The neuropeptides GnRH-II and GnRH-I are produced by human T cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs.

Can T cells be directly activated to de novo gene expression by gonadotropin-releasing hormone-II (GnRH-II), a unique 10-amino-acid neuropeptide conserved through 500 million years of evolution? GnRH-II, which has been identified in mammals, shares 70% homology with the mammalian hypothalamic neurohormone GnRH (GnRH-I), the primary regulator of reproduction, but is encoded by a different gene. Although both neuropeptides are produced mainly in brain, their localization and promoter regulation differ, suggestive of distinct functions. Indeed, GnRH-II barely affects reproduction and its role in mammalian physiology is unknown. We find here that human normal and leukemic T cells produce GnRH-II and GnRH-I. Further, exposure of normal or cancerous human or mouse T cells to GnRH-II or GnRH-I triggered de novo gene transcription and cell-surface expression of a 67-kD non-integrin laminin receptor that is involved in cellular adhesion and migration and in tumor invasion and metastasis. GnRH-II or GnRH-I also induced adhesion to laminin and chemotaxis toward SDF-1alpha, and augmented entry in vivo of metastatic T-lymphoma into the spleen and bone marrow. Homing of normal T cells into specific organs was reduced in mice lacking GnRH-I. A specific GnRH-I-receptor antagonist blocked GnRH-I- but not GnRH-II-induced effects, which is suggestive of signaling through distinct receptors. We suggest that GnRH-II and GnRH-I, secreted from nerves or autocrine or paracrine sources, interact directly with T cells and trigger gene transcription, adhesion, chemotaxis and homing to specific organs, which may be of clinical relevance.

Local production of the gonadotropic hormones in the rat ovary.

The gonadotropic hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are synthesized by and released from the anterior pituitary in response to the hypothalamic gonadotropin-releasing hormone (GnRH) signaling. In the female, LH and FSH affect folliculogenesis, ovarian steroid production, oocyte maturation, ovulation and corpus luteum formation. We have recently studied the expression of GnRH and its receptor in the rat ovary and found organ-specific, estrous cycle-dependant, fluctuations. Subsequently, we wished to determine whether rat ovaries also express gonadotropic hormones. Using RT-PCR, we detected LHbeta, FSHbeta and the common alpha-subunit mRNA's in intact follicles, theca cells, corpora lutea and in meiotically competent and incompetent oocytes. Granulosa cells, however, express mRNA's for LHbeta and the common alpha-subunit, but not for FSHbeta. We cloned and sequenced the ovarian LHbeta transcript and found it to be longer (2.3kb) than the one produced by pituitary gonadotropes (0.8kb), due to a longer 5'-UTR. We studied the regulation of ovarian LHbeta mRNA in sexually immature female rats administered with pregnant mare serum gonadotropin (PMSG) and in adult cyclic rats. PMSG administration caused a significant decrease in LHbeta mRNA expression, detected by real-time PCR. Similarly, LHbeta mRNA levels were lower on estrous morning versus proestrous evening. Interestingly, ovarian content of LH remained unchanged following hypophysectomy, although ovarian weight was immensely reduced. Taken together, it seems probable that ovarian LH is heterologously/homologously regulated by pituitary, and possibly also by local gonadotropins. Thus, these findings may imply the existence of a local GnRH-gonadotropin axis in the mammalian ovary that may be involved in the management of processes that lead to ovulation.

Conjugates of gonadotropin releasing hormone (GnRH) with carminic acid: Synthesis, generation of reactive oxygen species (ROS) and biological evaluation.

We synthesized two carminic acid (7-alpha-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracene carboxlic acid, CA)-GnRH conjugates to be used as a model for potential photoactive targeted compounds. CA was conjugated to the epsilon-amino group of [d-Lys(6)]GnRH through its carboxylic moiety or via a beta-alanine spacer (beta-ala). Redox potentials of CA and its conjugates were determined. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of CA and its CA-GnRH conjugates. Upon irradiation, the compounds stimulated the formation of reactive oxygen species (ROS), that is, singlet oxygen ((1)O(2)) and oxygen radicals (O(2)(-*) and OH(*)). Both conjugates exhibited higher ROS production than the non-conjugated CA. The bioactivity properties of the CA conjugates and the parent peptide, [d-Lys(6)]GnRH, were tested on primary rat pituitary cells. We found that the conjugates preserved the bioactivity of GnRH as illustrated by their capability to induce ERK phosphorylation and LH release.

Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat.

