. Beclin 1 expression of the cultured stromal cells and tissues was assessed by reverse transcription polymerase chain reaction and western blot analysis.
Beclin 1 messenger RNA (mRNA) expression in cultured stromal cells of eutopic endometria and endometrial tissues of women with adenomyosis was significantly lower than that of controls (P < 0.05). Beclin 1 protein expression in cultured stromal cells of eutopic endometria and endometrial tissues of adenomyosis was also significantly lower compared with that of controls (P < 0.01). Beclin 1 protein expression in eutopic endometrial tissues was negatively correlated with serum CA125 (r = -0.307, P = 0.015), and pelvic pain (r = -0.542, P = 0.000).
The study revealed Beclin 1 mRNA and protein expression were significantly decreased in eutopic endometria of women with adenomyosis. Moreover, Beclin 1 was negatively correlated with serum CA125 and pelvic pain. Beclin 1 might contribute to the pathogenesis and progression of endometriosis. Further research on autophagy of adenomyosis is required.
Gonadotropin releasing hormone (GnRH) has a pivotal role in the biology of reproduction processes. In extrapituitary compartments GnRH and its receptor act as a part of the autocrin regulatory system of cell proliferation, resulting in its anticancer activity. Here the anticancer activity of a new analogue of GnRH has been investigated. Results indicate that proliferation of human breast and ovarian cancer cell lines is dose‐dependently inhibited. The inhibitory efficiency of this new analogue is proved to be higher than the original triptorelin. In addition to its antimitogenic activity, evidence was found for the involvement of the apoptotic mechanism in the action of the new analogue. Furthermore the presence of chemical groups in the peptide sequence is thought to increase the protease stability of the new analogue in comparison with triptorelin. Consequently our new analogue can be considered as a good pharmaceutical candidate. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 292–297, 2010.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
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.
To investigate whether the GnRH agonist may reduce aromatase P450 and COX-2 in the eutopic endometrium of patients with endometriosis and ovarian endometrioma.
Endometrial specimens and ovarian endometrioma were obtained from 15 women with endometriosis undergoing laparoscopic surgery. The stromal cells of the eutopic endometrium and ovarian endometroma were cultured in the presence of the GnRH agonist (leuprolide acetate 0, 1, 5 and 10 microM) for 24 h. To investigate the effects of the GnRH agonist on the eutopic endometrium in vivo, biopsy samples of the endometrium (n = 5) among the patients who underwent laparoscopy were obtained after GnRH agonist therapy. The protein production of aromatase cytochrome P450 and COX-2 was examined by Western blot.
Proteins of aromatase P450 and COX-2 were reduced in the eutopic endometrium of patients with endometriosis treated with the GnRH agonist for 3 months. The stromal cells in the culture of endometrial explants and ovarian endometrioma which were treated with the GnRH agonist reduced the aromatase P450 and COX-2.
The GnRH agonist reduced aromatase P450 and COX-2 by direct action on the eutopic endometrium of patients with endometriosis and ovarian endometrioma.
The potential roles of GnRH I and GnRH II have been assigned in promoting the invasive capacity of human trophoblasts by regulating matrix metalloproteinases-2 and -9, type I tissue inhibitor of matrix metalloproteinase, and urokinase plasminogen activator/plasminogen activator inhibitor protease systems during human placentation, and GnRH II has been shown to be more potent than GnRH I. However, the mechanisms for the differential effects of these two hormones remain unclear. In this study, we examined the invasion-promoting effects and the signaling pathways of GnRH I and GnRH II in human trophoblasts. The data revealed that both GnRH I and GnRH II were key autocrine and/or paracrine regulators in facilitating trophoblast invasion. The GnRH receptor antagonist (Antide) and specific small interfering RNA for GnRH receptor inhibited the regulatory effects of GnRH I, but not GnRH II, on trophoblast invasion. Both GnRH I and II activated protein kinase C, ERK1/2, and c-Jun N-terminal kinase to mediate their effects on trophoblast invasion, whereas only GnRH II elicited invasion-promoting action through transactivating the tyrosine kinase activity of epidermal growth factor receptor in trophoblasts. Our observations elucidate a ligand-dependent selective cross-communication between GnRH receptor and epidermal growth factor receptor signaling systems in human trophoblastic cell, and this would further our understanding on the differentially biological significance of these two forms of GnRH in extrapituitary tissues.
