Lamprey gonadotropin hormone-releasing hormone-III has no selective follicle-stimulating hormone-releasing effect in rats.

Lamprey gonadotropin releasing-hormone (LGnRH)-III, a hypothalamic neurohormone recently isolated from sea lamprey, was reported to have a selective stimulatory effect on follicle-stimulating hormone (FSH) release in rats and suggested to be the mammalian FSH-releasing factor. In this study, we determined the relative luteinizing hormone (LH)- and FSH-releasing potency of LGnRH-III compared to mammalian gonadotropin-releasing hormone (LHRH) in normal female rats, ovariectomized (OVX) and oestrogen/progesterone substituted rats and the superfused rat-pituitary cell system. The specificity of LGnRH-III for the mammalian LHRH receptor was investigated by blocking the receptor with an LHRH antagonist, MI-1544. In vitro, LGnRH-III dose-dependently stimulated both LH and FSH secretion from rat pituitary cells at 10(-7) to 10(-5) M concentrations, while LHRH stimulated gonadotropin secretion at a 1000-fold lower doses (10(-10) to 10(-8) M). The difference between its LH- and FSH-releasing potency was similar to that of LHRH. LGnRH-III bound to high affinity binding sites on rat pituitary cells with a Kd of 6.7 nM, B(max)=113 +/- 27 fmol/mg protein. In vivo, LGnRH-III also stimulated both LH and FSH secretion in a dose-dependent manner and, similar to LHRH, induced a greater rise in the serum LH than the FSH level. In normal cycling rats, it showed 180-650-fold weaker potency than LHRH in stimulating LH secretion and 70-80-fold weaker effect in stimulating FSH secretion. In OVX rats, LGnRH-III demonstrated a similarly weak effect on both gonadotropins. It was found to be 40-210-fold less potent than LHRH regarding LH release and 50-160-fold weaker regarding FSH release. LHRH-receptor antagonist MI-1544 prevented both the LH- and the FSH-releasing effect of LGnRH-III both in vitro and in vivo. These results do not support the hypothesis that LGnRH-III might be the mammalian FSH-releasing factor but demonstrate that it is a weak agonist for the pituitary LHRH receptor and stimulates both gonadotropins in a dose-dependent fashion.

Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues.

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.

Interaction of the P-glycoprotein multidrug transporter (MDR1) with high affinity peptide chemosensitizers in isolated membranes, reconstituted systems, and intact cells.

P-glycoprotein-mediated multidrug resistance can be reversed by the action of a group of compounds known as chemosensitizers. The interactions with P-glycoprotein of two novel hydrophobic peptide chemosensitizers (reversins 121 and 205) have been studied in model systems in vitro, and in a variety of MDR1-expressing intact tumor cells. The reversins bound to purified P-glycoprotein with high affinity (77-154 nM), as assessed by a quenching assay using fluorescently labeled purified protein. The peptides modulated P-glycoprotein ATPase activity in Sf9 insect cell membranes expressing human MDR1, plasma membrane vesicles from multidrug-resistant cells, and reconstituted proteoliposomes. Both peptides induced a large stimulation of ATPase activity; however, higher concentrations, especially of reversin 205, led to inhibition. This pattern was different from that of simple linear peptides, and resembled that of chemosensitizers such as verapamil. In both membrane vesicles and reconstituted proteoliposomes, 1-2 microM reversins were more effective than cyclosporin A at blocking colchicine transport. Reversin 121 and reversin 205 restored the uptake of [3H]daunorubicin and rhodamine 123 in MDR1-expressing cells to the level observed in the drug-sensitive parent cell lines, and also effectively inhibited the extrusion of calcein acetoxymethyl ester from intact cells. In cytotoxicity assays, reversin 121 and reversin 205 eliminated the resistance of MDR1-expressing tumor cells against MDR1-substrate anticancer drugs, and they had no toxic effects in MDR1-negative control cells. We suggest that peptides of the reversin type interact with the MDR1 protein with high affinity and specificity, and thus they may be good candidates for the development of MDR1-modulating agents to sensitize drug resistance in cancer.

