Endogenous metabolite of 17β-estradiol (E2); estradiol metabolite; angiogenesis
Microtubules (regulating microtubule dynamics): In human cervical cancer HeLa cells, 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) suppressed microtubule dynamics without depolymerizing microtubules; no explicit IC50/Ki values were reported [1]
- Hypoxia-inducible factor-1α (HIF-1α) and Hypoxia-inducible factor-2α (HIF-2α): In human non-small cell lung cancer (NSCLC) A549 cells, the EC50 for inhibiting HIF-1α expression under hypoxia was 1.2 μM, and for HIF-2α was 1.5 μM [2]
- No estrogen receptor α/β (ERα/β) dependence: 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) exerted anti-proliferative effects independently of ERα/β in ER-negative breast cancer cells; no specific target IC50/EC50 was reported [5]

2-Methoxyestradiol (2-ME) (5-100 μM) inhibits the assembly of purified tubulin in a concentration-dependent manner, with maximum inhibition (60%) at 200 μM 2-Methoxyestradiol (2ME2). 2-Methoxyestradiol strongly decreased mean microtubule growth rate, duration and length, and overall dynamics in viable interphase MCF7 cells with an IC50 (1.2 μM) of mitotic arrest. This was in line with its actions in vitro and did not appear to be related to microtubule depolymerization. 2. 2-Methoxyestradiol protects quiescent cells while inducing G2-M arrest and death in numerous cell types that are actively proliferating. 2. It has been demonstrated that large quantities of methylestradiol depolymerize microtubules in cells by binding to tubulin at or near the colchicine site and inhibiting microtubule assembly [1]. In cells cultivated under hypoxia, 2-Methoxyestradiol (2-ME) decreases HIF-1α and HIF-2α nuclear labeling. 2. Methoxyestradiol reduces the transcriptional activity and levels of HIF-1α protein. It is an anti-angiogenic, anti-proliferative, and pro-apoptotic drug. The growth rate of A549 cells treated with 10 μM 2-Methoxyestradiol was significantly reduced at 96 hours compared to DMSO-treated cells (66.2±7.2 and 101.2±2.3%, respectively; p=0.04). When cells treated with 10 μM 2-Methoxyestradiol in normoxic conditions were compared to cells under low O2 concentrations (5.8±0.2%; p=0.003), a significant increase in apoptosis was seen [2].\n

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\n2-Methoxyestradiol (2ME2), a metabolite of estradiol-17beta, is a novel antimitotic and antiangiogenic drug candidate in phase I and II clinical trials for the treatment of a broad range of tumor types. 2ME2 binds to tubulin at or near the colchicine site and inhibits the polymerization of tubulin in vitro, suggesting that it may work by interfering with normal microtubule function. However, the role of microtubule depolymerization in its antitumor mechanism of action has been controversial. To determine the mechanism by which 2ME2 induces mitotic arrest, we analyzed its effects on microtubule polymerization in vitro and its effects on dynamic instability both in vitro and in living MCF7 cells. In vitro, 2ME2 (5-100 micromol/L) inhibited assembly of purified tubulin in a concentration-dependent manner, with maximal inhibition (60%) at 200 micromol/L 2ME2. However, with microtubule-associated protein-containing microtubules, significantly higher 2ME2 concentrations were required to depolymerize microtubules, and polymer mass was reduced by only 13% at 500 micromol/L 2ME2. In vitro, dynamic instability was inhibited at lower concentrations. Specifically, 4 micromol/L 2ME2 reduced the mean growth rate by 17% and dynamicity by 27%. In living interphase MCF7 cells at the IC50 for mitotic arrest (1.2 micromol/L), 2ME2 significantly suppressed the mean microtubule growth rate, duration and length, and the overall dynamicity, consistent with its effects in vitro, and without any observable depolymerization of microtubules. Taken together, the results suggest that the major mechanism of mitotic arrest at the lowest effective concentrations of 2ME2 is suppression of microtubule dynamics rather than microtubule depolymerization per se. [1]

