Intermittent aerobic-resistance interval training versus continues aerobic training: Improvement in cardiac electrophysiologic and anthropometric measures in male patients post myocadiac infarction, a randomized control trial.

PURPOSE: Exercise is a valuable intervention modality for patients post-myocardial infarction (MI). Aerobic and resistance training are both commonly used separately in cardiac rehabilitation. However, the effect of aerobic interval exercise combined with alternating sets of resistance training (super-circuit training, SCT) on cardiac electrophysiologic and anthropometric measures had not been thoroughly investigated. AIM: The primary objective of this study was to compare the effectiveness of moderate-intensity continuous-aerobic training (CAT) vs. SCT on cardiac electrical measures (resting electrocardiographic, ECG; a nd heart rate variability, HRV) in patients' post-MI presenting reduced left ventricular function. Second, to examine its effect on anthropometric measures. MATERIAL AND METHODS: Twenty-nine men post-MI with reduced left ventricular function were assigned randomly to either 12 weeks of CAT (n = 15) or SCT (n = 14). CAT group performed moderate-intensity activity. SCT group performed high-intensity exercise, alternating between resistance and aerobic training. Differences between CAT and SCT groups were done using independent t-tests, paired t-tests and effect size (ES). RESULTS: Participants in both groups improved their HRV measures (increase in HFnu; p < 0.05; ES > 0.51) and ECG (reduction in QT-dispersion; p < 0.05; ES > 0.51). Only the SCT group had significant improvements in waist circumference (p < 0.05). CONCLUSION: Exercise improves cardiac electrical measures post-MI. However, in comparison to CAT, SCT may yield greater anthropometric changes. In order to have improvements in cardiac electrical stability, clinicians working with post-MI patients may use both CAT and SCT. However, SCT might result in greater improvements.

The impact of COVID-19 lockdown on physical activity and weight gain among active adult population in Israel: a cross-sectional study.

BACKGROUND: The COVID-19 outbreak holds public health concerns. The stay-at-home increases sedentary behavior, with unintended adverse outcomes. Since organized recreation and sports facilities were closed, we aimed to study how the crisis of closure affected exercise habits and weight gain among the trainee population in Israel. We examined differences in weight gain among individuals with different PA activities and assessed their ability to adapt to digital media as an alternative training structure. METHODS: A cross-sectional survey consisted of a multiple-choice questionnaire obtained using a web-based survey application. Trainees (1202) who exercised steadily anonymously answered the questionnaire sent by their coaches regarding their activity and weight gain during lockdown times. RESULTS: Results confirmed that 70% of Israelis trained less than their usual routine, 60% used digital media for training, 55% gained weight. Half of the respondents gained more than 2 kg, with an average increase of 1.2 kg. However, those who exhibited a higher physical activity level gained less weight. Using digital media for training was associated with higher physical activity levels. The aged population was less likely to use digital media. CONCLUSIONS: Since increased sedentary behavior could increase the risk for potential worsening of health conditions, health agencies should look for strategies, including digital remote media training to promote physical activity and subsequently, preventing the increased burden of future comorbidities worsening by a sedentary lifestyle. Approval: by the Helsinki ethics committee of Sheba Medical Center (6504-19-SMC).

Detection of unexpected ischaemia due to left main disease during tele-rehabilitation using 12-lead electrocardiogram monitoring: a case report.

BACKGROUND: Cardiac rehabilitation (CR) reduces mortality and improves quality of life. Unfortunately, participation in CR remains low and studies have examined the use of home-based tele-monitoring to improve participation in CR. These studies generally utilized single- or three-lead electrocardiogram (ECG) channels with limited sensitivity to detect ischaemic changes. In this report, we describe detection of unexpected, significant left main coronary disease in a patient participating in a home CR programme using a unique 12-lead ECG monitoring system. CASE SUMMARY: A 68-year-old man was referred for CR after acute coronary syndrome and stent implantation to the obtuse marginal. Three months following this intervention, he began complaining of chest pain. Repeat coronary angiogram showed a borderline lesion in the left main not felt to be clinically significant. The patient performed CR and was monitored with the Master Caution® System, a digital platform including a garment with 10 textile electrodes (Master Caution garment) configuring a device which enables 12 lead ECG. While being monitored, the ECG technician detected 1-2 mm ST-depression in leads 1, L V2-V5. The patient was asymptomatic at the time the ST-depressions were noted. Repeat angiography revealed a significant increase in the severity of the left main lesion and the patient was referred for bypass surgery. DISCUSSION: This life-threatening diagnosis could easily have been missed using conventional ECG monitoring and our case demonstrates the potential clinical utility of tele-monitoring with a 12-lead digital home ECG monitoring device.

