Alpha-fetoprotein-derived antiestrotrophic octapeptide

Alpha-fetoprotein (AFP) is a major serum protein produced during fetal development. Experimental findings suggest that AFP has antiestrotrophic activity and that it can be developed as a therapeutic agent to treat existing estrogen-dependent breast cancer or to prevent premalignant foci from developing into breast cancer. The antiestrotrophic activity of AFP was reported to be localized to a peptide consisting of amino acids 447-480, a 34-mer peptide termed P447. A series of parsings and substitutions of amino acids in the P447 sequence was intended to identify the shortest analog which retained antiestrotrophic activity. Peptides related to P447 were generated using solid phase peptide synthesis. Several shorter peptides, including an 8-mer called P472-2 (amino acids 472-479, peptide sequence EMTPVNPG), retained activity, whereas peptides shorter than eight amino acid residues were inactive. The dose-related antiestrotrophic activity of AFP-derived peptides was determined in an immature mouse uterine growth assay that measures their ability to inhibit estradiol-stimulated uterine growth. In this assay, the maximal inhibitory activities exhibited by peptide P472-2 (49%), by peptide P447 (45%), and by intact AFP (35-45%) were comparable. The octapeptide P472-2 was also active against estradiol-stimulated growth of T47D human breast cancer cells in culture. These data suggest that peptide P472-2 is the minimal sequence in AFP, which retains the antiestrotrophic activity found with the full-length molecule. The synthetic nature and defined structure of this 8-mer peptide suggest that it can be developed into a new drug which opposes the action of estrogen, perhaps including the promotional effects of estradiol in the development of human breast cancer.     

A human placental hormone (UTPH) with uterine growth and DNA promoting effects.

A human placental protein previously described is studied in order to expand its biological characterization. Dose-response-curve of its action on uterine growth of prepuberal mice showed to be a significant logarithmic function and this effect is neutralized by its specific antiserum. It is also demonstrated that the uterine growth-promoting effect is not due either to estrogen, protein-bound estrogen, progesterone or chorionic gonadotrophin. Experimental evidence is given to demonstrate that the mechanism of action of this placental protein is to stimulate the synthesis and to increase the concentration of DNA in uterine tissue, differing then in this respect from the effect of estrogen. Previous work has shown that this placental protein is present in full-term placentas within similar ranges as HCG and HPL. It is also detected in blood during pregnancy and acts biologically in at least two target organs: uterus and mammary gland. Therefore the name of uterotrophic placental protein (UTPH) has been suggested for this substance.     

On the action of sulfanilamide

Summary A sulfanilamide activating principle was found to be present in red cells of the horse. This activator substance is active in the rather high dilution of 0.5% haemolysed red cells.The substance or substances are present in the red cells, not in their cell membranes. They seem to be of a protein nature or adsorbed to the protein (haemoglobin).In some media no sulfanilamide action is obtained without the activator. In other media sulfanilamide action, though clearly present, is markedly enhanced. So it must be emphasized, that the substance under discussion is an activator and not a conditio sine qua non for the sulfanilamide action and its characteristics.The substance is activating sulfanilamide against streptococci, staphylococci andB. coli.The substance is not present in human blood or in the red cells of sheep, rabbits or mice.Sixth communication: K. C.Winkler, Antonie van Leeuwenhoek8, 10, 1942.     

Methyl p-hydroxyphenyllactate. An inhibitor of cell growth and proliferation and an endogenous ligand for nuclear type-II binding sites

We previously described and partially characterized endogenous ligands for nuclear type II sites in normal and malignant tissues. Chromatography of these ligands on Sephadex LH-20 revealed that two peaks with binding activity (alpha and beta) could be resolved. The beta-peak component was present in all normal tissues that we examined, but not in malignant tissues, and it inhibited the growth of MCF-7 human breast cancer cells in vitro. Conversely, the alpha-peak component was found to be present in both normal and malignant tissues, and did not inhibit MCF-7 cell growth. The present studies describe the purification and identification of the alpha-peak and beta-peak components in bovine serum and an assessment of the effects of these compounds on normal and malignant cell growth. Gas chromatography-mass spectroscopy analysis of the purified beta-peak component demonstrated that the compound was methyl p-hydroxyphenyllactate (MeHPLA). Competition analysis revealed that MeHPLA binds to nuclear type II sites with a high binding affinity, while physiological levels of this compound blocked estradiol stimulation of uterine growth in vivo and inhibited the growth of MCF-7 human breast cancer cells in vitro. The alpha-peak component was found to be the corresponding acid, p-hydroxyphenyllactic acid (HPLA). This compound interacted with nuclear type II sites with a relatively low affinity and did not block uterotropic response to estradiol or inhibit MCF-7 cell growth. These studies demonstrate that HPLA and MeHPLA are ligands for nuclear type II sites and that MeHPLA may be a very important regulator of normal and malignant cell growth.     

