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[Figure: see text]. [ABSTRACT FROM AUTHOR]

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Cytochrome P450 1B1 protects against angiotensin II (Ang II)-induced hypertension and associated cardiovascular changes in female mice, most likely via production of 2-methoxyestradiol. This study was conducted to determine whether 2-methoxyestradiol ameliorates Ang II-induced hypertension, renal dysfunction, and end-organ damage in intact Cyp1b1-/-, ovariectomized female, and Cyp1b1+/+ male mice. Ang II or vehicle was infused for 2 weeks and administered concurrently with 2-methoxyestradiol. Mice were placed in metabolic cages on day 12 of Ang II infusion for urine collection for 24 hours. 2-Methoxyestradiol reduced Ang II-induced increases in systolic blood pressure, water consumption, urine output, and proteinuria in intact female Cyp1b1-/- and ovariectomized mice. 2-Methoxyestradiol also reduced Ang II-induced increase in blood pressure, water intake, urine output, and proteinuria in Cyp1b1+/+ male mice. Treatment with 2-methoxyestradiol attenuated Ang II-induced end-organ damage in intact Cyp1b1-/- and ovariectomized Cyp1b1+/+ and Cyp1b1-/- female mice and Cyp1b1+/+ male mice. 2-Methoxyestradiol mitigated Ang II-induced increase in urinary excretion of angiotensinogen in intact Cyp1b1-/- and ovariectomized Cyp1b1+/+ and Cyp1b1-/- female mice but not in Cyp1b1+/+ male mice. The G protein-coupled estrogen receptor 1 antagonist G-15 failed to alter Ang II-induced increases in blood pressure and renal function in Cyp1b1+/+ female mice. These data suggest that 2-methoxyestradiol reduces Ang II-induced hypertension and associated end-organ damage in intact Cyp1b1-/-, ovariectomized Cyp1b1+/+ and Cyp1b1-/- female mice, and Cyp1b1+/+ male mice independent of G protein-coupled estrogen receptor 1. Therefore, 2-methoxyestradiol could serve as a therapeutic agent for treating hypertension and associated pathogenesis in postmenopausal females, and in males. [ABSTRACT FROM AUTHOR]

OBJECTIVE: In this study, we sought to investigate the effects of 2-methoxyestradiol (2-ME) on cisplatin-induced apoptosis and growth inhibition in SKOV3 ovarian cancer cells. MATERIALS AND METHODS: Cells were treated with 2-ME, carboplatin, or both, the control group, and cell viability and growth inhibition assays were performed using the MTT method. Apoptosis was detected by flow cytometry analysis. Reverse transcription polymerase chain reaction and western blotting were used to monitor the mRNA and protein expression of the pro-apoptotic genes bax and caspase-3 and the anti-apoptotic gene bcl-2. The phosphorylation of Bcl-2 protein was monitored by western blotting. RESULTS: Cell viability was inhibited by all three treatments in a time-dependent manner. Importantly, the combination treatment resulted in significantly reduced cell growth compared with the other groups. The mRNA and protein expression of Bax and caspase-3 were increased in the combination treatment group, and the expression of Bcl-2 was decreased in the combination treatment group as compared with the other two groups. The ratio of bax to Bcl-2 mRNA in the combination treatment group was higher than that in the carboplatin-treated group. Finally, phosphorylation of Bcl-2 protein was increased stronger in the combination treatment group compared with the carboplatin-treated group. CONCLUSIONS: 2-ME promoted the growth inhibitory and apoptosis-inducing effects of platinum-based agents in SKOV3 ovarian cancer cells. The mechanism mediating this effect may be related to the phosphorylation of Bcl-2 protein, which reduces the formation of dimers and, thereby, increases apoptosis. Moreover, 2-ME promoted the mRNA and protein expression of Bax, thereby, increasing the Bax/Bcl-2 expression ratio and activating the mitochondrial apoptosis pathway. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is known anticancer approach used mainly for topical skin cancer. However, it could serve as an excellent alternative to traditional anticancer therapies, such as chemotherapy or radiotherapy. The study aimed to assess the effect of PDT, where the combination of cyanine with 2-methoxyestradiol (2-Me) was used on mammary and ovary adenocarcinoma human cell lines. The cyanine IR-775 was used as the photosensitizer. Two human malignant adenocarcinoma cell lines – ovary and mammary adenocarcinoma (MDA MB-231 and SKOV-3) were investigated in photodynamic reaction (PDR), with the enhancement of 2-Me. PDR efficiency was evaluated by the MTT test. Photosensitizer intracellular distribution was assessed by fluorescent microscopy. Additionally, apoptotic and oxidative stress markers were investigated by immunocytochemistry staining. It was observed that PDR enhanced by 2-Me is effective against two common but different types of cancer. The treatment decreased cells' viability by around 70%. Immunocytochemical staining of SOD2 and caspase-12 indicated apoptosis and oxidative stress induction in tested cell lines. The results suggest that the therapy could be involved in further oncological applications. [ABSTRACT FROM AUTHOR]

