Magnetic Resonance Imaging Reveals Distinct Roles for Tissue Transglutaminase and Factor XIII in Maternal Angiogenesis During Early Mouse Pregnancy.

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.

A daidzein-daunomycin conjugate improves the therapeutic response in an animal model of ovarian carcinoma.

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.

A sorafenib-sparing effect in the treatment of thyroid carcinoma cells attained by co-treatment with a novel isoflavone derivative and 1,25 dihydroxyvitamin D3.

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 µg/ml). This inhibition was similar to that of the generally used concentration of sorafenib (200 µg/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.

Synthesis and evaluation of the antiproliferative activities of derivatives of carboxyalkyl isoflavones linked to N-t-Boc-hexylenediamine.

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.

25 hydroxy-vitamin D(3)-1alpha hydroxylase expression and activity in cultured human osteoblasts and their modulation by parathyroid hormone, estrogenic compounds and dihydrotestosterone.

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.

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis.

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.

25-hydroxyvitamin D3-1alpha-hydroxylase is expressed in human vascular smooth muscle cells and is upregulated by parathyroid hormone and estrogenic compounds.

BACKGROUND: 1,25(OH)2 vitamin D3 exerts multiple effects in human vascular smooth muscle cells (VSMCs). We therefore tested the possibility that VSMCs possess an endogenous 25-hydroxyvitamin D3-1alpha-hydroxylase system, the final enzyme in the biosynthetic pathway of 1,25(OH)2D3. METHODS AND RESULTS: We assessed the expression and activity of 25-hydroxyvitamin D3-1alpha-hydroxylase by real-time polymerase chain reaction and the conversion of 25(OH)D3 into 1,25(OH)2D3. First, 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA was identified in cultured VSMCs by real-time polymerase chain reaction. Second, in cells treated daily (3 days) with parathyroid hormone (66 nmol/L), estradiol-17beta (30 nmol/L), raloxifene (3 micromol/L), and the phytoestrogens genistein (3 micromol/L), biochainin A (3 micromol/L), and 6-carboxy biochainin A (30 nmol/L), 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA increased by 43+/-13%, (P<0.05) 7+/-24% (P=NS), 176+/-28% (P<0.01), 65+/-11% (P<0.05), 152+/-24% (P<0.01), and 71+/-9% (P<0.05), respectively. Third, production of 1,25(OH)2D3 from 25(OH)D3 was seen with a Km of 25 ng/mL and increased dose dependently after treatment with parathyroid hormone, genistein, and the phytosetrogen derivative 6-carboxy biochainin A. Estradiol-17beta and biochainin A also increased the generation of 1,25(OH)2D3 by 40+/-23% (P<0.05) and 55+/-13% (P<0.05), respectively. CONCLUSIONS: We provide here the first evidence for the expression of an enzymatically active 25(OH)D3-1alpha-hydroxylase system in human VSMCs, which can be upregulated by parathyroid hormone and estrogenic compounds. Because exogenous vitamin D inhibits VSMC proliferation, the role of this system as an autocrine mechanism to curb changes in VSMC proliferation and phenotype is a subject for future investigation.

Responsiveness to estradiol-17beta and to phytoestrogens in primary human osteoblasts is modulated differentially by high glucose concentration.

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.

Responsiveness to phytoestrogens in primary human osteoblasts is modulated differentially by a "less-calcemic" analog of 1,25 dihydroxyvitamin D(3): JK 1624F(2)-2 (JKF).

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.

Estradiol-17ß increases 12- and 15-lipoxygenase (type2) expression and activity and reactive oxygen species in human umbilical vascular smooth muscle cells.

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 (∼50%) 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.

Role of putative membrane receptors in the effects of estradiol on human vascular cell growth.

The present study was designed to determine whether some of the effects of estrogen on human vascular cell growth are exerted through membrane-binding sites, using native as well as novel protein-bound, membrane non-permeant estrogenic complexes. We measured changes in DNA synthesis and creatine kinase-specific activity (CK), after treatment with estradiol-17beta (E(2)), estradiol-17beta-6-(O)-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (E(2)-BSA), 6-carboxymethyl genistein (CG) or 6- carboxymethyl genistein bound to the high molecular protein keyhole limpet hemocyanin (CG-KLH), and 7-(O)-carboxymethyl daidzein (CD) or 7-(O)-carboxymethyl daidzein linked to keyhole limpet hemocyanin (CD-KLH). High concentrations of either E(2) or E(2)-BSA inhibited DNA synthesis in vascular smooth muscle cells (VSMC) (-39% +/- 28% v -32% +/- 15%). Estradiol as well as CG and CD increased DNA synthesis dose dependently in endothelial ECV-304 cells. The CG and CD, as well as CG-KLH and CD-KLH, stimulated DNA synthesis dose dependently in VSMC (66% +/- 2%, 100% +/- 12%, 66% +/- 6%, and 41% +/- 8% at 300 nmol/L, respectively). In contrast all forms of protein-bound hormones were unable to affect DNA synthesis in ECV-304 cells or CK in either cell type. In VSMC, both free and bound hormones increased mitogen-activated protein-kinase (MAPK)-kinase activity, which was blocked by UO126, an inhibitor of MAPK-kinase. Furthermore, the effects of E(2), E(2)-BSA, or CG-KLH on DNA synthesis were inhibited by UO126. Using the E(2)-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for E(2) in VSMC and ECV 304 cells. Hence, the effects of E(2) on DNA synthesis in human VSMC, but not in endothelial cells, are apparently exerted by membrane-binding sites for E(2) and do not require intracellular entry of E(2) through the classic nuclear receptor route.

