ABSTRACT
OBJECTIVE: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood. Approach and Results: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast-enhanced-magnetic resonance imaging. Dynamic contrast-enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis. CONCLUSIONS: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.Visual Overview: An online visual overview is available for this article.
Subject(s)
Embryo Implantation/physiology , Factor XIII/physiology , GTP-Binding Proteins/physiology , Magnetic Resonance Imaging/methods , Neovascularization, Physiologic/physiology , Transglutaminases/physiology , Animals , Female , Fibrinogen/physiology , Mice , Pregnancy , Protein Glutamine gamma Glutamyltransferase 2ABSTRACT
BACKGROUND: Sorafenib improves progression-free survival in patients with progressive radioactive iodine-refractory differentiated thyroid carcinoma, but causes severe side effects. Estrogens may accelerate thyroid carcinoma cell growth. Our group recently reported that isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine (cD-tboc), a novel anti-estrogenic compound, retards the growth of both thyroid carcinoma cell lines and cultured human carcinoma cells. Vitamin D receptor (VDR) is expressed in malignant cells and responds to 1,25 dihydroxyvitamin D3 (1.25D) by decreased proliferative activity in vitro. The purpose of this study was to examine the effects of vitamin D metabolites (VDM) on the expression of estrogen receptors (ERs), VDR, and 1OHase mRNA, and to evaluate the inhibitory effect of low doses of sorafenib in combination with cDtboc and VDM on cell proliferation in cultured human papillary thyroid carcinoma (PTC). METHODS: In 19 cultured PTC specimens and 19 normal thyroid specimens, harvested during thyroidectomies from the same patients, expression levels of ERα, ERß, VDR, and 1 alpha-hydroxylase (1OHase) mRNA (by quantitative real-time PCR) were determined at baseline and after treatment with VMD. Cell proliferation was determined by measurement of 3[H] thymidine incorporation after treatment with sorafenib alone, sorafenib with added 1.25D or cD-tboc, and sorafenib with both 1.25D and cD-tboc added. RESULTS: 1,25D increased mRNA expression of all tested genes in the malignant and normal thyroid cells, while the ERα mRNA of the normal cells was unaffected. 1.25D dose-dependently inhibited cell proliferation in the malignant cells. The inhibitory effect of sorafenib on cell proliferation in the malignant cells was amplified after the addition of cDtboc and 1.25D, such that the maximal inhibition was not only greater, but also had been attained at a 10-fold lower concentration of sorafenib (20⯵g/ml). This inhibition was similar to that of the generally used concentration of sorafenib (200⯵g/ml) alone. CONCLUSIONS: The demonstration that low concentrations of cDtboc and 1.25D markedly amplify the inhibitory effect of sorafenib on the growth of human PTC supports the use of a 10-fold lower concentration of sorafenib. The findings may promote a new combination treatment for progressive radioactive iodine-refractory PTC.