A Novel Digital Platform for a Monitored Home-based Cardiac Rehabilitation Program.

Despite the evidence that cardiac rehabilitation (CR) reduces the risk of recurrent cardiac events, only a minority of eligible patients are willing to join existing programs at cardiac rehabilitation centers. The unique remote patient monitoring system presented here enables healthcare providers to monitor CR patients at home in real-time and at low cost. The system combines mobile technology, artificial intelligence, and supportive services, expanding the delivery of medical care to the patient's home. The primary aim of the study is to increase the long-term adherence to physical activity in patients who participate in CR via the addition of a home-based digitally monitored CR component to the standard CR program in patients with ischemic heart disease (IHD), with the idea of forming new habitual health behaviors and increasing the long-term motivation for physical exercise (PE) habits at home. Secondary aims are to assess the program's impact on the physical activity level measured by average steps per day, minutes of exercise per week, blood pressure, metabolic parameters, body mass index, and waist-to-hip ratio, as well as a quality-of-life (QoL) questionnaire.The study has two arms: (1) home-based monitored exercise using a smart digital garment and wristband, in addition to motivation and reinforcement via text messages; (2) standard CR facility-based exercise. The study design is a randomized, controlled trial comparing standard CR to a home-based monitoring and reinforcement program. The study program is designed for 12 weeks.Clinical tests and anthropometric measurements are performed before and after the study, measuring height, weight, waist circumference, visceral fat and body mass index (BMI), blood pressure, and HbA1c and lipid profile. Patients have to complete a baseline survey including socio-demographic characteristics and QoL questionnaire SF-36. At the end of the study, patients complete a survey regarding the use of the smart digital garment's benefits and usability. The study is currently underway.

Correction: Improvement in cardiac dysfunction with a novel circuit training method combining simultaneous aerobic-resistance exercises. A randomized trial.

[This corrects the article DOI: 10.1371/journal.pone.0188551.].

Improvement in cardiac dysfunction with a novel circuit training method combining simultaneous aerobic-resistance exercises. A randomized trial.

INTRODUCTION: Exercise is considered a valuable nonpharmacological intervention modality in cardiac rehabilitation (CR) programs in patients with ischemic heart disease. The effect of aerobic interval exercise combined with alternating sets of resistance training (super-circuit training, SCT) on cardiac patients' with reduced left ventricular function, post-myocardial infarction (MI) has not been thoroughly investigated. AIM OF STUDY: to improve cardiac function with a novel method of combined aerobic-resistance circuit training in a randomized control trial by way of comparing the effectiveness of continuous aerobic training (CAT) to SCT on mechanical cardiac function. Secondary to compare their effect on aerobic fitness, manual strength, and quality of life in men post MI. Finally, to evaluate the safety and feasibility of SCT. METHODS: 29 men post-MI participants were randomly assigned to either 12-weeks of CAT (n = 15) or SCT (n = 14). Both groups, CAT and SCT exercised at 60%-70% and 75-85% of their heart rate reserve, respectively. The SCT group also engaged in intermittently combined resistance training. Primary outcome measure was echocardiography. Secondary outcome measures were aerobic fitness, strength, and quality of life (QoL). The effectiveness of the two training programs was examined via paired t-tests and Cohen's d effect size (ES). RESULTS: Post-training, only the SCT group presented significant changes in echocardiography (a reduction in E/e' and an increase in ejection fraction, P<0.05). Similarly, only the SCT group presented significant changes in aerobic fitness (an increase in maximal metabolic equivalent, P<0.05). In addition, SCT improvement in the physical component of QoL was greater than this observed in the CAT group. In both training programs, no adverse events were observed. CONCLUSION: Men post-MI stand to benefit from both CAT and SCT. However, in comparison to CAT, as assessed by echocardiography, SCT may yield greater benefits to the left ventricle mechanical function as well as to the patient's aerobic fitness and physical QoL. Moreover, the SCT program was found to be feasible as well as safe.

