Jian-Pi-Yi-Shen decoction inhibits mitochondria-dependent granulosa cell apoptosis in a rat model of POF.

As a widely applied traditional Chinese medicine (TCM), Jian-Pi-Yi-Shen (JPYS) decoction maybe applied in curing premature ovarian failure (POF) besides chronic kidney disease (CKD). In vivo experiments, 40 female SD (8-week-old) rats were randomized into four groups, namely, control group (negative control), POF model group, JPYS treatment group, and triptorelin treatment group (positive control). JPYS group was treated with JPYS decoction (oral, 11 g/kg) for 60 days, and the triptorelin group was treated with triptorelin (injection, 1.5 mg/kg) for 10 days before the administration of cyclophosphamide (CTX) (50 mg/kg body weight) to establish POF model. We examined apoptosis, mitochondrial function, and target gene (ASK1/JNK pathway and mitochondrial fusion/fission) expression. In vitro experiments, the KGN human granulosa cell line was used. Cells were pretreated with CTX (20, 40, and 60 µg/mL) for 24 h, followed by JPYS-containing serum (2, 4, and 8 %) for 24 h. Thereafter, these cells were employed to assess apoptosis, mitochondrial function, and target gene levels of protein and mRNA. In vivo, JPYS alleviated injury and suppressed apoptosis in POF rats. In addition, JPYS improved ovarian function. JPYS inhibit apoptosis of granulosa cells through improving mitochondrial function by activating ASK1/JNK pathway. In vitro, JPYS inhibited KGN cell apoptosis through inhibited ASK1/JNK pathway and improved mitochondrial function. The effects of GS-49977 were similar to those of JPYS. During POF, mitochondrial dysfunction occurs in the ovary and leads to granulosa cell apoptosis. JPYS decoction improves mitochondrial function and alleviates apoptosis through ASK1/JNK pathway.

Development of a (68)Ga-peptide tracer for PET GnRH1-imaging.

OBJECTIVE: Total synthesis, quality control and preclinical evaluation of [(68)Ga]-DOTA-triptorelin ([(68)Ga]-DOTA-TRP) is reported as a possible PET radiotracer for GnRH receptor imaging. METHODS: DOTA-TRP was totally synthesized in two steps and after characterization went through radiolabelling optimization studies followed by tracer stability. The biodistribution of the tracer in normal male rats and 4T1 tumour-bearing mice was performed in 120 min after i.v. injection. RESULTS: The peptide and the conjugates were synthesized with >95 % chemical purity. [(68)Ga]-DOTA-TRP complex was prepared in high radiochemical purity (>99 %, ITLC, HPLC) and specific activity of 1400-2100 MBq/nM at 95 °C using 40-60 µg of the peptide in 5-7 min followed by solid phase purification. The IC50 [nM] DOTA-TRP was comparable to the intact peptide, 0.11 ± 0.01 and 0.22 ± 0.05, respectively. The biodistribution of the tracer demonstrated kidney, stomach, and testes significant uptake, all in accordance with GnRH receptor ligands. Significant tumour uptake was observed in 4T1 tumour-bearing female mice 30-120 min post-injection with tumour:blood and tumour:muscle ratios of 28 and >50 in 60 min, respectively. Kidney is rapidly washed from the tracer. [(68)Ga]-DOTA-TRP can be proposed as a possible tracer for GnRH-R imaging studies.

Atomic view of the histidine environment stabilizing higher-pH conformations of pH-dependent proteins.

External stimuli are powerful tools that naturally control protein assemblies and functions. For example, during viral entry and exit changes in pH are known to trigger large protein conformational changes. However, the molecular features stabilizing the higher pH structures remain unclear. Here we elucidate the conformational change of a self-assembling peptide that forms either small or large nanotubes dependent on the pH. The sub-angstrom high-pH peptide structure reveals a globular conformation stabilized through a strong histidine-serine H-bond and a tight histidine-aromatic packing. Lowering the pH induces histidine protonation, disrupts these interactions and triggers a large change to an extended ß-sheet-based conformation. Re-visiting available structures of proteins with pH-dependent conformations reveals both histidine-containing aromatic pockets and histidine-serine proximity as key motifs in higher pH structures. The mechanism discovered in this study may thus be generally used by pH-dependent proteins and opens new prospects in the field of nanomaterials.