Subject(s)
Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Isoflavones/pharmacology , Sorafenib/pharmacology , Thyroid Gland/drug effects , Thyroid Neoplasms/pathology , Vitamin D/analogs & derivatives , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Adolescent , Adult , Aged , Antineoplastic Agents/pharmacology , Case-Control Studies , Cells, Cultured , Drug Therapy, Combination , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Female , Humans , Male , Middle Aged , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Thyroid Gland/metabolism , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/metabolism , Vitamin D/pharmacology , Young AdultABSTRACT
Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe-/- mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe-/- mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Atherosclerosis/drug therapy , Hyaluronic Acid/therapeutic use , Macrophages/drug effects , Nanoparticles/therapeutic use , Plaque, Atherosclerotic/drug therapy , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacokinetics , Atherosclerosis/diagnostic imaging , Atherosclerosis/pathology , Hyaluronic Acid/chemistry , Hyaluronic Acid/pharmacokinetics , Macrophages/pathology , Male , Mice , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Plaque, Atherosclerotic/diagnostic imaging , Plaque, Atherosclerotic/pathology , Positron-Emission Tomography , Rabbits , Tissue DistributionABSTRACT
The net vascular effect of estrogens on the vasculature is still under debate. Here we tested the effects of estradiol- 17ß (E2) as well as estrogen-receptor subtype specific and non-specific agonists and antagonists on the expression and eicosanoid production of lipoxygenase (LO) enzymes expressed in culture human umbilical vascular smooth muscle cells (VSMC), the platelet type 12LO and 15LO type 2. E2 increased 12 and 15LO mRNA expression by 2-3 folds and elicited an acute 50% increase 12 and 15 hydroxyeicosatetraenoic acid (HETE) production. Neither estrogen receptor ERα nor ERß-specific agonists were able to reproduce the induction of LO expression, but E2-induced expression was effectively blocked by ER non-specific and receptor subtype specific antagonists. Because 12 and 15HETE can increase reactive oxygen species in other cell types, we tested the possibility that E2 could raise ROS through LO. Indeed, E2 as well as the LO products 12 and 15HETE increased reactive oxygen species (ROS) in VSMC. E2-dependent and HETE-induced ROS could be blocked by NAD (P) H-oxidase inhibitors and by the ER general antagonist ICI. E2-induced ROS was partially (â¼50%) blocked by the LO inhibitor baicalein, but the LO blocker had no effect on 12 or 15HETE- induced ROS formation, thus suggesting that part of E2-dependent ROS generation resulted from E2-induced 12 and 15HETE. Collectively these findings unveil an unrecognized effect of E2 in human VSMC, to induce 12 and 15LO type 2 expression and activity and suggest that E2-dependent ROS formation in VSMC may be partially mediated by the induction of 12 and 15HETE.
Subject(s)
Arachidonate 12-Lipoxygenase/genetics , Arachidonate 15-Lipoxygenase/genetics , Estradiol/pharmacology , Myocytes, Smooth Muscle/drug effects , Reactive Oxygen Species/metabolism , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Arachidonate 12-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Flavanones/pharmacology , Gene Expression Regulation , Humans , Hydroxyeicosatetraenoic Acids/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Nitriles/pharmacology , Phenols/pharmacology , Piperidines/pharmacology , Primary Cell Culture , Propionates/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Raloxifene Hydrochloride/pharmacology , Reactive Oxygen Species/agonists , Umbilical Veins/cytology , Umbilical Veins/drug effects , Umbilical Veins/metabolismABSTRACT
CGß subunits comprise a unique carboxyl-terminal peptide (CTP) that has multiple O-linked glycans and extends serum half-life of the protein. It has evolved by incorporating a previously untranslated region of the LHß gene into the reading frame. Although CTP-like sequences are encrypted in the LHß genes of several mammals, the CGß subunit developed only in primates and equids. To study this restriction in evolution, we examined whether the cryptic CTP decoded from the bovine LHß gene (boCTP) possesses key characteristics of the human (h) CGß-CTP. The boCTP does not impede several crucial aspects of hormone biosynthesis, but compared to the hCGß-CTP, the stretch lacks O-glycans and determinants for circulatory survival. O-glycan deficiency and the associated incapacity to extend serum half-life is a major drawback of the boCTP. This may explain why LH did not evolve into CG in ruminants and consequently alternative mechanisms evolved to delay luteolysis early in gestation.
Subject(s)
3' Flanking Region , Chorionic Gonadotropin, beta Subunit, Human/genetics , Evolution, Molecular , Luteinizing Hormone/genetics , Polysaccharides/chemistry , Protein Subunits/genetics , Amino Acid Sequence , Animals , CHO Cells , Cattle , Chorionic Gonadotropin, beta Subunit, Human/chemistry , Chorionic Gonadotropin, beta Subunit, Human/metabolism , Cricetulus , Female , Gene Expression Regulation , Half-Life , Horses , Humans , Luteinizing Hormone/chemistry , Luteinizing Hormone/metabolism , Molecular Sequence Data , Open Reading Frames , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Polysaccharides/metabolism , Pregnancy , Protein Subunits/chemistry , Protein Subunits/metabolism , RatsABSTRACT
We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7-treated animals had lower body weight (-9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (-51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (-40%) and serum aldosterone (-48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7-treated rats. WAT from Ang 1-7-treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7-treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7-treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.