Objective 2-Methoxyestradiol (2ME) deficiency leading to placental insufficiency has been related to pre-eclampsia (PE). Here we investigate whether 2ME is related to clinical profiles and vasoactive factors in early onset severe PE patients. Methods 28 severe PE patients and 20 uncomplicated normal pregnant women, with gestational weeks between 24 and 32weeks, were recruited. All cases and controls had singleton pregnancies and were matched for maternal age, parity, body mass index, and gestational weeks. Plasma levels of 2ME, estradiol (E2), soluble Fms-like tyrosine kinase-1 (sFLT-1), endothelin-1 (ET-1), nitric oxide (NO) were determined. Results PE patients had significant lower 2ME [906(422-1768) vs. 2032(1400-2910)pg/mL, P=0.002], higher sFLT-1 [5.55(3.24-11.22) vs. 3.13(2.17-5.36)ng/mL, P=0.015] and higher NO [122.40(72.92-168.23) vs. 45.83(25.52-61.46)μmol/L, P=0.0008] levels in their plasma than the controls. In the PE group, plasma 2ME level correlated negatively with systolic pressure (r=-0.48, P=0.012), diastolic pressure (r=-0.52, P=0.007) and mean arterial pressure (r=-0.54, P=0.005) even after controlling for maternal age; 2ME level did not correlate with proteinuria, plasma levels of E2, sFLT-1, ET-1 or NO. In the control group, plasma 2ME level did not correlate with any of the above clinical profiles or laboratory measurements. Conclusions 2ME levels were markedly lower in early onset severe PE and they correlated inversely with blood pressure only in women with PE. Although we cannot tell whether lower 2ME level is the causation or the result of PE, our study provides clinical evidences that 2ME deficiency is strongly related to hypertension in early onset severe PE patients. [ABSTRACT FROM AUTHOR]

determine the role of cytochrome P450 (CYP) IB 1 in the sex difference in response to angiotensin II (Ang II)-induced hypertension, female Cyplbl+/+ and Cyplbl-/- mice were infused with Ang II (700 ng/kg per minute) or vehicle with or without ovariectomy. In addition, mice were treated with the CYP1B1 inhibitor, 2,3/,4,5/-tetramethoxystilbene (TMS; 300 ng/kg IP, every third day), and 17-ß estradiol metabolites, 2-hydroxyestradiol (2-OHE), 4-OHE, or 2-methoxyestradiol (1.5 mg/kg per day IP, for 2 weeks) and systolic blood pressure (SBP) measured. Ang II increased SBP more in Cyplbl-/- than in Cyplbl+/+ mice (119±3-171±11 versus I20±4-149±4 mmHg; P<0.05). Ang II caused cardiovascular remodeling and endothelial dysfunction and increased vascular reactivity and oxidative stress in Cyplblbut not in Cyplbl+/+ imce. The Ang II-induced increase in SBP was enhanced by ovariectomy and TMS in Cyplbl+/+ but not in Cyplbl-/- mice. 2-OHE did not alter Ang II-induced increase in SBP in Cyplbl+/+ mice but minimized it in Cyplbl-/- mice, whereas 4-OHE enhanced Ang II-induced increase in SBP in Cyplbl+/+ mice but did not alter the increased SBP in Cyplbl-/- mice. 2-OHE-derived catechol-O-methyltransferase metabolite, 2-methoxyestradiol, inhibited Ang II-induced increase in SBP in Cyplbl-/- mice. Ang II increased plasma levels of 2-methoxyestradiol in Cyplbl+/+ but not in Cyplbl-/- mice and increased activity of cardiac extracellular signal-regulated kinase 1/2, p38 mitogen-activated kinase, c-Src, and Akt in Cyplbl-/- but not in Cyplbl+/+ mice. These data suggest that CYP1B1 protects against Ang II-induced hypertension and associated cardiovascular changes in female mice, most likely mediated by 2-methoxyestradiol-inhibiting oxidative stress and the activity of these signaling molecules. [ABSTRACT FROM AUTHOR]