A novel normalized retention factor in micellar electrokinetic chromatography.

Characteristic properties of the expression k'' = (t(m)-t(o))/(t(mc)-t(o)) and its applicability in micellar electrokinetic capillary chromatography (MEKC) were compared to the previous expression, k' = (t(m)-t(o))/t(o)(1-t(m)/t(mc)), introduced by Terabe. It was proved with theoretical calculations (curve shape analysis) that the properties of function k'' are in full accordance with the properties of the MEKC system and k'' could be applied advantageously to characterize hydrophobicity of the analytes. This conclusion is now supported by experimental data obtained with homolog series of alkylbenzenes and alkylphenones as well as with hydrophobic protected peptides. Migration times, k', k'' values, and software-calculated hydrophobicity data are summarized and analyzed in the present study. Since k'' is a normalized parameter, good relationships between the migration time, the software-calculated hydrophobicity, and the k'' values were obtained. Differences in hydrophobicity of the analytes could be estimated in a more realistic way with the aid of function k'' than by using function k'. Hydrophobicity data estimated on the basis of the k'' values proved to be in good accordance with the expectations based on the migration times and on the chemical structures of the compounds investigated.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Synthesis, conformation, biodistribution, and hormone-related in vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate.

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Comparative analysis of somatostatin analog peptides by capillary electrophoresis and micellar elektrokinetic chromatography.

Capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC) methods, utilizing uncoated silica capillary and triethyl ammonium phosphate or sodium borate buffers in the pH range of 2.25-11.0, containing sodium dodecyl sulfate (SDS) (0-100 mM) for analysis of somatostatin-analog peptides were developed. The method presented here was compared with the reversed-phase high performance liquid chromatographic (RP-HPLC) and CE methods developed for analysis of peptides. The peptides investigated in this work can be separated by CE on the basis of their electrophoretic mobility in aqueous buffer of low pH value (pH 2.25) or by MEKC on the basis of their hydrophobicity in SDS containing buffer of high pH value (pH 11.0). Optimal MEKC separation of the investigated peptides has been achieved at pH 11.0 in an Na-borate buffer containing 100 mM SDS. CE at pH 2.25 proved insensitive to the hydrophobicity of the peptides investigated. By contrast, results obtained with MEKC at pH 11.0 proved to be anologous to those obtained by RP-HPLC, with highly hydrophobic peptides-migrating slower than peptides without hydrophobic moieties.

Effect of gonadotropin-releasing hormone analogs and their conjugates on gonadotropin-releasing hormone receptor--positive human cancer cell lines.

The purpose of the present investigation was to develop new gonadotropin-releasing hormone (GnRH) antagonists and to increase their stability and antitumor effect by conjugation with carrier macromolecules. Antitumor effect was evaluated using clonogenic assay, cell counting for antiproliferation, and sulforhodamine B method. The presence of GnRH-binding sites in human cancer cell lines (MCF-7, MDA-MB-231, Ishikawa, LNCaP) was proved. The direct growth inhibition of tumor cell lines is achieved with relatively high analog concentrations (10(-10)- 10(-5) M). We have developed new GnRH analogs of human and chicken origin. MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)GnRH and the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3, D-Cpa2, Lys5, [beta-Asp(DEA)]6, Gln8, D-Ala10)-GnRH have stronger direct antitumor properties than the agonists. The antagonists inhibited proliferation of GnRH receptor-positive human cancer cell lines by 28 to 38%. GnRH peptide analogs were coupled with macromolecules through biodegradable groups, to enhance their antitumor effects. The antagonists reduced survival of MCF-7 and MDA-MB-231 cells by 38 to 48% and 20 to 41%, respectively. They showed less activity against human endometrial and prostate cancer cells (10-20%). The copolymer (P) as polyanionic carrier molecule reached only 15 to 20% survival reduction in all cell lines. However, the copolymer GnRH antagonist conjugates P-X-1892 and P-X-1544 killed 95 to 98% of cells at doses corresponding to the GnRH analog concentration. These compounds having antitumor activity could be tried for the treatment of prostate, breast, and endometrium cancer.