Gonadotropin-releasing hormone type II (GnRH-II) has an antiproliferative effect on human endometrial cancer cells. Apoptosis in cancer cells may play a critical role in regulating cell proliferation. However, more studies are necessary to elucidate the underlying molecular mechanisms and develop potential applications of GnRH-II. Therefore, we explored the mechanisms of GnRH-II-induced apoptosis and the effects of GnRH-II on GADD45alpha activation in human endometrial cancer cell lines. GnRH-II decreased cell viability in a dose- and time-dependent manner. Apoptosis was induced with increased terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling apoptotic cells after GnRH-II treatment. Knockdown of the endogenous GnRH-I receptor with small interfering RNA (siRNA) rescued the cells from GnRH-II-mediated cell growth inhibition and abolished the induction of apoptosis. GnRH-II activated extracellular signal-regulated kinase (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) in a time-dependent manner, and the activation was abolished by GnRH-I receptor siRNA and MAPK inhibitors. Cells pretreated with MAPK inhibitors were rescued from GnRH-II-mediated cell growth inhibition. Moreover, both inhibitors abolished GnRH-II-induced apoptosis. GnRH-II induced GADD45alpha expression, which was abolished by knockdown of endogenous GnRH-I receptors and MAPK inhibitors. GnRH-II-stimulated cell growth inhibition was rescued by knockdown of endogenous GADD45alpha with siRNA. Cells treated with GADD45alpha siRNA were refractory to GnRH-II-induced apoptosis. Thus, GnRH-II inhibits cell growth by inducing apoptosis through binding of the GnRH-I receptor, activation of the ERK1/2 and p38 MAPK pathways, and induction of GADD45alpha signaling. This finding may provide a new concept relating to the mechanism of GnRH-II-induced antiproliferation and apoptosis in endometrial cancer cells, indicating the possibility of GnRH-II as a promising therapeutic intervention for human endometrial cancer.
Type I GnRH (GnRH-I, GNRH1) and type II GnRH (GnRH-II, GNRH2), each encoded by separate genes, have been identified in humans. The tissue distribution and functional regulation of GnRH-I and GnRH-II clearly differ despite their comparable cDNA and genomic structures. These hormones exert their effects by binding to cell surface transmembrane G protein coupled receptors and stimulating the Gq/11 subfamily of G proteins. The hypothalamus and pituitary are the main origin and target sites of GnRH, but numerous studies have demonstrated that extra-hypothalamic GnRH and extra-pituitary GnRH receptors exist in different reproductive tissues such as the ovary, endometrium, placenta, and endometrial cancer cells. In addition to endocrine regulation, GnRH is also known to act in an autocrine and paracrine manner to suppress cell proliferation and activate apoptosis in the endometrium and endometrial cancer cells through several mechanisms. Both GnRH-I and GnRH-II exhibit regulatory roles in tissue remodelling during embryo implantation and placentation, which suggests that these hormones may have important roles in embryo implantation and early pregnancy. The presence of varied GnRH and GnRH receptor systems demonstrate their different roles in distinct tissues using dissimilar mechanisms. These may result in the generation of new GnRH analogues used for several hormone-related diseases.
GnRH-II has been shown to exert a strong antiproliferative action on tumors of the female reproductive system. The data so far reported on the effects of GnRH-II on prostate cancer growth are controversial. Moreover, it is still unclear through which receptor [type I or type II GnRH-receptor (GnRH-R)] GnRH-II might modulate cancer cell proliferation.