Purpose: Tissue level hypoxia has been noted in animal models of partial bladder outlet obstruction. The key mechanisms linking hypoxia and obstruction induced bladder dysfunction remain unknown. 2-Methoxyestradiol is a natural derivative of 17β-estradiol and is currently used as an oncologic agent for its ability to regulate the hypoxia pathway. We investigated the ability of 2-methoxyestradiol to modulate the hypoxia response in a mouse model of bladder obstruction. Materials and Methods: A group of 5 to 6-week-old female C57BL/6 mice underwent oophorectomy and partial bladder outlet obstruction. Obstructed animals received a subcutaneous pellet of cholesterol placebo (7) or 2-methoxyestradiol plus cholesterol (7). Age matched controls underwent oophorectomy only (8). After 4 weeks the bladders of mice with partial bladder outlet obstruction and of unobstructed animals were harvested. Bladder sections (5 μm) were immunostained for Hypoxyprobe™-1, glucose transporter 1 and hypoxia inducible factor-1α. Real-time polymerase chain reaction was performed for hypoxia inducible factor-1α and lysyl oxidase. Statistical analysis was performed using 1-way ANOVA and the Wilcoxon rank sum test. Results: Immunostaining for glucose transporter 1 and Hypoxyprobe-1 revealed the presence of tissue hypoxia after partial bladder outlet obstruction. Immunostaining and real-time polymerase chain reaction demonstrated the up-regulation of hypoxia inducible factor-1α in mice after partial bladder outlet obstruction compared to controls (p = 0.0394). Although not statistically significant, a trend toward lower gene expression of hypoxia inducible factor-1α was seen in mice receiving 2-methoxyestradiol compared to placebo (p = 0.0625). Compared to placebo, 2-methoxyestradiol treatment increased lysyl oxidase expression (p = 0.007). Conclusions: Murine partial bladder outlet obstruction resulted in hypoxia and up-regulation of the hypoxia inducible factor-1 pathway. Subcutaneous 2-methoxyestradiol administration attenuated this response and may be a viable tool to study the role of hypoxia after partial bladder outlet obstruction. [Copyright &y& Elsevier]

Abstract: Background: Delayed onset of cardiovascular disease (CVD) in female patients is not well understood, but could be due in part to the protective effect of estrogen before menopause. Experimental studies have identified the angiotensin type 1 receptor (AT1R) as a key factor in the progression of CVD. Objective: We examined the effects of the estrogen metabolite 2-methoxyestradiol (2ME2) on AT1R expression. Methods: Rat liver cells were exposed to 2ME2 for 24 hours, and angiotensin II (AngII) binding and AT1R mRNA expressions were assessed. Results: In the presence of 2ME2, cells exhibited significant down-regulation of AngII binding that was both dose and time dependent, independent of estrogen receptors (ERα/ERβ). Down-regulation of AngII binding was AT1R specific, with no change in receptor affinity. Under similar conditions, we observed lower expression of AT1R mRNA, significant inhibition of AngII-mediated increase in intracellular Ca2+, and increased phosphorylation of ERK1/2. Pretreatment of cells with the MEK inhibitor PD98059 prevented 2ME2-induced ERK1/2 phosphorylation and down-regulation of AT1R expression, which suggests that the observed inhibitory effect is mediated through ERK1/2 signaling intermediates. Similar analyses in stably transfected CHO (Chinese hamster ovary) cell lines with a constitutively active cytomegalovirus promoter showed no change in AT1R expression, which suggests that 2ME2-mediated effects are through transcriptional regulation. The effects of 2ME2 on AT1R down-regulation through ERK1/2 were consistently reproduced in primary rat aortic smooth muscle cells. Conclusions: Because AT1R has a critical role in the control of CVD, 2ME2-induced changes in receptor expression may provide beneficial effects to the cardiovascular and other systems. [Copyright &y& Elsevier]