Subject(s)
Angiotensin I/administration & dosage , Cardiovascular Agents/administration & dosage , Dietary Carbohydrates/administration & dosage , Fructose/administration & dosage , Metabolic Syndrome/prevention & control , Peptide Fragments/administration & dosage , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Animals , Disease Models, Animal , Drug Administration Schedule , Epididymis/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Male , Muscle, Skeletal , Oxidative Stress , Phosphorylation , Proto-Oncogene Mas , Proto-Oncogene Proteins/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species , Receptors, G-Protein-Coupled/metabolism , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolismABSTRACT
Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) α mRNA expression in TT cells was more abundant than ERß, with a ratio of 48:1. Estradiol-17ß (E2) increased DNA synthesis in a dose dependent manner. [(3)H]-thymidine incorporation was also stimulated by the ERß agonist DPN and the ERα agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on [(3)H]-thymidine incorporation could be blocked by the ERß antagonist PTHPP but not by the ERα antagonist MPP, suggesting that the antiproliferative effect of cD-tBoc on these cells is mediated through ERß. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.
Subject(s)
Antineoplastic Agents/pharmacology , Isoflavones/chemistry , Thyroid Neoplasms/drug therapy , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Carcinoma, Neuroendocrine , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Creatine Kinase/antagonists & inhibitors , Creatine Kinase/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/antagonists & inhibitors , Estrogen Receptor beta/genetics , Humans , Isoflavones/pharmacology , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathologyABSTRACT
BACKGROUND: Estrogens may enhance thyroid cancer cell growth. We have recently reported that a novel isoflavone-derived anti-estrogenic compound developed in our laboratory, the N-t-boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc), can induce apoptosis and retard growth in human thyroid carcinoma cell lines through inhibitory interaction on estrogen receptor ß. Here we tested the hypothesis that cD-tboc can likewise retard cell growth in cultured human thyroid papillary carcinoma cells, normal thyroid cells, and goiter cells removed during thyroidectomy. METHODS: In vitro experiments in cultured human thyroid normal, goiter, and papillary thyroid carcinoma (PTC) cells were performed. Estrogen receptors α and ß (ERα and ERß), DNA synthesis and creatine kinase (a marker of estrogenic genomic response), and the effects of cD-tboc on DNA synthesis in cultured human PTC cells were assessed. RESULTS: First, all cell types thus harvested and grown in culture expressed both ERα and ERß, with a variably higher abundance of ERß over ERα seen in the goiter and PTC cells, but not in the normal thyroid cells. Second, DNA synthesis and creatine kinase were increased in response to estradiol-17ß (E2), the ERα agonist propyl-pyrazole-trisphenol as well as the ERß agonist diarylpropionitrile. Third, cD-tboc dose-dependently inhibited DNA synthesis in cultured human PTC cells (-65%) and to a lesser extent in goiter cells (â¼-30%). CONCLUSION: This study provides the first evidence that cD-tboc can act to inhibit growth in primary cultures of human PTC cells and goiter cells removed during thyroidectomy. Whether this can be utilized for the treatment of human thyroid cancer and/or goiter remains to be explored.
Subject(s)
Carcinoma/drug therapy , Estrogen Receptor beta/biosynthesis , Isoflavones/therapeutic use , Thyroid Neoplasms/drug therapy , Carcinoma, Papillary , Cell Line, Tumor , Cell Proliferation/drug effects , Estradiol/pharmacology , Estrogen Receptor alpha/biosynthesis , Estrogen Receptor alpha/drug effects , Estrogen Receptor beta/drug effects , Goiter/pathology , Humans , Isoflavones/pharmacology , Thyroid Cancer, Papillary , Thyroid Gland/pathologyABSTRACT
Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE-777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.