The objective of this work was to investigate whether GnRH-II might affect the proliferation of prostate cancer cells and to identify the GnRH-R through which the peptide might exert its activity.
We investigated the effects of GnRH-II on prostate cancer cell proliferation. We then transfected PC3 cells with a small interfering RNA targeted to type I GnRH-R. After receptor silencing we evaluated the effects of GnRH-II on cell proliferation and on forskolin-induced intracellular cAMP accumulation. Similar experiments were performed by silencing type II GnRH-R.
GnRH-II exerted an antiproliferative activity on prostate cancer cells. Transfection of PC3 cells with a type I GnRH-R small interfering RNA resulted in a significant decrease of the expression of this receptor. After type I GnRH-R silencing: 1) the antiproliferative effect of GnRH-II was completely abrogated; and 2) GnRH-II lost its capacity to counteract the forskolin-induced cAMP accumulation. On the contrary, type II GnRH-R silencing did not counteract the antiproliferative effect of GnRH-II.
GnRH-II exerts a specific and significant antiproliferative action on prostate cancer cells. This antitumor effect is mediated by the activation of type I (but not of type II) GnRH-R and by its coupled cAMP intracellular signaling pathway.
This study presents the results of fresh and frozen-thawed embryo transfers in women undergoing IVF with gonadotrophin-releasing hormone (GnRH) agonists and GnRH antagonists. By evaluating cycle outcomes, the impact of two different protocols on the endometrium was indirectly evaluated. For 714 women, embryos were frozen following day-3 fresh embryo transfer and the outcome of those fresh cycles (329 agonist cycles and 290 antagonist cycles) and subsequent frozen-thawed embryo transfer (91 agonist cycles and 104 antagonist cycles) were evaluated. Peak oestradiol concentrations of both groups were similar; however, significantly more oocytes were retrieved and more embryos frozen in the agonist versus antagonist group (both P = 0.0001). In fresh embryo transfer cycles, implantation and pregnancy rates in the agonist versus antagonist group were 42.3% versus 32.0% (P = 0.0001) and 68.6% versus 58.2% (P = 0.009) respectively. However, neither implantation or pregnancy rate significantly differed among frozen-thawed embryo transfer cycles between the two groups (21.4% versus 23.5% and 52.2% versus 52.4% respectively). These results suggest that ovarian stimulation parameter outcomes of GnRH antagonist cycles were not inferior to GnRH agonist cycles, therefore reduced embryo implantation and pregnancy rates in GnRH antagonist cycles can be attributable to possible deleterious effects on the endometrium.
Several studies have previously reported the expression of the gonadotropin-releasing hormone receptor (GnRHr) in cases of endometrial cancer. However, the relationship between GnRHr expression and a variety of clinicopathologic parameters remains unclear. This study was conducted with 141 endometrial cancer patients, all of whom had undergone operations between 1993 and 2002. Paraffin-embedded tissue blocks were sectioned and immunostained with monoclonal anti-GnRHr antibody. Clinicopathologic variables were also evaluated, with 10% cutoff values for GnRHr positivity. Seventy specimens (49.6%) stained as GnRHr-positive. Mean parity was higher in the patients with GnRHr-positive tumors than those with GnRHr-negative tumors (2.50+/-1.92 versus 1.82+/-1.37, P=0.016). Body mass indices were also higher in the patients with GnRHr-positive tumors (26.6+/-4.6 versus 24.7+/-4.2, P=0.010). However, GnRHr positivity was not determined to be statistically significantly associated with any other clinicopathologic characteristics, including age, menopausal status, histotype, disease stage, tumor differentiation, lymph node metastasis, and myometrial invasion. The results of this study, although they may require further investigation, suggested that obese and multiparous women with endometrial cancer might be greatly influenced by endogenous gonadotropin-releasing hormone and/or exogenous gonadotropin-releasing hormone analogs.