2-Methoxyestradiol (2-ME; estradiol metabolite) inhibits vascular smooth muscle cell (VSMC) growth and protects against atherosclerosis and vascular injury; however, the mechanisms by which 2-ME induces these actions remain obscure. To assess the impact of 2-ME on biochemical pathways regulating VSMC biology, we used high-density oligonucleotide microarrays to identify differentially expressed genes in cultured human female aortic VSMCs treated with 2-ME acutely (4 hours) or long term (30 hours). Both single gene analysis and Gene Set Enrichment Analysis revealed 2-ME-induced downregulation of genes involved in mitotic spindle assembly and function in VSMCs. Also, Gene Set Enrichment Analysis identified effects of 2-ME on genes regulating cell-cycle progression, cell migration/adhesion, vasorelaxation, inflammation, and cholesterol metabolism. Transcriptional changes were associated with changes in protein expression, including inhibition of cyclin D1, cyclin B1, cyclin-dependent kinase 6, cyclin-dependent kinase 4, tubulin polymerization, cholesterol and steroid synthesis, and upregulation of cyclooxygenase 2 and matrix metalloproteinase 1. Microarray data suggested that 2-ME may activate peroxisome proliferator-activated receptors (PPARs) in VSMCs, and 2-ME has structural similarities with rosiglitazone (PPARγ agonist). However, our finding of weak activation and lack of binding of 2-ME to PPARs suggests that 2-ME may modulate PPAR-associated genes via indirect mechanisms, potentially involving cyclooxygenase 2. Indeed, the antimitogenic effects of 2-ME at concentrations that do not inhibit tubulin polymerization were blocked by the PPAR antagonist GW9662 and the cyclooxygenase 2 inhibitor NS398. Finally, we demonstrated that 2-ME inhibited hypoxia-inducible factor 1α. Identification of candidate genes that are positively or negatively regulated by 2-ME provides important leads to investigate and better understand the mechanisms by which 2-ME induces its vasoprotective actions. [ABSTRACT FROM AUTHOR]

2-Methoxyestradiol (2ME) is a major non-oestrogenic metabolite of oestfadiol. Our previous studies, performed in several models of cardiac and/or vascular injury, suggest that 2ME strongly inhibits both pressure-dependent and pressureindependent cardiac and vascular remodelling. Furthermore, recently we have shown that in male rats 2ME attenuates the development and retards the progression of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH); and in female rats 2ME eliminates the exacerbation of PAH and increased mortality due to ovariectomy. In the present study we compared the therapeutic effects of three different doses of 2ME (3, 10 and 30 Hg/kg/hour; 2ME-3, 2ME-10 and 2ME-30, respectively) in male rats with MCT-induced PAH. The animals were also monitored for plasma 2ME levels and potential oestrogenic effects. Treatments were initiated 12 days after administration of MCT (60 mg/kg, i.p.). Twenty-eight days post MCT, right ventricular peak systolic pressure (RVPSP) was measured and morphometric analysis was conducted. All three doses of 2ME produced beneficial therapeutic effects in pulmonary hypertensive animals, i.e. reduced pulmonary artery pressure and right ventricular hypertrophy, attenuated puhnonary vascular remodelling and inflammatory response, and had favourable effects on survival. Notably, none of the three doses had and effect on plasma testosterone levels or on seminal vesicle or testicle weight. Dose-dependent increases in 2ME plasma levels were observed only with 2ME-3 and 2ME-10; 2ME-30 protected 2ME plasma levels similar to those seen with 2ME10. Nonethclcss, 2ME-30 waz significantly more efficacious than 2ME-3 or 2ME-10 and eliminated the high mortality (34%) induced by MCT. In summary, the present study indicates that 2ME, used in doses that produce plasma levels similar to those seen in the last trimester of pregnancy (1000-3000pg/ml), is effective and safe (i.e. has no oestrogenic effects) in experimental PAH. These data also suggest that 2ME disposition, rather than plasma concentration, determines the therapeutic effects of 2ME in PAH. [ABSTRACT FROM AUTHOR]

Summary: Drugs that affect microtubule dynamics, including the taxanes and vinca alkaloids, have been a mainstay in the treatment of leukemias and solid tumors for decades. New, more effective microtubule-targeting agents continue to enter into clinical trials and some, including the epothilone ixapebilone, have been approved for use. In contrast, several other drugs of this class with promising preclinical data were later shown to be ineffective or intolerable in animal models or clinical trials. In this review, we discuss the molecular mechanisms as well as preclinical and clinical results for a variety of microtubule-targeting agents in various stages of development. We also offer a frank discussion of which microtubule-targeting agents are amenable to further development based on their availability, efficacy and toxic profile. [Copyright &y& Elsevier]

The article presents an assessment of the antiproliferative effects of 2-methoxyestradiol (2-ME) alone and in combination with various chemotherapeutic agents on human endometrial cancer cells. It examines the potential of 2-ME in the treatment of endometrial cancer. It concludes that 2-ME may selectively enhance the anticancer actions of certain chemotherapeutic agents in human endometrial cancer.