Identification of differentially expressed genes in human uterine leiomyomas using differential display

INTRODUCTIONUterine leiomyomas (ULs) have been consideredto be of uniceIIular origin[l1. It is one of the mostcommon benign tumors, occurring in 20% to 30% ofwomen[2], accounting for significant morbidity andusually need major surgery[3] which might causesome side effects afterwards[4]. Therefore, to de-velop certain drug treatments instead has been thehope of these patients for a long time. Using alter-native approaches fOr studying patients sufferingfrom leiomyoma in various ethnic gr…     

Substance P stimulates translocation of protein kinase C in brain microvessels

The action of substance P on protein kinase C in cerebral microvessels, isolated from bovine, was investigated. We found that in untreated microvessels 85% of the total activity was localized to the cytosolic fraction. Substance P caused a shift of activity to the membrane fraction, accompanied by a loss of activity in the cytosolic fraction. This effect resulted in dose-dependence and it was evident after 5 min treatment. These data suggest that substance P may be involved in the regulation of processes underlying protein phosphorylation in the blood-brain barrier.     

Substance P contracts the human iris sphincter: possible modulation by endogenous enkephalinase.

Substance P-immunoreactive neurons have been found in the irides of many species including humans. In several species, substance P has been shown to induce contraction of the sphincter muscle but this action of substance P has not been previously demonstrated in the human eye. Using an eye cup model in which the sensitivity of the iris muscle to substance P is increased compared to the isolated sphincter muscle, we have observed that nanomolar amounts of substance P induced contraction of the sphincter in the human iris. This contractile response was enhanced in eyes pretreated with thiorphan, an enkephalinase inhibitor, suggesting that endogenous enkephalinase (E.C. 3.4.24.11) may modulate the substance P contraction in the human iris. Further support for this hypothesis was the finding of enkephalinase-like immunoreactivity and enzyme activity in the human iris sphincter muscle.     

Growth regulation of human breast carcinoma occurs through regulated growth factor secretion

We describe studies on human breast cancer in which it is shown that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone-dependent breast cancer and secreted constitutively by hormone-independent cells. These growth factor activities can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta, a growth-inhibitory substance, when treated with antiestrogens. TGF beta functions as a negative autocrine growth regulator and is responsible for some of the growth-inhibitory effects of antiestrogens.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

Characterization of Niemann-Pick Type C2 Protein Expression in Multiple Cancers Using a Novel NPC2 Monoclonal Antibody

Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.     

Progesterone potentiating effect of Dipsacus mitis D. Don for its contraceptive action in hamster

A pure compound, isolated from ethyl acetate extract (root) of D. mitis D. Don, prevented pregnancy by 100% in adult female hamster but partially in rat when administered orally on Days 1-7 and 1-10 post-coitum respectively. The effective dose in both species was 150 mg/kg. Using uterine wet weight in ovariectomized immature rat as bioassay method, the compound was found to be devoid of estrogenic and antiestrogenic property. On examination for progestational and antiprogestational activity, using trauma-induced deciduoma formation in immature rat uterus as end points, the compound (per se) did not show the former activity but in a conjoint treatment with progesterone it augmented the action of latter. The compound was assumed to act by potentiating progesterone biosynthesis, the excess of which might be the cause for interruption of pregnancy in hamster. This is the first study to report contraceptive efficacy and mode of its action at the uterine level.     

Mullerian inhibiting substance inhibits breast cancer cell growth through an NFkappa B-mediated pathway

Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.     

Phenazine methosulfate induces a neurally-mediated contraction of the guinea-pig ileum

Phenazine methosulfate (PMS) and related phenazines are widely used in biochemistry and histochemistry and act as anti-bacterial agents, however, there is little information on their pharmacological actions. In the present paper the guinea-pig ileum was used as a model for studying the effects of PMS on nerve cells. PMS was found to contract intestinal muscle. This action appeared to be mediated by the activation of muscarinic receptors since it was blocked by atropine. Neostigmine potentiated the response to PMS. The nerve blocker tetrodotoxin prevented the effect of PMS and it is concluded that PMS causes the release of acetylcholine from nerve elements. The action of PMS on nerves is not mediated by nicotinic receptors. Receptors for serotonin, substance P or cholecystokinin also appear not to be involved. Of all the phenazines tested PMS was found to be the most potent and reversible.     