More than 80% of human ovarian cancers express LHRH and its receptor. The proliferation of human ovarian cancer cell lines is reduced by both LHRH agonists and antagonists. This study was designed to further clarify the possible biological function of this LHRH system.
As LHRH agonists and antagonists uniformly reduce proliferation of human ovarian cancer in a dose-dependent way, the effect of low concentrations of authentic LHRH was studied. In addition, longer periods of treatment (up to 9 days) were analyzed. To assess the physiological role of LHRH produced by ovarian cancer cells it was neutralized by adequate concentrations of a specific LHRH antiserum.
Human ovarian cancer cells EFO-21 and EFO-27, which express LHRH and its receptor, were incubated for 1-9 days with increasing concentrations (1pmol/l to 10 micromol/l) of authentic LHRH or with concentrations of LHRH antiserum capable of neutralizing at least 1nmol/l LHRH. Proliferation was assessed by counting cells.
Authentic LHRH reduced time- and dose-dependently proliferation (by maximally mean+/-s.e.m. 32.7 +/- 4.4%, Newman-Keuls, P < 0.001) of both ovarian cancer cell lines. At very low concentrations (1pmol/l) a marginal reduction of proliferation or no effect was observed. A mitogenic effect of authentic LHRH was never detected. Treatment of ovarian cancer cell cultures with antiserum to LHRH significantly increased (up to mean+/-s.e.m. 121.0 +/- 2.8% of controls, Newman-Keuls P <0.001) proliferation of EFO-21 and EFO-27 cells. These findings suggest that LHRH produced by human ovarian cancer cells might act as a negative autocrine regulator of proliferation.
The expression of GnRH and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary and endometrium. Dose-dependent antiproliferative effects of GnRH agonists in cell lines derived from these cancers have been observed by various investigators. GnRH antagonists also have marked antiproliferative activity in most breast, ovarian and endometrial cancer cell lines tested, indicating that the dichotomy of GnRH agonists and antagonists might not apply to the GnRH system in cancer cells. The classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of GnRH analogs in cancer cells. Rather, the GnRH receptor interacts with 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 downregulation of cancer cell proliferation. In addition, GnRH activates nuclear factor kappaB and protects the cancer cells from apoptosis. Furthermore, GnRH induces activation of the c-Jun N-terminal kinase/activator protein-1 (AP-1) pathway independent of the known AP-1 activators, protein kinase or mitogen activated protein kinase.
We have cloned and characterized two genomic loci encoding the human type II gonadotropin-releasing hormone (GnRH) receptor and RNA-binding motif protein-8 (RBM8A). In both loci the genes overlap and are in antisense orientation to each other. The locus on chromosome 1 encompasses the type II GnRH receptor gene (GNRHR2), which is composed of three exons. We found transcripts from this gene in a wide range of tissues, but they lacked a methionine initiation codon and had a stop codon in exon 2. In the antisense orientation, this locus contains RBM8A, which consists of six exons and directs the synthesis of an RBM8A protein of 173 or 174 amino acids by alternative splicing. A second locus on chromosome 14 contains pseudogenes of RBM8A and the type II GnRH receptor and probably originated from the chromosome 1 locus by retrotransposition.
The expression of LHRH and its receptor has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary, endometrium, and prostate. These findings suggest the presence of an autocrine regulatory system based on LHRH. Dose-dependent antiproliferative effects of LHRH agonists in cell lines derived from these cancers have been observed by various investigators. LHRH antagonists also have marked antiproliferative activity in most of the ovarian, breast, and endometrial cancer cell lines tested, indicating that the dichotomy of LHRH agonists and antagonists might not apply to the LHRH system in cancer cells. Findings from our laboratories suggest that the classical LHRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of LHRH analogues in cancer cells. Results obtained by several groups, including ours, instead suggest that LHRH analogues interfere with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity. The pharmacological exploitation of these direct antiproliferative actions of LHRH analogues might provide new therapeutic approaches to these cancers.