Oxytocin and vasopressin secretion from the rat hypothalamo-neurohypophysial system is stimulated by triptorelin.

INTRODUCTION: Several observations have suggested that the secretion of neurohypophysial hormones could be modified by gonadotropin- releasing hormone (GnRH). Since, in medical practice, more often than GnRH itself, its analogues are used, the present study was undertaken to investigate the influence of the GnRH agonist - triptorelin on oxytocin (OT) and vasopressin (AVP) release from the rat hypothalamo-neurohypophysial (H-N) system both in vitro and in vivo. MATERIALS AND METHODS: Male rats served as donors of the H-N explants, which were placed in 1 mL of Krebs-Ringer fluid (nKRF) and incubated successively in: 1 - nKRF (B1); 2 - incubation fluid as B1 enriched with an excess amount (56 mM) of K(+) (S1); 3 - incubation fluid as B1 enriched with an appropriate concentration of triptorelin, i.e., 10(-11) - 10(-5) M (B2); and 4 - incubation fluid as S1 enriched with the same concentrations of triptorelin (S2). After 20 minutes of incubation, each medium (B1, S1, B2, S2) was collected and frozen before OT and AVP estimation by the RIA. During in vivo experiment, animals were infused intracerebroventricularly (icv) with triptorelin, at a concentration of 10(-7) M, and 20 minutes later they were decapitated. The neurohypophysis was dissected from the brain and blood plasma samples were collected and frozen for further OT and AVP RIA assays. RESULTS: The GnRH agonist - triptorelin stimulates both OT and AVP release from isolated H-N system at concentrations of 10(-9)-10(-5) M. The strongest effect was displayed by triptorelin at a concentration of 10(-7) M. Under the conditions of K(+) stimulation, triptorelin affects neither OT, nor AVP secretion in vitro. When infused icv, triptorelin, at a concentration of 10(-7) M, significantly stimulated both OT and AVP secretion into the blood. CONCLUSIONS: Triptorelin may play a role as a neuromodulator contributing to the functional regulation of OT and AVP secretion in the rat.

Stability of triptorelin in the presence of dermis and epidermis.

An important issue with respect to the transdermal delivery of peptides is their stability during transit through the epidermis and dermis before entry into the systemic circulation. The objective of the present study was to evaluate the effect of epidermal and dermal tissue on the stability of the luteinizing hormone releasing hormone superagonist, triptorelin. The decapeptide was dissolved in PBS (pH 7.4) and placed in contact with (i) heat separated epidermis (HSE), (ii) dermatomed skin (0.75 mm; DS) and (iii) full thickness skin (FTS) and the extent of peptide biotransformation monitored as a function of time by HPLC. The results showed that triptorelin was metabolized when in contact with each of the skin tissues. However, there were marked differences with respect to the extent of peptide degradation. Triptorelin was least stable in the presence of FTS. After 3 h exposure to HSE, DS and FTS, the extent of triptorelin degradation was 15.0+/-6.0%, 64.8+/-9.9% and 100%, respectively. After 24 h, further triptorelin degradation had occurred in the samples in contact with HSE and DS--with 51.3+/-6.0% and 87.8+/-4.4%, respectively, of the peptide being degraded. The chromatograms revealed the presence of a degradation peak at a higher retention time than the parent molecule--most probably the free acid.

Differential expression of LHRH-receptor in bovine nasal tissue and its role in deslorelin delivery.

