Pancreatic Beta-Cell Proliferation Induced by Estradiol-17ß is Foxo1 Dependent.

Estradiol-17ß (E2) and the Foxo1 transcription factor have each been implicated in the regulation of ß-cell proliferation. Interaction between Foxo1and estrogen receptor alpha (ERα), effecting cell cycle, has been demonstrated in breast cancer cells, but has not been studied thus far in ß-cells. Using human islets and the INS1-E ß-cell line, this study investigated the contribution of Foxo1 to E2-mediated ß-cell replication. Foxo1 expression was knocked down in INS1-E cells using siRNA and Foxo1 activity was inhibited in human islets with a specific Foxo1 inhibitor (AS1842856). Cells were treated with E2 and the ERα agonist PPT and evaluated for proliferation by 3[H]-thymidine incorporation and for transcriptional activity through the estrogen response element by the luciferase assay. As Foxo1 activity is regulated by post-translational modifications, the effect of E2 on phosphorylation was also assessed. In INS1-E cells, knock down of Foxo1 abrogated the proliferative response to E2 and PPT. In human islets, inhibition of Foxo1 abrogated E2-mediated proliferation and attenuated the response to PPT. Foxo1 knock down and inhibition reduced activity through the estrogen response element by 25% (p<0.05) and 50% (p<0.01) respectively, in INS1-E cells. E2 increased Foxo1 phosphorylation in a time dependent manner in INS1-E and human islets (p<0.01, p<0.05, respectively). These findings suggest that Foxo1 is involved in E2-mediated proliferation in INS1-E cells and human islets. This may have implications vis-à-vis variations in circulating endogenous E2 concentrations in diabetes.

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.

Interaction between the effects of the selective estrogen modulator femarelle and a vitamin D analog in human umbilical artery vascular smooth muscle cells.

To further investigate the interaction between vitamin D system and estrogen-mimetic compounds in the human vasculature we studied the effect of the "less- calcemic" analog of 1,25(OH)2D3 (1,25D); JK 1624F2-2 (JKF) in the presence of selective estrogen modulator femarelle (F), the phytoestrogen daidzein (D) and estradiol-17b (E2) on 3[H] thymidine incorporation (DNA synthesis) and creatine kinase specific activity (CK) in human umbilical artery vascular smooth muscle cells (VSMC). F, D and E2, stimulated DNA synthesis at low concentrations, and inhibited it at high concentrations. All estrogen-related compounds increased CK dose- dependently. Daily treatment with JKF (1nM for 3days) resulted in decreased DNA synthesis, increased CK and up- regulation of the stimulation of DNA synthesis by low estrogen-related hormones whereas D- and E2- mediated inhibition of cell proliferation was abolished by JKF. In contrast, inhibition of cell proliferation by F could not be blocked by JKF. JKF also up-regulated the stimulatory effects on CK by F, E2 and D. VSMC expressed Estrogen Receptor (ER)a and ERb mRNA at a relative ratio of 2.7:1.0, respectively. JKF pretreatment increased ERa (∼50%) and decreased ERb (∼25%) expression. E2 did not affect ERs whereas both D and F up-regulated ERb (∼100%) and ERa (∼50%). Additionally, JKF increased the intracellular competitive binding of F (from ∼70 to ∼310%), of D (from ∼60 to ∼250%) and of E2 from (from∼70 to ∼320%). F reciprocally modulated the vitamin D system by up-regulating VDR- and 25 hydroxyy vitamin D 1-a hydroxylase (1OHase) mRNA expression (∼120%). F also stimulated 1OHase activity as indicated by an increase in the production of 1, 25D (∼250%). A similar increase was elicited by D (∼90%) but not by E2. In conclusion, F has unique effects on human VSMC in that it can sustain inhibition of cell growth even in the presence of the vitamin D analog JKF. That JKF increases ER expression and F increased the endogenous production of 1, 25D and VDR expression offer new opportunities to modulate VSMC growth. Whether or not these mutual effects of F and JKF can be exploited to promote vascular health, particularly in estrogen-deficient states (e.g., menopause) is under investigation.

Rivaroxaban significantly inhibits the stimulatory effects of bone-modulating hormones: In vitro study of primary female osteoblasts.