We recently described patterns of GnRH and GnRH receptor (GnRH-R) expression in the hypothalamus, pituitary and ovary throughout the rat estrus cycle. Here, we wished to distinguish between regulatory effects of ovarian factors and underlying circadian rhythmicity. We quantified GnRH and GnRH-R mRNA in the pituitary and hypothalamus of long-term ovariectomized (OVX) rats, at different times of day, using real-time PCR. Furthermore, we expanded our previous study of hypothalamic and pituitary GnRH and GnRH-R expression in intact rats by including more time points throughout the estrus cycle. We found different daily patterns of GnRH and GnRH-R expression in intact versus OVX rats, in both tissues. In the hypothalamus of OVX rats, GnRH mRNA peaked at 12, 16 and 20 h, whereas in the hypothalamus of intact rats we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred at 17 h. In this tissue, GnRH-R fluctuated less significantly and peaked at 16 h in OVX rats. During the estrus cycle, we observed higher levels in the afternoon of each day except on estrus. In OVX rats, pituitary GnRH mRNA rose sharply at 9 h, with low levels thereafter. In these animals, pituitary GnRH-R also peaked at 9h followed by a second rise at 22 h. In intact rats pituitary GnRH was high at noon of diestrus-II and on estrus, whereas GnRH-R mRNA was highest in the evening of diestrus-II. This is the first demonstration of daily GnRH and GnRH-R mRNA expression patterns in castrated animals. The observed daily fluctuations hint at underlying tissue-specific circadian rhythms. Ovarian factors probably modulate these rhythms, yielding the observed estrus cycle patterns.

Two forms of gonadotropin-releasing hormone (GnRH) are expressed in human breast tissue and overexpressed in breast cancer: a putative mechanism for the antiproliferative effect of GnRH by down-regulation of acidic ribosomal phosphoproteins P1 and P2.

Gonadotropin-releasing hormone (GnRH) analogues are used for the treatment of breast, prostate, and ovarian cancer. These analogues exert their antitumor effects indirectly by inhibiting the pituitary-gonadal axis, as well as by direct inhibition of proliferation of human breast cancer cells. However, the molecular mechanisms mediating these direct antiproliferative effects are not fully understood. We found that normal and malignant human breast tissue express two forms of the neuropeptide GnRH. Quantitative reverse transcription-PCR shows that mRNA encoding the GnRH-I and GnRH-II peptides are overexpressed in cancerous versus normal tissues obtained from the same patients. To elucidate the function of these peptides in breast cancer cells, we used the atlas human cDNA expression arrays technology and studied the differentially regulated genes after GnRH treatment of MCF-7 cells. We found that a wide range of GnRH-I or GnRH-II concentrations (0.1-10 nM) inhibit the expression of mRNA encoding the 60S acidic ribosomal phosphoproteins, P1 and P2. These results were confirmed by quantitative reverse transcription-PCR, as well as Western blot analysis and immunofluorescence staining. The P1 and P2 proteins interact with elongation factors EF1 and EF2, and the level of their phosphorylation is one of the regulatory mechanisms for the overall rate of protein elongation. Thus, reduced expression of P1 and P2 proteins can affect the rate of protein translation, thereby decreasing proliferation rate of cells. Our studies may therefore suggest a putative mechanism for the direct antiproliferative effect of GnRH in breast cancer cells.

Differential gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acid expression patterns in different tissues of the female rat across the estrous cycle.

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.

Novel methyl helianthrones as photosensitizers: synthesis and biological evaluation.

A combination of light, oxygen and a photosensitizer is used to induce death of cancer cells by photodynamic therapy. In this study, we have synthesized several new methyl helianthrone derivatives and compared their phototoxicity with that of hypericin. In contrast to hypericin, methyl helianthrones are soluble in aqueous solutions and have a broad range of light absorbance, which allows the use of polychromatic light. Structural modifications of methyl helianthrone demonstrated that substitution of hydrogen atoms of methyl helianthrone at Positions 2 and 5 with Br atoms or methylation of its phenolic hydroxyls, significantly increases the corresponding singlet oxygen quantum yield and their phototoxicity toward alphaT3-1, M2R and LNCaP cells. The phototoxicity of some of these compounds was similar to that of hypericin. Methyl helianthrones, like hypericin, accumulated mainly in the perinuclear region as evident by confocal microscopy. Irradiation of cells pretreated with methyl helianthrone derivatives generates intracellular reactive oxygen species and lipid free radicals, as shown by a fluorescentic probe and electron paramagnetic resonance methods, respectively. The phototoxicity of these methyl helianthrones as well as their ability to oxidize membrane lipids were significantly decreased on addition of specific Type-II inhibitors, suggesting the involvement of singlet oxygen as the main oxidant.

Receptor-mediated targeting of a photosensitizer by its conjugation to gonadotropin-releasing hormone analogues.

