Tommie W. Redding

Growth inhibition of MXT mammary carcinoma by enhancing programmed cell death (apoptosis) with analogs of LH-RH and somatostatin

BDF female mice inoculated with MXT mammary adenocarcinoma were treated for 30 days with microcapsules of the luteinizing hormone-releasing hormone (LH-RH) agonist D-Trp-6-LH-RH (releasing 25µg/day for 30 days), microcapsules of the somatostatin agonist RC-160 (liberating 25µg/day for one month), or the combination of these peptides. Bilateral surgical ovariectomy was performed in one group which served as an additional control. Tumor volume was measured weekly during the treatment period of 30 days. When tumor volume changes in the treated groups were compared to the corresponding changes in controls, the combination of D-Trp-6-LH-RH and RC-160 was the most effective in inhibiting tumor growth and approached the effect of surgical ovariectomy. At the conclusion of the experiment, tumor weights were also measured. All peptide analogs inhibited tumor weight by 42 to 63%. In the D-Trp-6-LH-RH treated group, ovarian weights and uterine weights decreased by 48% and 52%, respectively, as compared to controls. Histologically, the regressive changes in tumors caused by the treatment with RC-160, D-Trp-6-LH-RH and their combination were characterized by the coexistence of apoptosis (programmed cell death) and coagulation necrosis. The transition of apoptosis into coagulation necrosis was a common finding. The term ‘apoptotic index’ is proposed for the ratio of tumorous glands containing apoptotic cells. The apoptotic index was higher in the treated groups than in the control.

Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi.

Seven new antagonists of bombesin (Bn)/gastrin-releasing peptide (GRP) containing C-terminal Trp or Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-carboxylic acid) in a reduced peptide bond were synthesized by solid phase methods and evaluated biologically. The reduced bond in four [Leu13 psi(CH2NH)Trp14]Bn(6-14) analogs was formed by reductive alkylation at the dipeptide stage. In the case of three [Leu13 psi(CH2N)Tpi14]Bn(6-14) analogs, the Trp dipeptide with reduced bond was reacted with formaldehyde to form the corresponding Tpi derivative. These Tpi-containing analogs have a new reduced bond which is structurally more constrained. Leu13 psi(CH2N)Tpi14 analogs inhibit [125I][Tyr4]bombesin binding to Swiss 3T3 cells with IC50 values of 2-4 nM, compared to 5-10 nM for Leu13 psi(CH2NH)Trp14 analogs. Leu13 psi(CH2N)Tpi14 analogs are also more potent than Leu13 psi(CH2NH)Trp14 analogs in growth inhibition studies using Swiss 3T3 cells. The two best bombesin antagonists of this series, [D-Trp6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3415) and [Tpi6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3440), inhibited GRP-stimulated growth of Swiss 3T3 cells with IC50 values less than 1 nM. RC-3440 was also active in vivo, suppressing GRP(14-27)-stimulated serum gastrin secretion in rats. Bombesin/GRP antagonists, such as RC-3440, containing the new reduced bond (CH2N) reported herein are very potent.

Disappearance and excretion of labeled α-MSH in man

Abstract Despite the considerable evidence for the CNS actions of α-melanocyte-stimulating hormone (α-MSH) in man, little attention has been given to its half-time disappearance from plasma and urinary excretion in normal individuals. In the first experiment, a healthy man was given 15 μCi of tritiated ( 3 H)-α-MSH as a rapid IV injection. A plot of the disappearance time in plasma was characteristic of a multiexponential curve, the linear components of which were resolved by the subtraction method and half-time disappearance calculated directly from the slope of the regression line. The half-time disappearance was 1 min for the first component and 25 min for the second component. After the IV administration of 50 μCi of 125 I-α-MSH in the second experiment, the two components showed halftime disappearances of 1 min and 4.8 min respectively. These times were not changed by precipitation of the plasma with 10% trichloroacetic acid. Thirty-eight percent and 42% of the label appeared in the urine 4 hr after the injection of either 3 H-α-MSH or 125 I-α-MSH. The results suggest that the persistence of high levels of α-MSH in the blood after injection in man may be too short to fully explain the CNS effects of α-MSH.