BACKGROUND: Anticoagulant therapy is a mainstay of treatment subsequent to major orthopedic surgeries. Evidence linking anticoagulant therapy, osteoporosis, and delayed fracture healing is not conclusive. We have previously reported that rivaroxaban significantly inhibited cell growth and energy metabolism in a human osteoblastic cell line. This study analyzed the response of primary female osteoblast cells to rivaroxaban in combination with various bone-modulating hormones. METHODS: Bone samples were taken from both premenopausal (pre-Ob) and postmenopausal (post-Ob) women. Cells were isolated from each sample and cultured to sub-confluence. Each sample was then treated with Rivaroxaban (10 µg/ml) in combination with the following hormones or with the hormones alone for 24 hours: 30nM estradiol-17ß (E2), 390nM estrogen receptor α (ERα) agonist PPT, 420nM estrogen receptor ß (ERß) agonist DPN, 50nM parathyroid hormone (PTH), and 1nM of vitamin D analog JKF. RESULTS: No effects were observed after exposure to rivaroxaban alone. When pre-Ob and post-Ob cells were exposed to the bone-modulating hormones as a control experiment, DNA synthesis and creatine kinase (CK)-specific activity was significantly stimulated with a greater response in the pre-Ob cells. When the cells were exposed to rivaroxaban in combination with bone-modulating hormones, the increased DNA synthesis and CK-specific activity previously observed were completely attenuated. CONCLUSIONS: Rivaroxaban significantly inhibited the stimulatory effects of bone-modulating hormones in both pre-Ob and post-Ob primary human cell lines. This finding may have clinical relevance for patients at high risk of osteoporosis managed with rivaroxaban or other factor Xa inhibitors.

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.

Vitamin D receptor expression is linked to potential markers of human thyroid papillary carcinoma.

Genes regulated cell-cell and cell-matrix adhesion and degradation of the extracellular matrix (ECM) have been screened as potential markers of malignant thyroid nodules. The mRNA expression levels of two of them, the ECM protein-1 (ECM1) and the type II transmembrane serine protease-4 (TMPRSS4), were shown to be an independent predictor of an existing thyroid carcinoma. The vitamin D receptor (VDR) is expressed in epithelial cells of the normal thyroid gland, as well as in malignant dividing cells, which respond to the active metabolite of vitamin D by decreased proliferative activity in vitro. We evaluated the relationship between mRNA gene expressions of TMPRSS4, ECM1 and VDR in 21 papillary thyroid carcinoma samples and compared it to 21 normal thyroid tissues from the same patients. Gene expression was considered as up- or down-regulated if it varied by more or less than 2-fold in the cancer tissue relative to the normal thyroid tissue (Ca/N) from the same patient. We found an overall significant adjusted correlation between the mRNA expression ratio (ExR) of VDR and that of ECM1 in Ca/N thyroid tissue (R=0.648, P<0.001). There was a high ExR of VDR between Ca/N thyroid tissue from the same patient (3.06±2.9), which also exhibited a high Ca/N ExR of ECM1 and/or of TMPRSS4 (>2, P=0.05).The finding that increased VDR expression in human thyroid cancer cells is often linked to increased ECM1 and/or TPMRSS4 expression warrants further investigation into the potential role of vitamin D analogs in thyroid carcinoma.

The response of cells derived from the supraspinatus tendon to estrogen and calciotropic hormone stimulations: in vitro study.

PURPOSE: The most frequent complications after rotator cuff repair (RCR) are non-healing and re-tear. Age and gender are both proven risk factors for faulty RCR. This study analyzed the effects of female sex steroids and calciotropic hormones on tendon-derived cell characteristics. METHODS: Tendon-derived cells from rat supraspinatus were treated with estradiol-17ß (E2); soy isoflavones (daidzein, genistein, biochainin A); raloxifene and estrogen receptors α and ß agonists and antagonists; and less-calcemic vitamin-D analog, parathyroid hormone, and vehicle control for 24 h. Cell proliferation and mRNA expression of estrogen receptor α and ß, vitamin-D receptor (VDR), scleraxis, and collagen-1 were assessed. RESULTS: E2, Biochainin A, raloxifene, and vitamin-D significantly increased tendon-derived cell proliferation. Estrogen receptor α antagonists neutralized tendon-derived cells response to estradiol 17-ß; however, estrogen receptor ß antagonists did not have an effect. Scleraxis expression decreased following estradiol 17-ß and vitamin-D treatments. Vitamin-D significantly reduced collagen-1 expression, while estradiol 17-ß had no effect. Vitamin-D and estradiol 17-ß upregulated VDR expression. CONCLUSIONS: Significant tendon-derived cell proliferation can be achieved with commonly prescribed female sex and calciotropic hormones. However, collagen-1 expression remained constant or decreased following the administration of these hormones. Female sex steroids and vitamin-D promoted tendon-derived cell proliferation via estrogen receptor α and VDR, not estrogen receptor ß. Amplified cell proliferation was not associated with increased scleraxis and collagen-1 expression. These results have important implications to the properties of healing tendon and possible pharmaceutical therapies for patients with torn RC. Further research is warranted to expose the underling mechanisms of these effects.