Subject(s)
Antineoplastic Agents/pharmacology , Carbon-Sulfur Lyases/pharmacology , Cysteine/analogs & derivatives , Isoflavones/pharmacology , Ovarian Neoplasms/drug therapy , Prodrugs/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Carbon-Sulfur Lyases/chemistry , Carbon-Sulfur Lyases/pharmacokinetics , Carbon-Sulfur Lyases/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , Cysteine/chemistry , Cysteine/pharmacokinetics , Cysteine/pharmacology , Cysteine/therapeutic use , Drug Compounding , Female , Fluorescent Dyes/chemistry , Humans , Isoflavones/chemistry , Isoflavones/pharmacokinetics , Isoflavones/therapeutic use , Luciferases/genetics , Mice , Mice, Nude , Molecular Imaging , Ovarian Neoplasms/metabolism , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Prodrugs/therapeutic use , Tissue Distribution , Transfection , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
One of the major difficulties in the treatment of epithelial ovarian cancer (EOC) is the high rate of recurrent disease. This is thought to be due to the survival of a population of chemo-resistant cells within the tumor, the ovarian cancer stem cells (OCSCs), that are able to regenerate the tumor following chemotherapy. Therefore, the identification of a compund that can target the OCSCs is one of the main steps in improving overall survival of ovarian cancer patients. The objective of this study was to determine the effect of N-t-boc-Daidzein, a novel daidzain derivative, on OCSCs. The efficacy of this compound was evaluated in OCSC and mature ovarian cancer cell (mOCC) lines isolated from malignant ovarian cancer asicites. Cells were treated with increasing concentrations of N-t-boc-Daidzein (0.003-10 microM) and cell growth was monitored by "real time in vitro micro-imaging" using the IncuCyte system. Cell viability was measured using the CellTiter 96 Assay. Apoptosis was determined by Caspase-Glo 3/7, 8 and 9 assays. The components of the apoptotic cascade were characterized by western blot analysis. N-t-boc-Daidzein was able to significantly inhibit cell growth and decrease cell viability of OCSC as well as mOCC cells in a dose and time dependent maner. This effect was due to the induction of apoptosis, which is characterized by caspase activation, XIAP and AKT degradation, and mitochondrial depolarization. This study describes a novel compound that can target the OCSCs. These findings may provide vital aide in improving overall survival in patients with EOC.
Subject(s)
Apoptosis/drug effects , Carbamates/pharmacology , Cell Proliferation/drug effects , Chromones/pharmacology , Isoflavones/pharmacology , Neoplastic Stem Cells/drug effects , Blotting, Western , Carbamates/chemistry , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Chromones/chemistry , Dose-Response Relationship, Drug , Epithelial Cells/pathology , Female , Flow Cytometry , Humans , Isoflavones/chemistry , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/physiology , Models, Biological , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Oncogene Protein v-akt/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Time Factors , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)-based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells.
Subject(s)
Endocytosis/physiology , Ovarian Neoplasms/blood , Ovarian Neoplasms/pathology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Biological Transport , Female , Humans , Isoflavones/metabolism , Isoflavones/pharmacokinetics , Mice , Mice, Nude , Nystatin/metabolism , Ovarian Neoplasms/drug therapy , Pentetic Acid/metabolism , Serum Albumin, Bovine/pharmacokinetics , Tissue DistributionABSTRACT
Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (microCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new microCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.