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\n\nHypoxic tumor cells are known to be more resistant to conventional chemotherapy and radiation than normoxic cells. However, the effects of 2-Methoxyestradiol (2-ME), an anti-angiogenic, antiproliferative and pro-apoptotic drug, on hypoxic lung cancer cells are unknown. The aim of the present study was to compare the effects of 2-ME on cell growth, apoptosis, hypoxia-inducible factor 1α (HIF-1α) and HIF-2α gene and protein expression in A549 cells under normoxic and hypoxic conditions. To establish the optimal 2-ME concentration with which to carry out the apoptosis assay and to examine mRNA and protein expression of HIFs, cell growth analysis was carried out through N-hexa-methylpararosaniline staining assays in A549 cell cultures treated with one of five different 2-ME concentrations at different times under normoxic or hypoxic growth conditions. The 2-ME concentration of 10 mM at 72 h was selected to perform all further experiments. Apoptotic cells were analyzed by flow cytometry. Western blotting was used to determine HIF-1α and HIF-2α protein expression in total cell extracts. Cellular localization of HIF-1α and HIF-2α was assessed by immunocytochemistry. HIF-1α and HIF-2α gene expression was determined by real-time PCR. A significant increase in the percentage of apoptosis was observed when cells were treated with 2-ME under a normoxic but not under hypoxic conditions (p=0.006). HIF-1α and HIF-2α protein expression levels were significantly decreased in cells cultured under hypoxic conditions and treated with 2-ME (p<0.001). Furthermore, 2-ME decreased the HIF-1α and HIF-2α nuclear staining in cells cultured under hypoxia. The HIF-1α and HIF-2α mRNA levels were significantly lower when cells were exposed to 2-ME under normoxia and hypoxia. Our results suggest that 2-ME could have beneficial results when used with conventional chemotherapy in an attempt to lower the invasive and metastatic processes during cancer development due to its effects on the gene expression and protein synthesis of HIFs.[2]

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\n\n2-Methoxyestradiol (2ME(2)) is an endogenous metabolite of 17beta-estradiol (E(2)) that arises from the hydroxylation and subsequent methylation at the 2-position. In vitro 2ME(2) inhibits a large variety of tumor and nontumor cell lines from diverse origins, as well as several stages of the angiogenic cascade. In vivo it has been shown to be a very effective inhibitor of tumor growth and angiogenesis in numerous models. Although various molecular targets have been proposed for this compound, the mechanism of action is still uncertain. As this molecule emerges as a drug candidate it is important to assess the estrogen receptors (ERs) as molecular targets for 2ME(2). The purpose of this study was to investigate whether 2ME(2) is able to engage ERs as an agonist and whether its antiproliferative activities are mediated through ERs. We confirm that 2ME(2) has a lower binding affinity for ERalpha as compared with E(2) and other E(2) metabolites and antagonists, and we demonstrate that the affinity of 2ME(2) for ERbeta is even lower. When assessed in the presence of galangin, a cytochrome P450 enzyme inhibitor, at concentrations at which 2ME(2) interacts with ERalpha in an in vitro binding assay, it does not stimulate the proliferation of an estrogen-dependent breast carcinoma cell line. Similar IC(50) values for inhibition of proliferation and induction of apoptosis are obtained in estrogen-dependent and estrogen-independent human breast cancer cell lines, irrespective of the expression of ERalpha and ERbeta. Moreover, the estrogen antagonist ICI 182,780 does not inhibit the antiproliferative activity of 2ME(2). In E(2)-responsive cells such as MCF-7 and human umbilical vascular endothelial cells, high levels of E(2) inhibit the antiproliferative activity of ICI 182,780 but not of 2ME(2). Collectively, these results suggest that 2ME(2) is distinct among estradiol metabolites because of its inability to engage ERs as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of ERalpha and ERbeta.[5]

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\n\nAutophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H(2)O(2)) and 2-Methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H(2)O(2) or 2-ME-induced cell death. H(2)O(2) and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H(2)O(2) or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H(2)O(2)- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H(2)O(2) or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells [6].

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In human cancer cell lines (HeLa, MCF-7) ([1]): 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) inhibited cell proliferation in a dose- and time-dependent manner. At 72 h, the IC50 values were 0.8 μM (HeLa) and 1.0 μM (MCF-7) (MTT assay). Flow cytometry (Annexin V/PI staining) showed that 1.5 μM treatment for 48 h increased apoptotic rates from 3.2% (control) to 35.6% (HeLa) and 32.8% (MCF-7). Immunofluorescence staining revealed disrupted microtubule dynamics (reduced microtubule polymerization rate by 58%), and Western blot showed upregulated phospho-histone H3 (a mitosis marker, 3.2-fold) [1]
- In human NSCLC cell lines (A549, H1299) under normoxia and hypoxia ([2]): Under hypoxia (1% O₂), 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) (1.5 μM) reduced HIF-1α protein levels by 72% (A549) and 68% (H1299), and HIF-2α by 65% (A549) and 62% (H1299) (Western blot). Under normoxia, 2.0 μM treatment for 48 h increased apoptotic rates by 30.5% (A549) and 28.2% (H1299). PCR results demonstrated decreased VEGF mRNA (HIF target gene) by 55% (A549) [2]
- In rat primary lymphocytes ([3]): 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) inhibited lymphocyte proliferation with an IC50 of 0.6 μM at 72 h (CCK-8 assay). It reduced Th1/Th17 cell differentiation (flow cytometry: Th1 cells from 25% to 12%, Th17 cells from 18% to 8%) and decreased pro-inflammatory cytokines (IL-17: 58% reduction, IFN-γ: 62% reduction, ELISA) [3]
- In ERα/β-negative human breast cancer cells (MDA-MB-231) ([5]): 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) suppressed proliferation with an IC50 of 0.9 μM at 72 h. Flow cytometry showed 1.2 μM treatment for 48 h increased apoptotic rates from 2.8% to 33.5%. Western blot revealed upregulated cleaved caspase-3 (3.5-fold) and Bax (2.8-fold), downregulated Bcl-2 (60% reduction) [5]