Characterization of immunosuppressive substances in the basic protein fraction of uterine secretions from pregnant ewes

The basic protein fraction of ovine uterine secretions collected late in pregnancy (Days 125-140) contains a substance capable of inhibiting in vitro blastogenic responses of lymphocytes to phytohemagglutinin (PHA) or mixed lymphocyte reactions. In this study, the immunosuppressive substance in the basic protein fraction of uterine secretions was further defined by gel filtration. The immunosuppressive activity resided in a group of high molecular weight proteins eluting at the void volume of Sephacryl S-200 and Sepharose CL-6B columns. For example, incorporation of thymidine by PHA-stimulated lymphocytes incubated with 20, 40, 80, and 120 micrograms/ml of protein from the void volume of Sepharose CL-6B was 65, 28, 2, and 0 percent of control lymphocytes, respectively. Based on polyacrylamide-gel electrophoresis in the presence of sodium dodeylsulfate (SDS), the immunosuppressive fraction from Sepharose CL-6B chromatography contained aggregates of uterine milk proteins (UTM-proteins) and a pair of proteins running at the top of a 5% (w/v) polyacrylamide gel. Other protein peaks resolved by Sephacryl S-200 and Sepharose CL-6B contained aggregates of UTM-proteins but were not immunosuppressive. The substance inhibiting in vitro lymphocyte function was not of conceptus origin, because it was found in fluid from the ligated uterine horn of unilaterally pregnant ewes and from the uterus of an ovariectomized ewe treated for 60 days with progesterone and estrone.     

Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen.

The anti-estrogenic substance tamoxifen is effective in the adjuvant therapy applied in human breast cancer. Since it partly exhibits estrogenic activity and has serious side-effects, however, pure anti-estrogenic compounds are being sought. In our experimental study, we compared the anti-proliferative effect of estradiol and 13 endogenous estradiol metabolites on human breast cancer cells with the effect of tamoxifen. We used MCF-7 and MDA-MB 231, the well-established estrogen receptor-positive and -negative cell lines. 4-hydroxytamoxifen, the active metabolite of tamoxifen, estradiol and 13 estradiol metabolites were tested in concentrations ranging from 3.1 to 100 microM. Incubation time was 4 days and cell proliferation was measured by means of the ATP chemosensitivity test. 4-hydroxytamoxifen showed an IC50 value of 27 microM and 18 microM in MCF-7 and MDA-MB 231 cells, respectively. Estradiol and its metabolites were anti-proliferative in both cell lines. A few A-ring metabolites were more effective in inhibiting cell proliferation than D-ring metabolites and the parent substance 17beta-estradiol. 4-OHE1, 2-MeOE1 and 2-MeOE2 were as effective in both cell lines as tamoxifen. For the first time it has been demonstrated that endogenous estradiol metabolites are equally anti-proliferative as tamoxifen in the context of human breast cancer cells. Since some of these metabolites exhibit no estrogenic activity, they are likely to be valuable in clinical studies of chemoprevention and adjuvant therapy of breast cancer.     

Effect of new organophosphates on the membrane of identified central neurons of Helix pomatia L. (Mollusca, Gastropoda)

Effects of two newly synthesized organophosphates were studied on identified neurons of Helix pomatia by microelectrophysiological methods. The single intracellular spikes were processed by computer using a "phase-plane trajectory" method. Dimethoate was also involved as reference substance. The mechanism of action of the substance NE-79297 was found to be similar to that of dimethoate resulting in prolongation of action potentials due to a delayed rectification of the outward current. Phosmethylan (NE-79168), a much more selective compound, altered the membrane parameters in a different way: it affected the slow--mainly calcium-mediated--inward current.     

Modulatory effect of three antibiotics on uterus bovine contractility in vitro and likely therapeutic approaches in reproduction

This in vitro study investigates the modulatory effect of three antibiotics (amoxicillin, enrofloxacin, and rifaximin) on contractility of the bovine uterine tissue in follicular and luteal phases. The effects of these antibiotics at three single doses (10⁻⁶, 10⁻⁵, and 10⁻⁴ M) on their basal contractility were evaluated in isolated organ bath. The functionality of the strip throughout the experiment was evaluated by a dose of carbachol (10⁻⁵ M); the obtained effect had to be repeatable (difference of ≤20%) that is comparable to that induced by the previous administration of the same substance. The results demonstrate the different modulatory activities of these antibiotics on uterine contractility in follicular and luteal phases. The effects induced by amoxicillin and enrofloxacin are opposite: the first relaxes and the second increases the uterine contractility in both cycle phases. Instead, the activity of rifaximin varies depending on the phase of estrous cycle: it increases in the follicular phase and relaxes in the luteal phase. The obtained data provide the hypothesis of possible implications of these drugs in the pharmacologic modulation of uterine contractions. Their action at this level, associated with their specific antimicrobial effects, could suggest using these antibiotics for the treatment of diseases related to postpartum or infections that may occur in pregnant cattle, by virtue of their effects on myometrial contractility too.