In vivo studies of the new gonadotropin-releasing hormone antagonist--copolymer conjugates having antitumor activity.

The aim of the study was to test in vivo the gonadotropin-releasing hormone (GnRH) antagonists and their conjugates showing antitumor activities in vitro. The in vivo experiments with the human GnRH antagonist MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)-GnRH, the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3,D-Cpa2,Lys5,/beta-Asp(DEA)/6,Gln8,D-Al a10)-GnRH, and their copolymer conjugates were carried out on MCF-7 and MDA-MB-231 human breast tumors xenografted in immunosuppressed CBA/Ca HRIJ-T6 female mice and on MXT mouse mammary tumors in BDF1 mice. The P-X-1544 and P-X-1892 conjugates were prepared by coupling the GnRH antagonists to macromolecule copolymer through biodegradable spacers. MI-1544 and its conjugate had strong, whereas MI-1892 and its conjugate had slight, castration effect in rats. All of them showed selective antitumor activity. The conjugates, given daily, inhibited both types of xenografts by 42 to 49%. Their activity was stronger in MXT mammary tumor (72 to 61%). The in vivo effect of GnRH antagonists was largely increased by coupling them to nonbiodegradable macromolecule carriers of polyanionic character. P-X-1544 and P-X-1892 GnRH antagonist-macromolecule conjugates might become important therapeutic agents for the treatment of breast cancer.

Synthesis of GnRH analogs having direct antitumor and low LH-releasing activity.

New chicken I GnRH agonists and antagonists have been synthesized and tested for their biological activities. The common feature of these analogs was that the molecules had a beta-L-aspartyl residue inserted in position 6. The agonist bound to the pituitary still had low endocrinological activity. On the other hand, it exhibited direct antitumor effect in in vitro assays. The endocrinological activity of the antagonist was low; however, it showed potent, direct antitumor activity. These observations might lead to the development of new GnRH analogs with selective antitumor effect.

Interaction of bioactive hydrophobic peptides with the human multidrug transporter.

In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1). The stimulation of the MDR1-ATPase by these agents does not correlate with their known biochemical or pharmacological activities but rather with their hydrophobicity. The peptides that show high-affinity interaction with the MDR1-ATPase also interfere strongly with fluorescent dye extrusion catalyzed by the multidrug transporter in intact cells and some have been shown to reverse drug resistance in cultured cells. These data suggest that several hydrophobic peptides behave as substrates of the multidrug transporter and may be used to modulate the chemotherapy resistance of tumor cells.

Improvement of the reproductive performance of sows by treatment with a GnRH superactive analogue.

The effect of a Hungarian-made superactive analogue of GnRH (Ovurelin, D-Phe6-GnRH-EA, Reanal, Hungary) on the postpartal sexual function of sows was monitored. GnRH treatment was carried out on day 19 before weaning. The sows were inseminated at the first oestrus after weaning. GnRH treatment markedly increased litter size at weaning, substantially reduced (to 25 and 50%, respectively) the number of sows failing to come into oestrus after weaning, and increased the number of sows coming into oestrus within one week after weaning by 42.5% and 9%, respectively. These beneficial effects were particularly apparent on the far using closed management technology.

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts.

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.

Effect of treatment with Prolan or with a GnRH superactive analog on the sexual function of sows after weaning.