Deslorelin, a luteinizing hormone releasing hormone (LHRH) agonist, is transported via the LHRH-receptor (LHRH-R) and exhibits regional variation as follows: inferior turbinate posterior (ITP)>medium turbinate posterior (MTP)>medium turbinate anterior (MTA) of the bovine nasal mucosa. Differential LHRH-R expression in various regions of the nose is a potential explanation for regional variation in deslorelin transport. Thus, the objective was to determine whether LHRH-R expression exhibits regional variation in bovine nasal mucosa. LHRH-R density (B(max)) and affinity constant (K(d)) were determined by saturation experiments using 0.5mg tissue in the presence of increasing amounts of I(125)-deslorelin (100-100,000 cpm) at 4 degrees C for 4h. The 50% inhibitory concentration (IC(50)) was determined by competition experiments using various amounts of unlabelled deslorelin (0.01-3000 ng) at 4 degrees C for 4h. LHRH-R mRNA and protein expressions were determined using real-time PCR and Western blot analysis, respectively. LHRH-R B(max) and K(d) varied between the regions of excised bovine nasal mucosa: ITP>MTP>MTA. The inhibition experiments yielded two IC(50) concentrations which exhibited trends similar to B(max) and K(d). Real-time PCR and Western blot analysis indicated that LHRH-R expression exhibits similar trends: ITP>MTP>MTA. We identified two deslorelin binding sites in the nasal tissues, with high affinity sites representing approximately 60-70% of the total sites available. In summary, regional differences in nasal deslorelin transport correlate with regional differences in LHRH-R expression, with LHRH-R expression, peptide binding, and transport being the highest in the inferior turbinate posterior region of the nose.

Development of novel 68Ga- and 18F-labeled GnRH-I analogues with high GnRHR-targeting efficiency.

A large majority of tumors of the reproductive system express the gonadotropin releasing hormone receptor (GnRHR). Blockade and activation of this receptor with various antagonistic and agonistic analogues of native GnRH-I (pGlu(1)-His(2)-Trp(3)-Ser(4)-Tyr(5)-Gly (6)-Leu(7)-Arg(8)-Pro(9)-Gly(10)-NH2), respectively, has shown efficient suppression of tumor growth. In this study, the GnRH-receptor system has been evaluated with respect to its suitability as a target for in vivo peptide receptor targeting using radiolabeled GnRH-analogues, and in parallel, new (18)F- and (68Ga)-labeled GnRH analogues have been developed. In vitro radioligand binding assays performed with various GnRHR-expressing human cell lines using [(125)I]Triptorelin (D-Trp(6)-GnRH-I) as the standard radioligand revealed a very low level of GnRH receptor expression on the cell surface. Generally, total cellular activity was very low (approximately 3% of the applied activity), and only a small fraction (max. 40%) of cell-associated activity could be attributed to receptor-specific radioligand binding/internalization. However, substitution of fetal calf serum by NU serum in the culture medium led to increased and stable GnRHR-expression, especially in the ovarian cancer cell line EFO-27, thus allowing for a stable experimental setup for the evaluation of the new radiolabeled GnRH-I analogues. The new radiolabeled GnRH-I analogues developed in this study were all based on the D-Lys(6)-GnRH-I-scaffold. For (68)Ga-labeling, the latter was coupled with DOTA at D-Lys(6). To allow (18)F-labeling via chemoselective oxime formation, D-Lys(6)-GnRH-I was also conjugated with Ahx (aminohexanoic acid) or beta-Ala, which in turn was coupled with Boc-aminooxyacetic acid. (18)F-labeling via oxime formation with 4-[(18)F]fluorobenzaldehyde was performed using the Boc-protected precursors. Receptor affinities of [(68)Ga]DOTA-GnRH-I, D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I, and D-Lys(6)-betaAla([(18)F]FBOA)-GnRH-I (FBOA = fluorobenzyloxime acetyl) were determined using GnRHR-membrane preparations, and internalization efficiency of the new radioligands was determined in EFO-27 cells. Both quantities were highest for D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I (IC 50 = 0.50 +/- 0.08 nM vs 0.13 +/- 0.08 nM for Triptorelin; internalization: 86 +/- 16% of the internal reference [(125)I]Triptorelin), already substantially reduced in the case of the -betaAla([(18)F]FBOA)-derivative (IC 50 = 0.86 +/- 0.13 nM; internalization: 42 +/- 3% of [(125)I]Triptorelin), while the [(68)Ga]DOTA-analogue showed almost complete loss of binding affinity and ligand internalization (IC50 = 13.3 +/- 1.0 nM; internalization: 2.6 +/- 1.0% of [(125)I]Triptorelin). Generally, the lipophilic residue [(18)F]FBOA is much better tolerated as a modification of the D-Lys(6)-side chain, with receptor affinity of the respective analogues strongly depending upon spacer length between the D-Lys(6)-side chain and the [(18)F]FBOA-moiety. In summary, D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I shows the highest potential for efficient GnRHR-targeting in vivo of the compounds investigated. Unfortunately, however, the very low cell surface expression of GnRH-receptors and thus very low radioligand uptake by GnRHR-positive tumor cells found in vitro was also confirmed by a preliminary biodistribution study in OVCAR-3 xenografted nude mice using the standard GnRHR radioligand [(125)I]Triptorelin. Tumor uptake was lower than blood activity concentration at 1 h p.i. (0.49 +/- 0.05 vs 0.96 +/- 0.13 for tumor and blood, respectively). These data seriously challenge the suitability of the GnRHR-system as a suitable target for in vivo peptide receptor imaging using radiolabeled GnRH-I derivatives, despite the availability of high-affinity radiolabeled receptor-ligands such as D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I.