The effect of gastrointestinal hormones on the incorporation of tritiated thymidine in the pancreatic adenocarcinoma cell line (WD PaCa)

SummaryIn view of the trophic action of gastrointestinal hormones on the exocrine pancreas, the effects of secretin, octapeptide of cholecystokinin (CCK-β), and desglugastrin on the growth of hamster pancreatic well differentiated adenocarcinoma were investigated in vitro. Desglugastrin exhibited the greatest effect on thymidine incorporation into these cells after a lag period of 96 h. Doses of desglugastrin in the range from 30 to 270 ng/mL caused a significant and dose-dependent increase in thymidine incorporation. Higher doses of this peptide led to a decreased response. Secretin also increased thymidine incorporation, but the response was less than that induced by gastrin. Prolonged incubation with secretin for 96 h increased tritiated thymidine incorporation in a log-dose fashion in the range of 30 to 270 ng/mL. Doses of CCK-8 in the range of 90 to 810 ng/mL significantly increased thymidine incorporation after 48 h of incubation. Following 72 h of incubation, only the dose of 270 ng/mL continued to exhibit a significant stimulation. Our study suggests that the gastrointestinal hormones could directly increase the growth of pancreatic carcinoma cells, act synergistically with endogenous growth factors, or stimulate the local production of these factors. In any event, our results that gastrin, secretin, and CCK can stimulate the growth of pancreatic ductal tumor cells in tissue cultures, support, earlier findings on normal and malignant pancreatic parenchyma.

Treatment of experimental ovarian carcinoma with monthly injection of the agonist D-Trp-6-LH-RH: A preliminary report

Hormones, particularly gonadotropins, have been implicated in the development of ovarian cancer. Chronic administration of agonistic analogs of luteinizing-hormone releasing-hormone (LH-RH) induces an inhibition of the pituitary-gonadal axis. The blockade of the release of luteinizing-hormone and follicle-stimulating hormone (FSH) may exert a possible therapeutic effect on ovarian cancer. We examined the results of prolonged administration of D-Trp-6-LH-RH, an agonistic analog of LH-RH in experimental ovarian cancer. We used the recently developed ovarian cancer model in rats, which is produced by treatment of pregnant rats with N-nitrosobis(2-oxopropyl)amine (BOP), following which a high incidence of ovarian tumors are induced in the offspring. In morphologic aspects the induced tumor resembles human ovarian neoplasms. Once a month administration of a delayed release preparation of microcapsules of D-Trp-6-LH-RH prolonged the survival and decreased tumor growth and the incidence of metastases. Additional experimental and clinical studies are needed to determine the efficacy of the treatment with LH-RH analogs in ovarian cancer.

Synthesis of luteinizing hormone-releasing hormone containing tritium-labelled pyroglutamic acid

Abstract A method of preparing luteinizing hormone-releasing hormone (LH-RH) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 , by a combination of solid-phase and classical reactions was employed to conveniently synthesize a tritium-labelled hormone by incorporation of 4-[ 3 H]-pyroglutamic acid into position I of the peptide chain. The tritiated LH-RH possessed a specific radioactivity of 18.3 Ci/mmole and a maximal biological potency.

Thyroid stimulating and pigmentary effects of synthetic peptides related to α-MSH and ACTH