Statins enhance rotator cuff healing by stimulating the COX2/PGE2/EP4 pathway: an in vivo and in vitro study.

BACKGROUND: Statins are lipid-lowering drugs with many beneficial pleiotropic effects. Cyclooxygenase (COX2) selective inhibitors that are commonly prescribed in orthopaedic patients may effect healing. Evidence indicates that statins stimulate COX2 activity. HYPOTHESIS: Atorvastatin (ATV) administration will enhance tendon healing by stimulating the acute inflammatory phase via increasing the production of prostaglandin E2 (PGE2). STUDY DESIGN: Controlled laboratory study. METHODS: After experimental rotator cuff (RC) tearing and suturing, 48 Wistar rats were randomly allocated into 4 groups: (1) ATV (20 mg/kg), (2) celecoxib (CEL; COX2 inhibitor) (10 mg/kg), (3) ATV + CEL (20 mg/kg + 10 mg/kg), and (4) saline alone. Animals were sacrificed 3 weeks after RC tears and repair, and tendon integrity was tested biomechanically in tension. To further evaluate the underlying mechanism of action, human and rat primary tenocytes were obtained from the supraspinatus tendon. Cultures were treated with a therapeutic dosage of 5 commonly used statins: CEL, ATV + CEL, PGE2, and a selective antagonist of PGE2 receptor 4 (EP4). Cell proliferation (thymidine incorporation), migration (wound healing assay), and adhesion (iCELLigence) were evaluated. The expression of all PGE2 receptors (EPs) was determined by quantitative reverse transcription polymerase chain reaction. RESULTS: Tension testing of healed tendons demonstrated significantly higher maximal loading and stiffness in the ATV group as compared with the saline (+30% and +20%, respectively; P < .001) and CEL groups (+33% and +50%, respectively; P < .005). Celecoxib alone did not affect tendon healing (P = .88). In line with these in vivo results, tenocytes treated with statins demonstrated significantly higher proliferation rates; CEL abrogated this effect, and PGE2 treatment stimulated tenocyte proliferation even in the presence of CEL. Also, ATV stimulated the migration (wound healing) and adhesion of tenocytes. Among all PGE2 receptors, tenocytes mainly express EP4, and an EP4 selective antagonist blocked the effect of ATV. CONCLUSION: Results indicate that ATV enhances tendon healing by stimulating tenocyte proliferation, migration, and adhesion via increased COX2 activity and autocrine/paracrine PGE2 signaling. Findings also demonstrate that this effect is mediated by EP4 signaling. CLINICAL RELEVANCE: Although chronic inflammation contributes to the development of tendinopathy, study results advocate for a positive role of PGE2 in tendon healing during the acute inflammatory phase that follows tendon surgical repair. It is therefore suggested that ATV should be further investigated as a possible modality to improve tendon healing.

Age- and sex-dependent hormonal modulation of the expression of vitamin D receptor and 25-hydroxyvitamin D 1α hydroxylase in human bone cells.

BACKGROUND: Human cultured osteoblasts from pre-Ob, post-Ob or m-Ob express different mRNAs and respond to different hormones. AIMS: To test expression and hormonal modulation of VDR and 1OHase and 1,25D production in hObs. METHODS: hObs obtained from bone explants were prepared, treated and analyzed as before. RESULTS: (i) VDR and 1OHase were expressed in all hObs. (ii) 1,25D was produced similarly. (iii) Treatment with E2, DPN (ER agonist), but not PPT (ER agonist) increased VDR. (iv) These hormones increased 1OHase. (v) Vitamin D analog JKF and PTH 1-84 increased similarly mRNAs. (vi) Treatment with E2, DPN and PPT increased 1,25D. (vii) JKF and PTH increased similarly 1,25D. (viii) DNA synthesis and CK were stimulated by all hormones in hObs. CONCLUSIONS: Hormonal modulation of VDR and 1OHase and 1,25D production is important for bone physiology but their correlation, activity and bone physiology is not yet clear.