Subject(s)
Bone Development/drug effects , Bone and Bones/drug effects , Oligopeptides/chemistry , Oligopeptides/pharmacology , Animals , Dose-Response Relationship, Drug , Estrogens/chemistry , Female , Mice , Mice, Inbred Strains , Molecular Mimicry , Ovariectomy , X-Ray MicrotomographyABSTRACT
We reported previously that high concentrations of either estradiol-17beta (E(2)) or dihydrotestosterone (DHT) inhibit growth of human cultured vascular smooth muscle cells (VSMC), mediated by cell membrane receptors and MAP-kinase-kinase activity (MEK). We now tested whether the presence of the opposite gender's dominant sex hormone modifies these effects. We incubated VSMC with various concentrations of E(2) and DHT or protein bound hormones (E(2)-BSA or T-BSA), alone or in various combinations. High concentration of E(2) or E(2)-BSA inhibited VSMC growth and stimulated MEK. In the presence of 3 nM DHT, high concentration of E(2) no longer inhibited (3)[H] thymidine incorporation or increased MEK. Moreover, when high DHT concentration (300 nM) was added to VSMC exposed to high E(2), VSMC growth actually increased without change in MEK. DHT at 300 nM suppressed VSMC growth and increased MEK while 0.3 nM E(2) had only marginal effect on this interaction, and 30 nM E(2) reversed the inhibitory effect of DHT on cell growth. The inhibitory effects of both E(2) and DHT on VSMC cell growth and the stimulation of MEK was apparently mediated by cell membrane receptors, as it persisted when bovine serum albumin (BSA)-bound hormones were used. Further, inhibition of VSMC growth induced by E(2)-BSA was reversed in the presence of T-BSA and vice versa. These results suggest that while female and male sex hormones affect VSMC growth similarly, they interfere in a dose-, hormone- and MEK-dependent manner with each other's effect.
Subject(s)
Dihydrotestosterone/metabolism , Estradiol/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/physiology , Animals , Cattle , Cells, Cultured , DNA/biosynthesis , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Female , Humans , MAP Kinase Signaling System/physiology , Male , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Myocytes, Smooth Muscle/cytology , Serum Albumin, Bovine/metabolismABSTRACT
The use of daunomycin against neoplasms is limited due to its severe cardiotoxicity. The cytotoxicity of daunomycin can be minimized by linking it to an affinity tag. Since ovarian cancer cells are sensitive to isoflavone action, we synthesized a daidzein daunomycin conjugate. In MLS human ovarian cancer cells, the conjugate was shown to have a larger cytotoxic effect than daunomycin per se at a low concentration. The conjugate was then tested in vivo in mice carrying MLS xenografts. Tumour growth in the groups of conjugate and daunomycin was inhibited by >50% as compared to vehicle treated mice. In contrast to daunomycin treated mice, no weight reduction or death was seen in mice treated with the conjugate. In vivo imaging of the fluorescence signal generated by daunomycin indicated uptake of both conjugate and daunomycin by the tumour. Tumour fluorescence was, however, higher in the conjugate treated mice than in the daunomycin treated mice, thus suggesting specific delivery of the drug to the tumour. Histological examination of myocardial tissue indicated that only the daunomycin, but not conjugate treated mice showed cardiac damage. These results indicate that targeting of daunomycin via carboxymethyldaidzein retains daunomycin's cytotoxic effects while averting its toxicity in an ovarian xenograft.
Subject(s)
Daunorubicin/pharmacology , Isoflavones/pharmacology , Ovarian Neoplasms/drug therapy , Xenograft Model Antitumor Assays/methods , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , Daunorubicin/chemistry , Daunorubicin/therapeutic use , Female , Humans , Isoflavones/chemistry , Isoflavones/therapeutic use , Mice , Mice, Nude , Molecular Structure , Ovarian Neoplasms/pathology , Phytoestrogens/chemistry , Phytoestrogens/pharmacology , Phytoestrogens/therapeutic use , Tumor Burden/drug effectsABSTRACT
The isoflavones biochanin A ( 1a), genistein ( 1b), and daidzein ( 4) at concentrations >20 microM inhibit cell growth of various cancer cell lines. To enhance the antiproliferative activities of these compounds, we synthesized three analogs, 2-[3-carboxy-(6-tert-butoxycarbonylamino)-hexylamino-propyl]-7,5-dihydroxy-4'-methoxyisoflavone ( 3a), 2-[3-[N-[6-(tert-butoxycarbonyl)-aminohexyl]]-caboxamidopropyl]-5,7,4'-trihydroxyisoflavone ( 3b), and 5-{2-[3-(4-hydroxy-phenyl)-4-oxo-4 H-chromen-7-yloxy]-acetylamino}-pentyl)-carbamic acid tert-butyl ester ( 6). When cancer cells expressing predominantly estrogen receptor mRNA of the beta- relative to alpha-subtype were treated with 3a, 3b, or 6, DNA synthesis was inhibited in a dose-dependent manner, ranging from 15 to 3000 nmol/L, with little inhibitory effect in normal vascular smooth muscle cells. Compound 6 was the most potent one, and its antiproliferative effect in cancer cells was modulated by estrogen and by the apoptosis inhibitor Z-VADFK. When tested in vivo, compound 6 decreased tumor volume of ovarian xenografts by 50%, with no apparent toxicity. Compound 6 may be a promising agent for therapy of cancer either alone or in combination with chemotherapeutic agents.