LHRH receptor targeted therapy for breast cancer.

Breast cancer remains the most common cancer among women, with an estimated 212,920 new cases and 40,970 deaths in the United States in 2006. The present work extends the studies of nanoparticles targeted to the luteinizing hormone-releasing hormone (LHRH) receptor which is overexpressed in breast, ovarian, endometrial and prostate cancer cells. In contrast, LHRH receptors are not expressed, or expressed at a low level in most visceral organs. In our studies, we conjugated Fe3O4 nanoparticles (20-30 nm) with [D-Trp6]LHRH (Triptorelin), a decapeptide analog of LHRH currently used for treatment of sex-hormone-dependent tumors. Conjugation of [D-Trp6]LHRH to Fe3O4 particles retained its binding affinity and biological activity for the LHRH receptor. Treatment of two separate breast tumor cell lines (MCF-7 and MDA-MB231) with these conjugated nanoparticles resulted in 95-98% cell death and loss of viability within 24 h whereas no change in cell proliferation or cell apoptosis was observed in cells treated with equal amounts of either [D-Trp6]LHRH or unconjugated Fe3O4 nanoparticles. These studies provide critical and important information regarding use of LHRH receptor targeted therapy for breast cancer.

Transdermal iontophoretic delivery of triptorelin in vitro.

The feasibility of delivering triptorelin ([D-Trp6]LHRH) by transdermal iontophoresis was evaluated in vitro. Peptide electrotransport at different current densities and donor concentrations was measured across porcine ear skin. The concomitant delivery of an electroosmotic marker enabled calculation of the respective contributions of electromigration (EM) and electroosmosis (EO) to iontophoretic delivery. At a given concentration (3 mM), a threefold increase in current density produced a corresponding increase in the cumulative amount of peptide present in the receptor compartment. Conversely, doubling the concentration to 6 mM produced a twofold reduction in the amount of peptide delivered, partly due to a concentration-dependent inhibition of EO. EM was revealed to be the predominant transport mechanism, accounting for 80% of overall delivery. Finally, despite the inhibition of EO, the results indicate that application of an iontophoretic current of 0.8 mA over a relatively small contact area (4 cm2) would provide a delivery rate of 36 microg/h, largely sufficient for therapeutic requirements.

Expression of receptors for luteinizing hormone-releasing hormone in human ovarian and endometrial cancers: frequency, autoregulation, and correlation with direct antiproliferative activity of luteinizing hormone-releasing hormone analogues.