Estrogens and hyperglycemic modulation of mRNAs expressions involved in bone metabolism: an overshadowed association?

Human bone cell line (SaOS2) express different mRNAs involved in bone biology and physiology such as estrogen receptor α (ERα), estrogen receptor ß (ERß), vitamin D receptor (VDR), 1α, 25 hydroxy vitamin D(3) hydroxylase (1OHase) as well as 12 and 15 lipoxygenases (12LO and 15LO). These mRNAs are modulated by estrogenic compounds. Since the skeletal protective effects of estrogens are not discernible in diabetic women, we tested whether the expression of the parameters measured here and their modulations by estrogens, in SaOS2 cells grown in growth medium containing high glucose (HG; 9.0 g/L; 44 mM) compared to normal glucose (NG; 4.5 g/L; 22 mM). High Glucose (HG) significantly increased DNA synthesis and creatine kinase (CK) specific activity in SaOS2 cells. Stimulations of DNA but not of CK by E(2), by 4, 4', 4''-[4-propyl-(1H)-pyrazol-1, 3, 5- triyl] tris-phenol (PPT, ERα specific agonist), or by 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN, ERß specific agonist), were abolished by HG. HG itself upregulated the expression of mRNA of 12LO and 15LO and upregulated to much less extent of ERß and VDR, but had no effect on the expression of mRNA of ERα and 1OHase. The different hormonal treatments modulated the expressions of 12LO and 15LO mRNAs which was reduced in HG, whereas the induction of their products 12HETE and 15HETE was only slightly affected by HG. The exact mechanism of HG effects on bone cell responses is yet to be investigated and its relationship to human bone physiology is not yet clear.

Estrogens and Hyperglycemic Modulation of mRNAs Expressions Involved in Bone Metabolism: An Overshadowed Association?

Human bone cell line (SaOS2) express different mRNAs involved in bone biology and physiology such as estrogen receptor α (ERα), estrogen receptor ß (ERß), vitamin D receptor (VDR), 1α, 25 hydroxy vitamin D(3) hydroxylase (1OHase) as well as 12 and 15 lipoxygenases (12LO and 15LO). These mRNAs are modulated by estrogenic compounds. Since the skeletal protective effects of estrogens are not discernible in diabetic women, we tested whether the expression of the parameters measured here, and their modulations by estrogens, in SaOS2 cells grown in growth medium containing high glucose (HG; 9.0g/L; 44mM) compared to normal glucose (NG; 4.5g/L; 22mM). HG significantly increased DNA synthesis (DNA) and creatine kinase specific activity (CK) in SaOS2 cells. Stimulations of DNA but not of CK by E(2), by 4, 4', 4"-[4-propyl-(1H)-pyrazol-1, 3, 5- triyl] tris-phenol (PPT; ERα specific agonist), or by 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERß specific agonist), were abolished by HG. HG Itself up regulated the expression of mRNA of 12LO and 15LO and up regulated to much less extent ERß and VDR, but had no effect on the expression of mRNA of ERα and 1OHase. The different hormonal treatments modulated the expressions of 12LO and 15LO mRNAs which was reduced in HG, whereas the induction of their products 12 and 15HETE was only slightly affected by HG. The exact mechanism of HG effects on bone cell responses is yet to be investigated and its relationship to human bone physiology is not yet clear.

Rivaroxaban, a direct inhibitor of the coagulation factor Xa interferes with hormonal-induced physiological modulations in human female osteoblastic cell line SaSO2.

The use of anticoagulants has been associated with systemic osteoporosis and increased risk for poor fracture healing but is inevitable following major orthopedic surgery of lower limbs. Rivaroxaban A (R) is an anticoagulant recently introduced in the clinical setting, which is a specific factor Xa inhibitor. We reported previously that R significantly inhibited cell growth, energy metabolism and alkaline phosphatase activity in human osteoblastic cell line SaOS2, with no effect on mineralization, indicating transient inhibition of bone formation. We now investigated the effects of R on SaOS2 response to osteoblast-modulating hormones. At sub-confluence cells were treated with: estradiol-17ß (E2), the phytoestrogens daidzein (D) and biochainin A (BA), the carboxy-pytoestrogenic derivative carboxy-D (cD), the estrogen receptor α (ERα) agonist PPT, the estrogen receptor ß (ERß) agonist DPN, parathyroid hormone (PTH) and several vitamin D metabolites and analogs with/without R for 24h. All hormones tested stimulated significantly DNA synthesis (DNA), creatine kinase (CK) and alkaline phosphatase (ALP) specific activities, but all these stimulations were totally inhibited when given together with R. R had no effect on mRNA expression of ERα, ERß and 25 Hydroxy-vitamin D3-1α hydroxylase (1OHase), but inhibited hormonal modulations of mRNA expressions. In conclusion R inhibited significantly hormonal stimulation of different parameters indicating inhibition of not only the early stages of bone formation, but also the stimulatory effects of bone modulating hormones with a yet unclear mechanism. The relevance of these findings to human bone physiology is yet to be investigated.

