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.

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.

Vitamin D modulation of the activity of estrogenic compounds in bone cells in vitro and in vivo.

Vitamin D analogs modulate different organs, including modulation of energy metabolism, through the induction of creatine kinase (CK) activity. Skeletal organs from vitamin D-depleted rats showed lower constituent CK than those from vitamin D-replete rats. Moreover, estradiol-17beta (E2) or dihydrotestosterone (DHT), which increased CK in organs from intact female or male rats, respectively, stimulated much less CK in vitamin D-depleted rats. Treatment of intact female rats with noncalcemic vitamin D analogs significantly upregulated E2- and DHT-induced CKresponse. These analogs upregulated the CK response to selective estrogen receptor modulators (SERMs) in organs from intact or ovariectomized (Ovx) female rats but abolished SERMs' inhibitory effect on E2-induced CK. These analogs significantly increased estradiol receptor alpha (ERalpha) protein in skeletal organs as well as histomorphological and biochemical changes due to this treatment followed by E2 or DHT. The analogs alone markedly altered the growth plate and the trabeculae and increased trabecular bone volume (%TB V) and trabecular width. The addition of E2 or DHT to this treatment restored all parameters as well as increased %TBV and cell proliferation. Treatment of Ovx female rats with JK 1624 F2-2 (JKF) decreased growth-plate width and increased %TB V, whereas QW1624 F2-2 (QW) restored growth-plate width and %TB V. Treatment of E2 with JKF restored %TBV and growth-plate width, whereas E2 with QW restored all parameters, including cortical width. There was also upregulation of the response of CK to E2 in both combined treatments. Our human-derived osteoblast (hObs)-like cell cultures respond to estrogenic compounds, and pretreating them with JKF upregulated the CK response to E2, raloxifene (Ral), and some phytoestrogens. ERalpha and ERbeta proteins, as well as mRNA, were modulated by CB 1093 (CB) and JKF. JKF increased specific nuclear E2 binding in female hObs but inhibited specific membranal E2 binding. hObs express 25 hydroxyvitamin D3-1alpha hydroxylase (1-OHase)-mRNA and its biological activity, which are both modulated by parathyroid hormone (PTH) and estrogenic compounds. Our results demonstrate mutual interaction between vitamin D and estrogenic compounds. We therefore conclude that combined treatment with less-calcemic analogs of vitamin D and estrogenic compounds might be superior for treatment of bone damage caused by ovariectomy in female rats, with possible application for postmenopausal osteoporosis.

DT56a (Femarelle): a natural selective estrogen receptor modulator (SERM).

A selective estrogen receptor modulator (SERM) is defined as a substance with dissimilar effects on different tissues: agonist in some and antagonists in others. The natural compound DT56a (Femarelle) was shown to activate estrogen receptors in human cultured female derived osteoblasts. It was also shown to relieve menopausal symptoms and to increase bone mineral density with no effect on sex steroid hormone levels and on the endometrial thickness. DT56a, similarly to estradiol-17beta (E2), stimulated the specific activity of creatine kinase (CK) in skeletal and vascular tissues of female rats, as a marker of estrogen receptor (ER) activation. However, in the uterus, CK was activated only by E2 but not by DT56a. In order to prove that DT56a is a SERM, we examined the mutual interaction between DT56a and E2, at supra physiological doses, in different tissues in both intact and ovariectomized female rats, as well as in human cultured vascular and bone cells. Administration of DT56a or E2 stimulated CK in all tissues tested, but when given simultaneously, in intact immature female rats, DT56a completely abolished E2 stimulation of CK in all organs except in the diaphyseal bone where the inhibition was partial. In ovariectomized female rats, DT56a abolished E2's stimulation of CK in diaphyseal bone, thymus, uterus and pituitary but caused a partial inhibition in aorta, left ventricle and epiphyseal cartilage. In human bone cells E2 stimulation of CK, of alkaline phosphatase (AP) activity and of DNA synthesis was completely abolished by DT56a in post-menopausal cells and partially inhibited in pre-menopausal cells. In human vascular cells, inhibition of DNA synthesis by E2 was completely abolished by DT56a and E2-induced CK was partially inhibited by DT56a. The results support the finding that DT56a is a SERM; it stimulated different parameters similar to E2, but when given simultaneously, at supra physiological doses, inhibited these E2's effects. Further investigations regarding intra and extra cellular mechanism of action of DT56a are currently performed.

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.

DT56a stimulates gender-specific human cultured bone cells in vitro.