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.

Curcumin induces apoptosis and inhibits growth of orthotopic human non-small cell lung cancer xenografts.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality. Curcumin is involved in various biological pathways leading to inhibition of NSCLC growth. The purpose of this study was to evaluate the effect of curcumin on expression of nuclear factor κB-related proteins in vitro and in vivo and on growth and metastasis in an intralung tumor mouse model. H1975 NSCLC cells were treated with curcumin (0-50 µM) alone, or combined with gemcitabine or cisplatin. The effects of curcumin were evaluated in cell cultures and in vivo, using ectopic and orthotopic lung tumor mouse models. Twenty mice were randomly selected into two equal groups, one that received AIN-076 control diet and one that received the same food but with the addition of 0.6% curcumin 14 days prior to cell implantation and until the end of the experiment. To generate orthotopic tumor, lung cancer cells in Matrigel were injected percutaneously into the left lung of CD-1 nude mice. Western blot analysis showed that the expressions of IkB, nuclear p65, cyclooxygenase 2 (COX-2) and p-ERK1/2 were down-regulated by curcumin in vitro. Curcumin potentiated the gemcitabine- or cisplatin-mediated antitumor effects. Curcumin reduced COX-2 expression in subcutaneous tumors in vivo and caused a 36% decrease in weight of intralung tumors (P=.048) accompanied by a significant survival rate increase (hazard ratio=2.728, P=.036). Curcumin inhibition of COX-2, p65 expression and ERK1/2 activity in NSCLC cells was associated with decreased survival and increased induction of apoptosis. Curcumin significantly reduced tumor growth of orthotopic human NSCLC xenografts and increased survival of treated athymic mice. To evaluate the role of curcumin in chemoprevention and treatment of NSCLC, further clinical trials are required.

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.

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.

Pyrimethamine increases ß-hexosaminidase A activity in patients with Late Onset Tay Sachs.

OBJECTIVE: To assess whether or not pyrimethamine (PMT) can be used to enhance ß-hexosaminidase A activity (HexA) in subjects with Late Onset Tay Sachs (LOTS), we studied the effect of incremental doses of PMT in vivo in 9 LOTS patients carrying the αG269S/c.1278insTACT mutations. METHODS: PMT treatment was initiated at a dose of 6.25 mg, increasing gradually up to a maximal allowable dose of 75 mg daily at 4-6 weeks intervals for a total of up 10 months. Mean patients' age was 37.9±16.1 yrs (range 20-67 years). RESULTS: Lymphocyte HexA activity rose in all subjects, peaking at 78±30% over baseline activity (mean±SD; range 36-114%). The optimal PMT dose varied considerably, averaging at 30±24.1 mg (range-6.25-75 mg, daily). Further increase in PMT beyond the optimal dose was associated with gradual loss of effect on lymphocyte HexA. Improvement in speech was seen within several weeks in 4 out of 9 subjects, mostly paralleling the initial increment in HexA. Mood stabilization was also perceived in 3 subjects, but this was more difficult to assess due to the concomitant use of psychotropic/mood stabilizing agents. Reversible decline in motor activity manifesting predominantly in more frequent falls was seen in 3 subjects when the PMT dose was increased beyond the peak effect generating dose. CONCLUSIONS: PMT therapy can increase HexA activity in LOTS in vivo. Optimal doses should be tailored individually to avoid loss of biochemical effects. Clear cut neurological and psychiatric effects are difficult to discern at this time, mostly due to short term study follow up and large inter-individual variability.

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.

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.

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.