On a large, closed pig farm using artificial insemination (AI), 29 sows were treated with Prolan S-öl injection (Bayer, FRG) and 31 sows with a GnRH superactive analog (Ovurelin, D-Phe6-GnRH-EA, Reanal, Hungary) 48 h after weaning. The effect of treatment on the sows' sexual function was monitored by serum progesterone radioimmunoassay (RIA). The conception rate in the control group (36 sows) was 69.4%. In the groups treated with Prolan and Ovurelin it was 79.3 and 71%, respectively. The use of Prolan S-öl injection markedly reduced the number of acyclic sows and of those having an irregular oestrous cycle. Treatment with the GnRH analog inhibited the manifestation of weaning-induced heat; subsequently, however, it induced a regular cycle in 30 out of the 31 sows treated.

Effects of long-term administration of a superactive agonistic and an antagonistic GnRH analog on the pituitary-gonad system.

A powerful GnRH antagonist: [Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]-GnRH (MI-1544) and a superactive GnRH agonist: [D-Phe6,desGly10]-GnRH(1-9)EA (OVURELIN) were used in long-term administration to compare their effects on the inhibition of ovulation, LH and progesterone (P) release, LH content of pituitaries as well as on the recovery period. Both analogs showed 100% inhibitory effects on ovulation in very low doses during the daily treatment for 21 days. The antagonist prevented LH release already after the first injection, decreased the serum P level to 40%, and increased the LH content of the pituitary up to 180%, inhibiting only the release but not the synthesis of LH. The agonist showed marked LH-releasing effects on the first day of the treatment, which were reduced to 12% on the 7th day. Serum P concentration was dropped to 68% by the end of the treatment. No change was found in the LH content of pituitaries in the group treated with the agonist. Ovaries showed polifollicular pictures in the antagonist-treated group, and persistent corpora lutea were seen in the ovaries from the agonist-treated group. Regular estrous cycles returned 13-15 days after ceasing the treatment with the antagonist and 3-5 days after ceasing the treatment with the agonist. No edema-inducing effect was observed after the injections of the antagonist in doses of 100 times higher than the single antiovulatory dose.

High-performance liquid chromatographic monitoring of transpeptidation reactions in analogues of gonadotropin releasing hormone containing aspartic acid derivatives in position six.

High-performance liquid chromatographic systems were used for monitoring the hydrolysis of five potent agonistic gonadotropin releasing hormone analogues containing aspartic acid derivatives in position six. To separate the closely related nonapeptides formed during the hydrolysis, columns with reversed-phase packings were used under isocratic conditions. The mobile phases were methanol containing ammonium acetate or triethylammonium phosphate buffers. Good separations of the hydrolysis products from the investigated peptides allowed the reaction rate constants for the transformations examined to be calculated.

Two components of type III protein kinase C with different substrate specificities and a phospholipid-dependent but Ca2+-inhibited protein kinase in rat brain.

The activities of rat brain protein kinase C isoenzymic fractions separated by hydroxyapatite chromatography were measured with histone H1 or the oligopeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide as substrates. The oligopeptide was a better substrate than histone H1 for nearly all of the protein kinase C fractions. Two subfractions of type III isoenzyme were resolved (IIIa and IIIb); type IIIb was characterized by a very low histone kinase activity compared to its peptide kinase activity. In some brain extracts a phospholipid-dependent but Ca2+-inhibited protein kinase was also observed which was eluted from the hydroxyapatite column between type II and III isoenzymes of protein kinase C.

Isoenzyme patterns of protein kinase C and a phospholipid-dependent but Ca2+-inhibited enzyme fraction in the crude extracts of different tissues.

We compared the protein kinase C isoenzyme patterns of crude extracts of rabbit brain, cerebellum, spleen, thymus and human and pig granulocytes. The isoenzymes were fractionated by hydroxyapatite chromatography and the protein kinase C activity was determined with a synthetic oligopeptide substrate. In the extracts of several tissues we also observed an enzyme fraction which was activated by phosphatidylserine + diacylglycerol but inhibited by Ca2+.

The phosphorylation site of Ca(2+)-dependent protein kinase from alfalfa.

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca(2+)-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.