Photodynamic therapy uses a combination of light, oxygen, and a photosensitizer to induce the death of malignant cells. To improve the selectivity of a photosensitizer toward cancerous cells that express gonadotropin-releasing hormone (GnRH) receptors, protoporphyrin IX (PpIX) was conjugated to a GnRH agonist, [d-Lys6]GnRH, or to a GnRH antagonist, [d-pGlu1, d-Phe2, d-Trp3, d-Lys6]GnRH. The condensation of the peptide with PpIX was carried out in a homogeneous solution using benzotriazole-1-yloxytris(pyrrolidinophosphonium) hexafluorophosphate as a coupling reagent. Although these conjugates had lower binding affinity to rat pituitary GnRH receptors than their parent analogues, they fully preserved their agonistic or antagonistic activity in vitro and in vivo. The GnRH agonist conjugate proved to be long-acting in vivo. Thus, 24 h after its administration to rats (2 nmol/rat), serum LH concentrations were significantly higher than in rats treated with the same amount of the parent peptide. The conjugates, notably the agonist, were more phototoxic toward pituitary gonadotrope alphaT3-1 cell line than was unconjugated PpIX. In contrast to PpIX, the phototoxicity of the conjugates toward alphaT3-1 cells or to human breast cancer cells (MCF-7 cells that were transfected with human GnRH receptors) was alleviated by co-incubation with the parent peptide, indicating that phototoxicity is receptor-mediated. The selectivity of the GnRH antagonist conjugate to gonadotrope cells in a primary pituitary culture was approximately 10 times higher than that of the unconjugated PpIX. Thus, GnRH-based conjugates may affect cancer cells not only by acting as classic GnRH analogues to reduce the plasma levels of steroids by desensitization of the pituitary gland but also by selective photodamage of cells that express GnRH receptors.

The transcription of the hGnRH-I and hGnRH-II genes in human neuronal cells is differentially regulated by estrogen.

Gonadotropin releasing hormone-I (GnRH-I), a decapeptide serves as a key regulator of reproduction. Recently, several groups have identified in the mammalian brain a second form of GnRH, of unknown function, designated GnRH-II. The human neuronal medulloblastoma cells (TE-671) were recently demonstrated to express the two forms of GnRH (GnRH-I and GnRH-II). We used this cell line, as a model system, to investigate the regulation of human GnRH-I and GnRH-II genes by estrogen. Estrogen is one of the principal regulators of GnRH-I in hypothalamic neurons, acting as a classic homeostatic feedback molecule between the gonads and the brain. In this study, we investigated the regulation of the two GnRH forms by estrogen, in the human neuronal cell line TE-671. We demonstrate, for the first time, that the hGnRH-II and hGnRH-I genes are differentially regulated by estrogen. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern hybridization, we found that estrogen increases endogenous hGnRH-II mRNA levels and decreases endogenous hGnRH-I mRNA levels. Furthermore, we found these effects to be promoter-mediated. We cloned the hGnRH-I and hGnRH-II promoter constructs upstream to a luciferase reporter plasmid, and cotransfected these constructs with an estrogen receptor alpha into the TE-671 neuronal cells. Luciferase activity of GnRH promoter constructs treated with estrogen demonstrates that the differential regulation of the GnRH genes by estrogen is mediated at the transcription level.

Backbone metal cyclization: novel 99mTc labeled GnRH analog as potential SPECT molecular imaging agent in cancer.

Gonadotropin-releasing hormone (GnRH) is a decapeptide secreted to the pituitary where it binds to specific receptors on the gonadotropes to regulate gonadotropic hormones (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) synthesis and secretion. Specific GnRH receptors are overexpressed in breast, prostatic, ovarian, and other tumors. The aim of this study was to synthesize a cyclic GnRH analog with high affinity to GnRH receptors that can be radiolabeled with 99mTc. A precyclic GnRH analog, [Cys-Gly]1[D-Ala]6[N(alpha)(eta-Cys-amino hexyl)]10GnRH (Gn-2), containing two hemi-chelator groups was synthesized. It was cyclized applying the recently reported backbone metal cyclization (BMC) approach, to obtain cyclo(Re(O)1-10)[Cys-Gly]1[D-Ala]6[N(alpha)(eta-Cys-amino hexyl)]10GnRH (cyclo[Re(O)-Gn-2]). For comparative evaluations, Gn-2 was oxidized on-resin to yield cyclo(S-S,1-10)[Cys-Gly]1[D-Ala]6[N(alpha)(eta-Cys-amino hexyl)]10GnRH, (cyclo[S-S-Gn-2]). The binding affinity of cyclo[Re(O)-Gn-2] to rat pituitary membranes showed IC50 of 50 nM, compared to IC50 = 10 nM in the native GnRH. Cyclo(99mTc(O)1-10)[Cys-Gly]1[D-Ala]6[N(alpha)(eta-Cys-amino hexyl)]10GnRH (cyclo[99mTc(O)-Gn-2]) was synthesized from Gn-2 and showed similar chromatographic behavior to its rhenium surrogate.