DT56a found to have SERM-like properties is used for the treatment of menopausal symptoms and osteoporosis. In vivo experiments demonstrated that DT56a displayed selective estrogenic activity; it stimulated creatine kinase (CK) specific activity in the skeletal tissues but not on the uterus of ovariectomized rats. DT56a, when applied together with estradiol-17beta (E(2)), completely inhibited the E(2)-stimulated CK, as demonstrated by other SERMs. DT56a stimulated bone formation in a rat model as measured by histological and histomorphometrical parameters. In a clinical study, administration of DT56a (Femarelle) resulted in a considerable elevation of bone mineral density and relief of menopausal symptoms. The aim of the present study was to analyze the effects of DT56a in vitro on human-derived bone cultured osteoblasts (Ob), by measuring its effects, at different concentrations, on DNA synthesis, CK and alkaline phosphatase (ALP) specific activities as well as changes in intracellular [Ca(2+)](i) concentrations. DT56a stimulated CK and DNA synthesis in both pre- and post-menopausal female Ob with maximal effect at 100 ng/ml for both age groups. In addition, DT56a stimulated ALP in Ob from both pre- and post-menopausal women with maximal effect at lower dose of 50 ng/ml, with higher response of pre-menopausal cells. Raloxifene (Ral) inhibited all DT56a-stimulated changes in Ob from both age groups. DT56a, when given together with E(2), completely antagonized E(2)-stimulated effects demonstrating its nature as a phyto-SERM. DT56a also, dose dependency, stimulated the intracellular levels of [Ca(2+)](i) with maximal effect at 10 ng/ml. Male-derived Ob did not respond to DT56a in any parameter. In summary, DT56a stimulated sex-specifically female-derived Ob, indicating its unique nature compared to the compounds currently used for postmenopausal osteoporosis by being bone-forming and not only an anti-resorptive agent.

DT56a (Femarelle) stimulates bone formation in female rats.

OBJECTIVE: DT56a is a natural compound for the treatment of menopausal symptoms and osteoporosis. The aim of this study was to examine the effects of long term treatment (two months) with DT56a on the skeletal tissues of intact and ovariectomised (OVX) adult rats. DESIGN: Thirty rats were divided into two groups, in one of which the rats were ovariectomised. The rats in each group were then treated for two months with DT56a, oestrogen or vehicle. SETTING: University and hospital laboratories. POPULATION: Thirty rats. METHODS: Histomorphometric measurements of trabecular bone volume (expressed as a percentage of total bone volume), trabecular and cortical thickness and growth plate width were recorded by a computerised system. In addition, creatine kinase (CK)-specific activity, as marker of oestrogen receptor activation, was measured in skeletal tissues and in the uterus. MAIN OUTCOME MEASURES: The changes in the histomorphometric measurements. RESULTS: OVX rats developed noticeable signs of osteoporosis, namely, significant decrease in trabecular bone volume and in trabecular and cortical thickness. DT56a, like oestrogen, restored the bone structure measurements of all tested parameters in the OVX rats to the values obtained in the intact rats. In skeletal tissues, CK activity was elevated in both treatment groups. However, in the uterus DT56a did not activate oestrogen receptors while oestrogen did elevate CK activity. CONCLUSIONS: DT56a was as effective as oestrogen in reversing the bone changes caused by OVX in rats.

DT56a (Tofupill/Femarelle) selectively stimulates creatine kinase specific activity in skeletal tissues of rats but not in the uterus.

The novel natural product DT56a (Tofupill/Femarelle), derived from soybean, has been shown to relieve menopausal vasomotor symptoms and to increase bone mineral density with no effect on sex steroid hormone levels or endometrial thickness. In the present study, we compared the effects of DT56a and estradiol-17beta (E2) on bone and cartilage (Ep) of immature or ovariectomized female rats, by measuring the changes in the specific activity of the BB isozyme of creatine kinase (CK). Single short-term injection of high doses of DT56a induced estrogenic activity in bones and uterus similar to that of E2. When administered in multiple oral doses, DT56a stimulated skeletal tissues similarly to E2, but whereas E2 increased CK specific activity in the uterus, DT56a did not. The selective estrogen receptor modulator (SERM) raloxifene (Ral) blocked the stimulation of CK by either DT56a or by E2 in all tissues tested. Our findings suggest that DT56a acts as a selective estrogen receptor modulator stimulating skeletal tissues without affecting the uterus. The effect of DT56a on other systems, such as the vascular and the central nervous system, are currently under investigation.