OBJECTIVE: Several recent reports have demonstrated the expression of luteinizing hormone-releasing hormone receptors by human ovarian and endometrial cancers. Controversy persists on the relevance of this finding, in particular whether these receptors mediate direct antiproliferative effects of luteinizing hormone-releasing hormone analogues. We correlated the expression of luteinizing hormone-releasing hormone receptors by well-characterized ovarian and endometrial cancer cell lines with the ability of luteinizing hormone-releasing hormone analogues to reduce their proliferation and studied the autoregulation of luteinizing hormone-releasing hormone receptor expression by luteinizing hormone-releasing hormone agonist triptorelin and antagonist cetrorelix. The expression of luteinizing hormone-releasing hormone receptors was assessed in a series of specimens from primary ovarian and endometrial cancers. STUDY DESIGN: Luteinizing hormone-releasing hormone receptor expression was assessed by semiquantitative reverse transcriptase-polymerase chain reaction and radioligand binding assay. Antiproliferative effects were ascertained by proliferation assays in the absence or presence of luteinizing hormone-releasing hormone analogues. RESULTS: Ovarian (4/6 cell lines) and endometrial (5/6 cell lines) cancer cell lines expressed luteinizing hormone-releasing hormone receptors. The proliferation of these luteinizing hormone-releasing hormone receptor-positive cell lines was dose- and time-dependently reduced by agonistic and antagonistic luteinizing hormone-releasing hormone analogues. Luteinizing hormone-releasing hormone receptor density was reduced to 80% of controls (control, 100 %; P <.001) by luteinizing hormone-releasing hormone analogues. Seventy percent of primary ovarian cancers and 83% of primary endometrial cancers expressed luteinizing hormone-releasing hormone receptors. CONCLUSION: These findings suggest that luteinizing hormone-releasing hormone receptors that are expressed by human ovarian and endometrial cancer cell lines mediate direct antiproliferative effects of luteinizing hormone-releasing hormone analogues. Because most respective primary cancers expressed luteinizing hormone-releasing hormone receptors, these receptors might be used for novel antiproliferative therapeutic approaches and should be further evaluated.

Residues within transmembrane helices 2 and 5 of the human gonadotropin-releasing hormone receptor contribute to agonist and antagonist binding.

To understand the ligand binding properties of the human GnRH receptor (hGnRH-R), 24 site-specific mutants within transmembrane helices (TMH) 1, 2, and 5 and the extracellular loop 2 (E2) were generated. These mutants were analyzed by using a functional reporter gene assay, monitoring receptor signaling via adenylate cyclase to a cAMP-responsive element fused to Photinus pyralis luciferase. The functional behavior of 14 receptor mutants, capable of G-protein coupling and signaling, was studied in detail with different well described agonistic and antagonistic peptide ligands. Furthermore, the binding constants were determined in displacement binding experiments with the antagonist [125I]Cetrorelix. The substitution of residues K36, Q204, W205, H207, Q208, F20, F213, F216, and S217 for alanine had no or only a marginal effect on ligand binding and signaling. In contrast, substitution of N87, Eg9, D9, R179, W206, Y211, F214, and T215 for alanine resulted in receptor proteins neither capable of ligand binding nor signal transduction. Within those mutants affecting ligand binding and signaling to various degrees, W101A, N102A, and N212Q differentiate between agonists and antagonists. Thus, in addition to N102 already described, the residues W101 in TMH2 and N212 in TMH5 are important for the architecture of the ligand-binding pocket. Based on the experimental data, three-dimensional models for binding of the superagonist D-Trp6-GnRH (Triptorelin) and the antagonist Cetrorelix to the hGnRH-R are proposed. Both decapeptidic ligands are bound to the receptor in a bent conformation with distinct interactions within the binding pocket formed by all TMHs, E2, and E3. The antagonist Cetrorelix with bulky hydrophobic N-terminal amino acids interacts with quite different receptor residues, a hint at the failure to induce an active, G protein-coupling receptor conformation.