Abstract: Emerging data suggest a significant involvement of estrogens in the development and clinical course of asthma. Yet, little is known regarding the effects of 17β-estradiol (estradiol; E2) metabolism in asthma. E2 is metabolized by 2-hydroxylation and subsequent O-methylation to 2-methoxyestradiol (2ME) and by 16-hydroxylation to estriol (16α-hydroxyestradiol; 16HE2) and to 16α-estrone (16HE1). 2ME is a non-estrogenic metabolite which exhibits anti-mitogenic, anti-inflammatory and anti-angiogenic effects, whereas 16HE2 and 16HE1 are estrogenic metabolites with mitogenic, pro-inflammatory, and angiogenic properties. We hypothesize that estradiol metabolites from the 2-hydroxylation and 16-hydroxylation pathways have opposing effects in asthma, and that the net effect of E2 in asthma reflects the balance of these two metabolic pathways. In this regard, we propose that 2ME and E2/16HE1 divergently affect mitogenic and synthetic activity of cells involved in airway inflammation, angiogenesis and remodeling. Finally, we suggest that an “anti-cancer approach”, i.e., the use of anti-inflammatory, anti-angiogenic and anti-mitogenic agents such as 2ME, may have therapeutic effects in asthma. [Copyright &y& Elsevier]

17β-Estradiol is metabolized to 2-hydroxyestradiol, and 2-hydroxyestradiol is converted to 2-methoxyestradiol. These steroids reduce proliferation of, migration of and collagen production by vascular smooth muscle cells with an order of potency of 2-methoxyestradiol > 2-hydroxyestradiol > 17β-estradiol, i.e. the converse of their estrogenic potency. Since cAMP also inhibits cell growth, it is conceivable that these steroids alter cAMP synthesis or degradation, and this hypothesis was addressed in this study. In cultured preglomerular vascular smooth muscle cells pretreated with a high concentration of a broad spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, 1 mM) to eliminate phosphodiesterase activity as a confounding variable, high (100 µM), but not low (10 µM), concentrations of 17β-estradiol, 2-hydroxyestradiol and 2-methoxyestradiol attenuated isoproterenol-induced cAMP by 15%, 19% and 55%, respectively. 2-Hydroxyestradiol (100 µM) also attenuated forskolin-induced cAMP by 31% in cells treated with 3-isobutyl-1-methylxanthine. In cells not pretreated with 3-isobutyl-1-methylxanthine and using concentrations of steroids (10 µM) that did not interfere with adenylyl cyclase, 17β-estradiol decreased by 34%, 2-hydroxyestradiol did not alter and 2-methoxyestradiol increased by 60% isoproterenol-induced cAMP. These results indicate that in preglomerular vascular smooth muscle cells, high concentrations of 2-hydrxoyestradiol markedly inhibit adenylyl cyclase whereas 17β-estradiol and 2-methoxestradiol only modestly inhibit adenylyl cyclase even at high concentrations. Our results also indicate that lower concentrations of 17β-estradiol, 2-hydroxyestradiol and 2-methoxyestradiol activate, have no effect on and inhibit, respectively, phosphordiesterase activity. The effects of these compounds on phosphodiesterase activity may account in part for the greater potency of 2-methoxyestradiol as an anti-growth compound compared with 17β-estradiol and 2-hydroxyestradiol. [ABSTRACT FROM AUTHOR]

Global cerebral ischemia is an important clinical problem with few effective treatments. The hippocampus, which is important for memory, is especially vulnerable during global ischemia. Brain-specific knockout of hypoxia inducible factor-1α (HIF-1α) has been shown to be protective in focal ischemia in vivo. 2-methoxyestradiol (2ME2) is a natural metabolite of estrogen that is known to inhibit HIF-1α. We tested 2ME2 in a rat model of global cerebral ischemia. Global ischemia was induced with the two-vessel occlusion model (2VO) which entailed hemorrhagic hypotension to a mean arterial pressure of 38–42 mmHg with simultaneous bilateral common carotid artery occlusion for 8 minutes. Sprague–Dawley rats (male, 280–350 g) were randomly assigned to three groups: global ischemia (GI, n=17), global ischemia with 2ME2 treatment (GI + 2ME2, n=17) and sham surgery (sham, n=12). 2ME2 treatment (15 mg/kg in 1% DMSO) was rendered 10 minutes after reperfusion. Rats in the GI and sham groups received similar doses of the DMSO solvent. Rats were killed 24 hours, 72 hours and 7 days after reperfusion. Quantitative CA1 hippocampal cell counts demonstrated significantly lower cell survival in the GI + 2ME2 group compared to either the GI or sham groups, in spite of a statistically significant reduction in HIF-1α by Western blotting analysis of the GI + 2ME2 group. We conclude that 2ME2 worsens outcomes after global ischemia in rats. [ABSTRACT FROM AUTHOR]