Abstract Seven synthetic peptides related to α-MSH and ACTH and eleven stereoisomeric fragments of α-MSH (sequence 6–10) were tested for their thyroid stimulating effects. This TSH-like activity was determined by following 131 I release in mice injected with 131 I and treated with thyroxine. N-α-acetyl-β-corticotropin-(1–24)-tetracosapeptide and β-corticotropin-(1–24)-tetracosapeptide hydrazide had the highest MSH activity and elicited the greatest effects on the blood levels of 131 I. The α-MSH-(7–13)-heptapeptide which had a moderate MSH activity, also caused a significant stimulation of 131 I release in mice. On the other hand, β-corticotropin-(11–24)-tetradecapeptide, which had virtually no MSH activity, possessed some TSH-like activity. D-His-D-Phe-D-Arg-D-Trp-Gly and the analogous L-pentapeptide, which were essentially free of MSH activity, had no effect on the thyroid at doses up to 100 μg. per mouse. Seven MSH-ACTH stereoisomers, which individually assayed up to 1,200 MSH U/mg., similarly had no effect on the thyroid, but L-His-D-Phe-L-Arg-L-Trp-Gly and three stereoisomers which had a low but definite MSH activity, did exert some TSH-like effects. These studies reveal that, although some relationship between MSH and TSH-like activity can be observed among peptides of the MSH-ACTH group, there is no definite correlation between these activities.

Observations on the growth hormone, insulin, and glucagon release-inhibiting activities of somatostatin analogues

Abstract Somatostatin was isolated from extracts of ovine 1 and porcine 2 hypothalamic tissue and was found to be a cyclic tetradecapeptide with the following sequence: Subsequent studies revealed that the peptide not only had inhibiting effects on growth hormone (GH) release, but also on pituitary thyrotropin (TSH) and a large array of gastrointestinal and pancreatic hormones, including insulin, glucagon, gastrin and gastric acid, secretin, CCK, GIP, VIP, and motilin. These activities have been recently reviewed. 3 The therapeutic value of somatostatin in several important areas is diminished by its broad spectrum of activities. As with most naturally occurring peptides, somatostatin also has a short biologic half-life, 4 making its long-term use impractical. Therefore, analogues are being designed for enhanced, prolonged, and selective activities. In addition, competitive inhibitors of somatostatin could be of great interest and pharmacologic value.

The effect of porcine thyrotropin (TSH)-releasing hormone (TRH) on the release of TSH from sheep and goat anterior pituitary tissue in vitro.

Abstract Highly purified porcine TRH and porcine SME extract increased the release of TSH from anterior tissue of sheep and goats in vitro . These studies extend to two more species the evidence of responsiveness to TRH and indicate a general lack of species specificity to porcine TRH among mammals.

Effect of hypothalamic preparations on human omental adipose tissue in vitro.

Abstract The lipolytic responses of rat and human adipose tissue to several pituitary hormones, catecholamines, and purified preparations from human and porcine hypothalami have been compared. Fragments of human omental adipose tissue, obtained at surgery, or rat epididymal adipose tissue were incubated in vitro for 2 hr in 2 ml of Krebs-Ringer bicarbonate medium containing 3% bovine albumin. Acetic acid extracts of human and porcine stalk median eminence tissue were purified by gel filtration on Sephadex G-25. Lipid-mobilizing factor (LMF) activity emerged in an area occupied by peptides with a molecular weight of 3000–5000. This lipolytic fraction was found to be free of thyroid-stimulating hormone (TSH) and catecholamines. The ACTH content of this fraction was found to be less than 10 mU/mg. Addition of 20–60 μg/ml of porcine or human LMF to the medium significantly increased lipolysis, as measured by glycerol release into the medium, from human and rat adipose tissue. Human LMF elicited a greated lipolytic response in human omental adipose tissue than did the procine LMF, but porcine LMF was more potent in rat epididymal adipose tissue. This suggests that a species specificity may exist for the lipolytic activity of hypothalamic lipid mobilizers. Responsiveness of human omental adipose tissue to adrenalin was lower than that of rat epididymal adipose tissue. Addition of corticotropin A (10–20 μg/ml), human growth hormone (10–30 μg/ml), and alpha or beta melanocyte-stimulating hormone (MSH) (10–30 μg/ml) had no effect on lipolysis when incubated with human omental adipose tissue. However, the addition of 1.5–3.0 mU/ml of human TSH significantly increased the release of glycerol from human omental adipose tissue. These data suggest that human hypothalamic extracts, like those of porcine hypothalamic extracts, contain a lipid-mobilizing factor or factors that may differ chemically from corticotropin, TSH, growth hormone, and the catecholamines.