Presence and characteristics of receptors for [D-Trp6]luteinizing hormone releasing hormone and epidermal growth factor in human ovarian cancer.

This study was undertaken to establish the presence and characteristics of receptors for [D-Trp6]LH-RH on the membranes of human ovarian cancer. Specific binding of [125I, D-Trp6]LH-RH was found in 29 of 37 (78.4%) ovarian cancers and in 6 of 11 (54.5%) non-malignant human ovaries. Ligand binding was dependent on time and plasma membrane concentration in a fashion expected of a peptide hormone. Saturation, kinetic and displacement data were consistent with the presence of a highly specific, single class of non-cooperative binding site. On the basis of receptors affinity, LH-RH-receptor-positive ovarian cancers could be divided into two groups: high affinity group (Kd=2.71 +/- 0.60 nM; Bmax=0.46 +/- 0.07 pmol/mg membrane protein) comprising 55% of tumors, and low affinity group (Kd=78.0 +/- 19.6 nM; Bmax=9.44 +/- 2.68 pmol/mg membrane protein) which included 45% of tumors. LH-RH antagonist Cetrorelix showed an affinity to LH-RH receptors on ovarian cancers 14 times higher than the agonist [D-Trp6]LH-RH. Using 125I-epidermal growth factor, specific high affinity receptors were also detected in membranes from 13 of 24 (54%) ovarian cancers and 5 of 11 (45%) non-malignant ovaries. The demonstration of LH-RH receptors in human ovarian cancers provides a rationale for the use of therapeutic approaches based on LH-RH analogues in this malignancy. The probable involvement of growth factors in the development of ovarian cancers suggests the merit of trying a combined therapy based on analogs of LH-RH and somatostatin for this carcinoma.

Inhibition of corneal metabolism of deslorelin by EDTA and ZnCl2.

It was the aim of this study to determine whether deslorelin is degraded by the rabbit corneal tissue and to further delineate the mechanisms. Deslorelin was incubated with intact cornea either alone or in the presence of 0.1 mM ouabain, 0.1% 2,4-dinitrophenol, 0.1 mM phosphoramidon, 0.1 mM N-tosyl-L-phenylalanine chloromethylketone (TPCK), 0.1-2% EDTA, 0.1-1% ZnCl2, 0.1% dithiothreitol (DTT), or 0.1% N-ethylmaleimide (NEM) at 37 degrees C. In addition, deslorelin alone was incubated with cornea at 4 degrees C. Following a 90-min incubation, the supernatants were analyzed using a reversed-phase HPLC. Metabolite peaks observed in controls at 37 degrees C were not detected in the low-temperature study, suggesting inhibition of metabolism at low temperature. Intact drug remaining in the supernatant was not altered by ouabain and dinitrophenol, suggesting that energy-dependent corneal uptake is not likely for deslorelin. Phosphoramidon and TPCK failed to alter deslorelin levels, indicating that phosphoramidon and TPCK-sensitive endopeptidases did not contribute to the observed metabolism. DTT and NEM also failed to affect deslorelin levels. However, 2% EDTA and 1% ZnCl2 significantly elevated the intact deslorelin levels by 44 and 60%, respectively, and the metabolite peaks almost completely disappeared. These observations are consistent with the corneal metabolism of deslorelin by either metallo-peptidases or metal-dependent peptidases.

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: III. Changes in GnRH binding levels with stage of gestation.

We have measured the levels of gonadotrophin-releasing hormone (GnRH) binding sites in human placental villous membrane fractions obtained at different stages of gestation. There was a marked decrease in the specific activity of 125I-labelled GnRH binding to membrane fractions obtained between 10-20 weeks gestation, but there was no change in either affinity or ligand specificity of these binding sites. The observed decrease in binding was not due to contamination of placental villous membranes by membranes from other tissues, since there was no gestation-dependent decrease in the specific activity of epidermal growth factor receptor or alkaline phosphatase activity in villous membrane fractions between 10-20 weeks of gestation. Furthermore, incubation of GnRH tracer with membranes from different stages of gestation, followed by re-incubation of the unbound tracer fraction with fresh membranes, demonstrated unequivocally that decreased GnRH binding to 10-20 week membranes was not due to increased degradation of GnRH tracer. We conclude that the observed changes in GnRH receptor levels between 10-20 weeks gestation must reflect either decreased expression/synthesis (or increased catabolism) of placental GnRH receptors, or increased occupancy (or down-regulation) of placental GnRH receptors by an endogenous GnRH-like ligand.