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.

The effects of direct factor Xa inhibitor (Rivaroxaban) on the human osteoblastic cell line SaOS2.

Thromboprophylaxis reduces the risk of surgery-related deep vein thrombosis, but anticoagulants were associated with systemic osteoporosis, a known risk factor for poor fracture healing. Rivaroxaban (XARELTO(®)) is a novel anticoagulant with specific ability to inhibit factor Xa, a serine endopeptidase, which plays a key role in coagulation. This study investigated the direct effects of rivaroxaban on bone biology using an in vitro cell culture model from the human female osteoblastic cell line SaOS2. Cells at subconfluence were treated for 24 hr with different concentrations of rivaroxaban and analyzed for DNA synthesis and creatine kinase- and alkaline phosphatase-specific activities, and were treated 21 days for analyzing mineralization. Rivaroxaban (0.01-50 µg/ml) dose-dependently inhibited up to 60% DNA synthesis of the cells. Creatine kinase-specific activity was also inhibited dose-dependently to a similar extent by the same concentrations. Alkaline phosphatase-specific activity was dose-dependently inhibited but only up to 30%. Cell mineralization was unaffected by 10 µg/ml rivaroxaban. This model demonstrated a significant rivaroxaban-induced reduction in osteoblastic cell growth and energy metabolism, and slight inhibition of the osteoblastic marker, alkaline phosphatase, while osteoblastic mineralization was unaffected. These findings might indicate that rivaroxaban inhibits the first stage of bone formation but does not affect later stages (i.e., bone mineralization).

Sex specific response of cultured human bone cells to ERα and ERß specific agonists by modulation of cell proliferation and creatine kinase specific activity.

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17ß (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERß specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERß mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERß specific agonist) and 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERß pathways in the effects of estrogens on hObs, with a yet unknown mechanism.

Dihydrotestosterone and estradiol-17beta mutually neutralize their inhibitory effects on human vascular smooth muscle cell growth in vitro.

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.

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.

Lipoxygenase metabolites are mediators of PTH-dependent human osteoblast growth.

PTH-induced osteoblast proliferation may contribute to its anabolic effects in bone. Since PTH-dependent osteoblast-like cell (Ob) growth is mediated via protein kinase C (PKC) and MAP kinase-kinase (MEK) and since lipoxygenase (LO) products activate PKC in a number of cell types, we assessed the expression of LO pathways in primary human cultured Ob. Ob from pre- or post-menopausal women were cultured and were treated with PTH and assayed for the expression of 12-LO and both type I and type II 15-LO mRNA and for the release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Cells were also treated with PTH for stimulation DNA synthesis. First, Ob express platelet type- 12-LO and both type I and type II 15-LO mRNA and release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Second, in female Ob, PTH induced a rapid increase in 12-HETE (50 fold increase) and 15-HETE (80 fold increase) and increased the expression of 12-LO mRNA but not of the two isoforms of 15-LO. PTH as well as 12 and 15-HETE stimulated DNA synthesis in Ob. The LO inhibitor baicalein inhibited PTH-stimulated DNA synthesis, which was reversed in the presence of either 12- or 15-HETE. A PKC inhibitor (bisindolylmaleimide I) as well as a MEK inhibitor (PD 98059) completely inhibited the stimulation of DNA synthesis by PTH, 12-HETE and the combination of PTH and 12-HETE. In contrast, 15-HETE-induced DNA synthesis was not abolished by these inhibitors. Further, 15-HETE partially restored the stimulatory effect of PTH on DNA synthesis in cells treated with PKC or MEK inhibitors. Finally, PTH- induced ERK1/2 phosphorylation, was blocked by a MEK inhibitor. These results demonstrate a novel mechanism of PTH-induced human bone cell proliferation operating through LO enzymes.

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.