Hormonal regulation of GnRH and LHbeta mRNA expression in cultured rat granulosa cells.

We have recently demonstrated that the rat ovary expresses LHbeta, FSHbeta, and the common alpha subunit mRNA. In the present report, we studied the regulation of LHbeta and of gonadotropin-releasing hormone (GnRH) mRNA expression in granulosa cells that were isolated from immature rats treated with either estrogen or pregnant mare serum gonadotropin (PMSG). In both cell types, GnRH agonist treatment resulted in a decrease in LHbeta mRNA expression. However, only in cells derived from PMSG-treated rats, GnRH treatment produced an increase in GnRH mRNA expression. A markedly increased GnRH mRNA expression was also obtained in granulosa cells derived from PMSG-primed rats in response to LH. In addition, FSH reduced the expression of LHbeta mRNA in granulosa cells from estrogen-primed rats. These results suggest that the expression of LHbeta in the ovary is regulated by locally produced GnRH and by FSH from either the ovary or the pituitary.

Synthesis and active oxygen generation by new emodin derivatives and their gonadotropin-releasing hormone conjugates.

In an attempt to develop efficient chemotherapeutic agents targeted at malignant cells that express receptors, we synthesized five new emodin derivatives and their gonadotropin-releasing hormone (GnRH) conjugates to be used as potential photoactive conjugates. Emodin was modified at its hydroxy groups and included different spacers for conjugation of the peptide. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of the emodin derivatives and their GnRH conjugates. Upon irradiation, all new emodin derivatives and their conjugates stimulated the formation of singlet oxygen, that is, (1)O(2), and oxygen radicals, that is, O(2)(-)(*) and OH(*). However, substantial differences were found between the tested derivatives as to the efficacy of reactive oxygen species (ROS) production. Because of its superior ROS production properties, [d-Lys(6)(MeoEmo)]GnRH was selected as a leading conjugate. En-route to evaluate its targeting capacity, this potentially cytotoxic conjugate was tested in vitro to determine its hormonal activity and binding affinity to GnRH receptors.

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.

beta-Sulfonamido gonadotropin-releasing hormone analogs: synthesis and evaluation of several parent hormone properties.

With the aim of producing long-acting analogs of gonadotropin releasing hormone (GnRH), four analogs, containing -X(6) (aa)psi(CH(2)SO(2)NH)-Leu(7) building unit (X(aa)=Gly, Ala, Val or Phe), and a reduced-size analog [Des-Tyr(5)]-GnRH which includes the unit Phe(5)psi(CH(2)SO(2)NH)-Leu(6), and [beta-Ala(6)]-GnRH were synthesized. The peptides were evaluated for their capacity to induce LH-release from rat pituitary cells and to withstand proteolysis by pituitary-derived enzymes, compared with the parent peptide GnRH. Albeit stable toward enzymatic degradation, the sulfonamido containing peptides were only marginally bioactive. [beta-Ala(6)]-GnRH, however, induced LH-release and bound to pituitary receptors nearly as efficiently as GnRH. This analog was also highly stable toward proteolysis suggesting that it may serve as a long-acting GnRH-analog.

Chemical and photochemical electron transfer of new helianthrone derivatives: aspects of their photodynamic activity.

Helianthrones 2-4 are a new class of synthetic photosensitizers, which have a molecular skeleton related to that of hypericin. We established that irradiation of heliantrones with visible light leads to the formation of semiquinone radicals and reactive oxygen species. The structures of the paramagnetic anion species produced by electron transfer were calculated on the density functional level and investigated by cyclovoltammetry, UV/vis, and EPR/ENDOR spectroscopy. As with hypericin, the pi system of the helianthrones was found to be considerably deviated from planarity, and, upon electron transfer, deprotonation in the bay region occurs. The structure of the semiquinone radicals was found to be identical in THF, DMF, and aqueous buffered solutions regardless of the means by which reduction was achieved. Semiquinone radicals can be formed via self-electron transfer between the excited state and the ground state or via electron transfer from an electron donor to the excited state of helianthrone. Therefore, the presence of an electron donor significantly enhanced the photogeneration of semiquinone and superoxide radical. The kinetic studies showed that no significant photochemical destruction of helianthrones occurred upon irradiation. Generation of superoxide and singlet oxygen upon irradiation of helianthrones was established by spin trapping techniques. This shows that both type I and type II mechanisms are of importance for the photodynamic action of these compounds.