Subject(s)
Antineoplastic Agents/chemical synthesis , Carbamates/chemistry , Chromones/chemistry , Isoflavones/chemical synthesis , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Colonic Neoplasms , DNA/biosynthesis , Drug Interactions , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/biosynthesis , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/biosynthesis , Estrogen Receptor beta/genetics , Female , Humans , Isoflavones/chemistry , Isoflavones/pharmacology , Male , Mice , Mice, Nude , Muscle, Smooth, Vascular/cytology , Neoplasm Transplantation , Ovarian Neoplasms , Ovary/cytology , Prostate/cytology , RNA, Messenger/biosynthesis , Transplantation, HeterologousABSTRACT
Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.
Subject(s)
25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Dihydrotestosterone/pharmacology , Estrogens/pharmacology , Osteoblasts/drug effects , Parathyroid Hormone/pharmacology , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Base Sequence , Cells, Cultured , DNA Primers , Humans , Osteoblasts/enzymology , RNA, Messenger/geneticsABSTRACT
The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to alpha-actin, a component of the cytoplasmic myofilaments. 12-LO/alpha-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to alpha-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein alpha-actin.
Subject(s)
Actins/metabolism , Arachidonate 12-Lipoxygenase/physiology , Muscle, Smooth, Vascular/metabolism , Angiotensin II/physiology , Humans , Immunohistochemistry , Isoenzymes/physiology , Microscopy, Electron , Muscle, Smooth, Vascular/cytology , Protein Transport , Subcellular Fractions/enzymology , Umbilical Arteries/cytology , Umbilical Arteries/metabolismABSTRACT
BACKGROUND: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women. METHODS: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction. RESULTS: Comparison of monoclonal TR-FIA, commercial polyclonal antibody-based TR-FIA, and gas chromatography-mass spectrometry showed correlations (r, 0.911-0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied. CONCLUSIONS: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status.
Subject(s)
Antibodies, Monoclonal , Coronary Disease/prevention & control , Dietary Supplements , Genistein/analysis , Isoflavones/analysis , Animals , Biological Availability , Equol , Female , Fluoroimmunoassay/methods , Gas Chromatography-Mass Spectrometry , Genistein/blood , Genistein/urine , Humans , Isoflavones/blood , Isoflavones/urine , Mice , Mice, Inbred BALB C , Postmenopause , Reproducibility of ResultsABSTRACT
We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and to raloxifene (Ral), whereas male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. In cells derived from pre-menopausal women, E(2), G, D and Ral stimulated CK to higher extent compared to post-menopausal bone cells, whereas quecertin (Qu), carboxy-biochainin A (cBA) and carboxy-genistein (cG) stimulated CK in both age groups similarly, and biochainin A (BA) stimulated post-menopausal cells to a bit higher extent than pre-menopausal cells. Since the skeletal protective effects of estrogens are not discernable in diabetic women, we tested in this study, the effects of high glucose concentration in the growth medium, on the effects of estrogenic compounds on CK in human-derived bone cells (hObs). Female-derived hObs were grown either in normal (4.5 g/l; 22 mM, NG) or high glucose (9.0 g/l; 44 mM, HG) for 7 days. HG increased constitutive CK, but attenuated E(2)- and DHT-induced CK in female or male hObs, respectively. HG also inhibited genistein (G) and daidzein (D) stimulated CK in female hObs, but not the effects of biochainin A (BA), quecertin (Qu) or