High affinity binding and direct antiproliferative effects of luteinizing hormone-releasing hormone analogs in human endometrial cancer cell lines.

Although specific binding sites for LH-releasing hormone (LHRH) and its analogs have been demonstrated in biopsy samples of human endometrial cancer, their biological significance remains obscure. In this study we evaluated whether binding sites for LHRH are also present in the human endometrial cancer cell lines HEC-1A and Ishikawa and if such sites could mediate antiproliferative effects of LHRH analogs. Using [125I,D-Trp6]LHRH as a ligand, a high affinity/low capacity binding site was detected in both lines: HEC-1A line, dissociation constant (Kd)1 = 5.7 x 10(-9) mol/L, binding capacity (Bmax)1 = 78 fmol/10(6) cells; Ishikawa line, Kd1 = 4.2 x 10(-9) mol/L, Bmax1 = 29 fmol/10(6) cells. In addition, a second class of low affinity/high capacity binding sites for LHRH was demonstrated (HEC-1A line, Kd2 = 1.4 x 10(-6) mol/L, Bmax2 = 21 pmol/10(6) cells; Ishikawa, Kd2 = 4 x 10(-6) mol/L, Bmax2 = 32 pmol/10(6) cells). In the presence of 10(-5) mol/L agonist [D-Trp6]LHRH (triptorelin), the proliferation of HEC-1A and Ishikawa cell lines was significantly reduced to 76 +/- 2% and 88 +/- 4% of controls, respectively, after 24 h and to 64 +/- 2% and 62 +/- 2%, respectively, after 6 days. Dose-response experiments showed that lower concentrations (10(-9) mol/L) of the agonist decreased the proliferation to 80 +/- 1% for the HEC-1A line and 71 +/- 2% of controls for the Ishikawa line after 6 days. Antiproliferative effects are enhanced by increasing the doses of triptorelin and were maximal in this series of experiments at 10(-5) mol/L, the proliferation in the HEC-1A line being 62 +/- 1% and in the Ishikawa line 52 +/- 2% of controls, respectively. Similar time- and dose-dependent antiproliferative effects were obtained in both cell lines with the LHRH antagonist SB-75 (cetrorelix). These data suggest that LHRH analogs can directly inhibit the proliferation of human endometrial cancer cells in vitro. This direct action could be mediated through the high affinity LHRH binding sites.

High affinity binding and direct antiproliferative effects of LHRH analogues in human ovarian cancer cell lines.