High glucose blocks the effects of estradiol on human vascular cell growth: differential interaction with estradiol and raloxifene.

Because diabetic women appear not to be protected by estrogen in terms of propensity to cardiovascular disease, we tested the possibility that chronic hyperglycemia modulates the effects of E(2) on vascular cell growth in vitro. Human endothelial cells (E304) and vascular smooth muscle cells (VSMC) were grown in normal glucose (5.5 mmol/l), high glucose (22 mmol/l) or high manitol (22 nmol/l; an osmotic control) for 7 days. In endothelial cells glucose per se stimulated DNA synthesis. However E(2)- (but not RAL-) stimulated [3H] thymidine incorporation was attenuated in the presence of high glucose. In parallel, E(2)-dependent MAP-kinase-kinase activity was blocked in the presence of high glucose. High glucose increased basal creatine kinase (CK) specific activity, but E(2)-stimulated CK was not significantly impaired in the presence of high glucose. In VSMC, high glucose prevented the inhibitory effect of high E(2) (but not of high RAL) concentrations on DNA synthesis. High glucose also prevented E(2)-induced MAP-kinase-kinase activity. In contrast, while high glucose augmented basal CK, the relative E(2)-induced changes were roughly equal in normal and high high glucose media. Hence, high glucose blocks several effects of E(2) on vascular cell growth, which are mediated, in part, via the MAP-kinase system and are likely contributors to E(2)'s anti-atherosclerotic properties. Since RAL's estrogen-mimetic effects on human vascular cell growth were independent of MAP-kinase activation and were not affected by hyperglycemia, the potential use of RAL to circumvent the loss of estrogen function induced by hyperglycemia and diabetes in the human vasculature should be further explored.

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells.

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.

Modulation of response to estrogens in cultured human female bone cells by a non-calcemic Vitamin D analog: changes in nuclear and membranal binding.

Estradiol17beta (E2) and the phytoestrogens genistein (G), and daidzein (D) increase creatine kinase (CK) specific activity in primary cell cultures of human female to a greater extent in cells from pre-menopausal than post-menopausal women. Pretreatment with the non-calcemic analog of Vitamin D, JK 1624 F2-2 (JKF), upregulated this estrogenic response at all ages. In contrast, biochainin A (BA) and quercertin (Qu) increased CK with no age dependence or modulation by JKF pretreatment. Both ERalpha and ERbeta present in the cells were upregulated by pretreatment with JKF, as measured by Western blot analysis. Real time PCR showed no significant change in ERalpha mRNA but a marked decrease in ERbeta mRNA in both age groups after JKF treatment. Cells from both age groups had surface binding sites for E2, shown by assays using cell impermeable Europium labeled ovalbumin-E2 conjugate (Eu-Ov-E2). Binding of [3H]-E2 to intracellular E2 receptors (ERs) was similar in both age groups with differences in phytoestrogenic competition. JKF pretreatment increased nuclear but decreased membranal binding in both age groups. These results provide evidence for membranal, in addition to nuclear estrogen receptors which are differentially modulated by a Vitamin D analog.

A synthetic peptide with estrogen-like activity derived from a phage-display peptide library.

We describe a novel approach to develop peptides with estrogen like activity using a monoclonal antibody specific to estradiol (mAb E2-15) for the affinity selection of phage displayed peptides from a combinatorial peptide library. Based on the sequences of the selected phage, we synthesized a 15-mer linear peptide LPALDPTKRWFFETK which was derivatized to a 23 mer cyclic peptide CAELPALDPTKRWFFETKPPPPC. Both peptides displayed estrogen-like activity according to the following criteria:(i) in inhibiting the binding of [3H]estradiol to mAb E2-15 and to estrogen receptor (ER)alpha; (ii) in inducing transcriptional activity in MCF7 human breast cancer cells transfected with an estrogen receptor element luciferase construct and (iii) in causing an increase in creatine kinase specific activity in rat tissues in vivo. This approach can be employed to design peptide mimetic for other hormones as well.

The role of the 3' region of mammalian gonadotropin ß subunit gene in the luteinizing hormone to chorionic gonadotropin evolution.

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.

Angiotensin 1-7 as means to prevent the metabolic syndrome: lessons from the fructose-fed rat model.

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.

Growth inhibition of human thyroid carcinoma and goiter cells in vitro by the isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine.

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 (∼-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.

Anti-proliferative effects of a novel isoflavone derivative in medullary thyroid carcinoma: an in vitro study.

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.

Conjugates of daidzein-alliinase as a targeted pro-drug enzyme system against ovarian carcinoma.

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.