Ral. Intracellular, mainly nuclear binding of (3)[H]E(2) was characteristic of the different phytoestrogens in female hObs, was abolished by HG. Membranal binding of Eu-Ov-E(2), was displaced only by E(2)-Ov, ICI, cG-Ov or cD-Ov but decreased total binding of Eu-Ov-E(2) in both age groups and completely abolished the competition with E(2)-Ov or ICI in both age groups, but the competition with cD-Ov and cG-Ov was decreased only slightly but not statistically significant. HG also abolished Eu-BSA-T, which bound similarly male-derived hObs. All hObs expressed mRNA for ERalpha and ERbeta with higher abundance of ERalpha. HG increased mRNA for both ERs in female-derived hObs, but decreased mRNA for both ERs in male-derived hObs. Hence, human bone cells, which express specific nuclear and membranal binding sites for estrogenic compounds, are modulated by HG, leading to altered hormonal responsiveness, which might block important effects of estrogenic compounds, contributing probably to their decreased skeletal preserving properties under hyperglycemia.
Subject(s)
Estradiol/pharmacology , Glucose/pharmacology , Osteoblasts/drug effects , Phytoestrogens/pharmacology , Adult , Age Factors , Aged , Aged, 80 and over , Binding, Competitive/drug effects , Cell Membrane/metabolism , Cells, Cultured , Creatine Kinase, BB Form/metabolism , Dihydrotestosterone/pharmacology , Dose-Response Relationship, Drug , Estradiol/metabolism , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/genetics , Female , Gene Expression/drug effects , Genistein/analogs & derivatives , Genistein/pharmacology , Humans , Male , Middle Aged , Osteoblasts/cytology , Osteoblasts/metabolism , Phytoestrogens/metabolism , Quercetin/pharmacology , Sex FactorsABSTRACT
We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and raloxifene (Ral), and male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. We also found that pre-treatment with the less-calcemic analog of Vitamin D, JK 1624 F(2)-2 (JKF), upregulated the response of CK to E(2) and Ral. In this study, we analyzed the response of human bone cells from pre- and post-menopausal females and males, to phytoestrogens. Bone cells derived from pre-menopausal women showed greater stimulation of CK than cells from post-menopausal women, after treatment with E(2) (30 nM), daidzein (D, 3000 nM), genistein (G, 3000 nM) and Ral (3000 nM); whereas the responses to biochainin A (BA 3000 nM), quecertin (Qu 3000 nM) or the carboxy derivative of G (cG 300 nM) were not age-dependent. Male-derived cells did not respond to phytoestrogens. When cells derived from female bones at both age groups were pre-treated with JKF, by daily addition of 1nM, for 3 days, there was an upregulation of the response to E(2), Ral, G and D but not to BA or Qu. Nuclear binding of (3)[H] E(2) was characteristic of the different phytoestrogens, with increase of the specific binding after pre-treatment with JKF. In contrast, the membranal binding of E(2)-Ov-Eu, which was specific for the estrogenic compounds except Ral, was inhibited by pre-treatment with JKF except for ICI 161480 (ICI). Male bone cells did not bind E(2)-Ov-Eu but bound T-BSA-Eu; this binding was abolished by pre-treatment with JKF. Pre-treatment with JKF increased mRNA for ERalpha and decreased mRNA for ERbeta in bone cells from both age groups of females and from males, all of which expressed both ERs, with a ratio of ERalpha to ERbeta of 121:1 in pre- and 78:1 in post-menopausal and 105:1 in male bone cells. This study raises the possibility of combined Vitamin D analog and phytoestrogen for hormonal replacement therapy to prevent post-menopausal osteoporosis, which is a subject of ongoing research in animal models.