Recently, specific binding sites for luteinizing hormone releasing hormone (LHRH) and its analogues have been demonstrated in biopsy samples of human epithelial ovarian cancer. Their biological significance remained obscure. In this study we ascertained whether such LHRH-binding sites are also present in the human epithelial ovarian cancer cell lines EFO-21 and EFO-27 and if they could mediate antiproliferative effects of LHRH analogues. Using [125I, D-Trp6]LHRH, a high affinity/low capacity binding site was detected in both lines: EFO-21 (Kd1 = 1.5 x 10(-9) M; binding capacity (Bmax1) = 4.9 fmol/10(6) cells) and EFO-27 (Kd1 = 1.7 x 10(-9) M; Bmax1 = 3 fmol/10(6) cells). In addition, a second class of low affinity/high capacity binding sites (EFO-21: Kd2 = 7.5 x 10(-6) M; Bmax2 = 24 pmol/10(6) cells; EFO-27: Kd2 = 4.3 x 10(-6) M; Bmax2 = 14.5 pmol/10(6) cells) was demonstrated. Specific binding of [125I, D-Trp6]LHRH was displaced with nearly equal efficiency by unlabeled [D-Trp6]LHRH, the LHRH-antagonists SB-75 and Hoe-013, and by native LHRH but not by unrelated peptides such as oxytocin and somatostatin. In the presence of 10(-5) M agonist [D-Trp6]LHRH, the proliferation of both cell lines was significantly reduced to 77% of controls after 24 h and to approx. 60% after 6 days. Lower concentrations (10(-9) M) of the agonist, significantly decreased the proliferation to 87.5% for EFO-21 and 86% for EFO-27 after 6 days. These antiproliferative effects were enhanced by increasing doses of [D-Trp6]LHRH and were maximal at 10(-5) M (EFO-21: 65.5% of control, EFO-27: 68% of control). Similar dose-dependent antiproliferative effects were obtained in EFO-21 line with the LHRH-antagonists SB-75 and Hoe-013, while these analogues had no effects on the proliferation of EFO-27 cells. SB-75 partly antagonized the antiproliferative effect of [D-Trp6]LHRH in a dose dependent way in the EFO-27 line. These data suggest that LHRH analogues can directly inhibit the in vitro proliferation of human ovarian cancer cells. This effect might be mediated through the high affinity LHRH binding sites.

Evaluation of binding of cytotoxic analogs of luteinizing hormone-releasing hormone to human breast cancer and mouse MXT mammary tumor.

The binding characteristics of several cytotoxic analogs of luteinizing hormone-releasing hormone (LH-RH) developed in our laboratory were examined in membranes from human breast cancer and estrogen independent MXT mammary cancer. Specific binding of [125I]D-Trp6-LH-RH and the cytotoxic LH-RH analog [125I]T-98 ([D-Lys6]LH-RH coupled to glutaryl-2-(hydroxymethyl)anthraquinone) (HMAQG) was demonstrated in membrane preparations from human breast and MXT mammary tumor cells. Ligand binding of T-98 was specific, saturable, and dependent on temperature, time, and plasma membrane concentration. Analysis of the binding data showed that in human breast cancer, interaction of [125I]T-98 was consistent with the presence of two classes of LH-RH receptors, one class showing high affinity and low capacity, and the other class showing low affinity and high capacity binding. In membranes from MXT mammary cancer, T-98 bound to one class of saturable, specific, noncooperative binding sites with high affinity and low capacity. The rates of association and dissociation for [125I]T-98 were calculated to be 4.757 x 10(8) M-1 min-1 and 0.016 min-1 (t1/2 = 38.7) in membranes from MXT mammary cancer. In human breast cancer, association rate constants (K1a and K1b) were 2.3 x 10(6) M-1 min-1 for binding to high affinity and 1.8 x 10(4) M-1 min-1 for binding to low affinity binding sites. Dissociation rate constants were K-1a = 0.0801 min-1 (t1/2a = 63.4 min) and K-1b = 0.0467 min-1 (t1/2b = 23.5 min), respectively. [125I]T-98 was not displaced by either unlabeled somatostatin or epidermal growth factor, but was displaced completely by unlabeled T-98 or [D-Trp6]LH-RH. The analysis of displacement curves of [D-Trp6]LH-RH by cytotoxic agonists and antagonists of LH-RH synthesized in our laboratory showed that T-121, AJ-11, T-120, T-133, and T-98 were the most potent in displacing [125I]D-Trp6-LH-RH from breast and MXT cancer membranes. Binding kinetics and analyses of displacement curves of [125I]D-Trp6-LH-RH and [125I]T-98 in membranes of human breast cancer and estrogen independent MXT mouse mammary cancer suggest that binding of the cytotoxic analog T-98 to the LH-RH receptor proceeds reversibly like that of its congeners without cytotoxic radicals. Our findings may provide a stimulus for further studies with LH-RH analogs carrying cytotoxic radicals.(ABSTRACT TRUNCATED AT 400 WORDS)