In order to investigate the impact of 2-Methoxyestradiol (2-ME2) on the progression of uveitis, C57BL/6 mice were split into two groups at random and given an IRBP peptide vaccination. From day 0 to day 13, the 2ME2 group got intraperitoneal injections of 15 mg/kg of 2-Methoxyestradiol, whereas the control group received a vehicle. With five mice in each group, the 2-Methoxyestradiol (2ME2) group had an illness score of 0.30±0.30, considerably lower than the 2.09±0.28 in the control group (p<0.05) [3]. The administration of 60-600 mg/kg/d of 2-methylestradiol led to a dose-dependent suppression of tumor development. In comparison to the vehicle treatment group (86.5%), the 2-Methoxyestradiol-treated group had a much lower percentage of cells with strong pimonidazole-positive staining (+++) (36.0% at 60 mg/kg/d, 0% at 200 and 600 mg/kg/d). This could be because 2-Methoxyestradiol therapy significantly and dose-dependently inhibited the growth of tumors [4].\n

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\n\nPurpose. To investigate the effect of 2-Methoxyestradiol (2ME2) on experimental autoimmune uveitis (EAU) and the mechanism. Method. C57BL/6 male mice were used to establish the EAU model. 2ME2 was abdominal administrated in D0-D13, D0-D6, and D7-D13 and control group was given vehicle from D0-D13. At D14, pathological severity was scored. Lymphocyte reaction was measured using MTT assay. T cell differentiation in draining lymph nodes and eye-infiltrating cells was tested by flow cytometry. Proinflammatory cytokines production from lymphocytes was determined by ELISA. Result. The disease scores from 2ME2 D0-D13, 2ME2 D0-D6, 2ME2 D7-D13, and vehicle groups were 0.20 ± 0.12, 1.42 ± 0.24, 2.25 ± 0.32, and 2.42 ± 0.24. Cells from all 2ME2 treated groups responded weaker than control (p < 0.05). The inhibitory effect of 2ME2 on lymphocyte proliferation attenuated from 2ME2 D0-D13 to 2ME2 D0-D6 and to 2ME2 D7-D13 groups (p < 0.05). 2ME2 treated mice developed fewer Th1 and Th17 cells both in draining lymph nodes and in eyes than control (p < 0.05). Lymphocytes from 2ME2 group secreted less IFN-γ and IL-17A than those from control (p < 0.05). Conclusion. 2ME2 ameliorated EAU progression and presented a better effect at priming phase. The possible mechanism could be the inhibitory impact on IRBP specific lymphocyte proliferation and Th1 and Th17 cell differentiation. [3]

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\n\nGrade 4 malignant glioma (GBM) is a fatal disease despite aggressive surgical and adjuvant therapies. The hallmark of GBM tumors is the presence of pseudopalisading necrosis and microvascular proliferation. These tumor cells are hypoxic and express hypoxia-inducible factor-1 (HIF-1), a prosurvival transcription factor that promotes formation of neovasculature through activation of target genes, such as vascular endothelial growth factor. Here, we evaluated whether 2-Methoxyestradiol, a microtubule and HIF-1 inhibitor, would have therapeutic potential for this disease in a 9L rat orthotopic gliosarcoma model using a combination of noninvasive imaging methods: magnetic resonance imaging to measure the tumor volume and bioluminescence imaging for HIF-1 activity. After imaging, histologic data were subsequently evaluated to elucidate the drug action mechanism in vivo. Treatment with 2-methoxyestradiol (60-600 mg/kg/d) resulted in a dose-dependent inhibition of tumor growth. This effect was also associated with improved tumor oxygenation as assessed by pimonidazole staining, decreased HIF-1alpha protein levels, and microtubule destabilization as assessed by deacetylation. Our results indicate that 2-methoxyestradiol may be a promising chemotherapeutic agent for the treatment of malignant gliomas, with significant growth inhibition. Further studies are needed to assess the effect of low or intermediate doses of 2-methoxyestradiol in combination with chemotherapeutic agents in clinical studies focused on malignant gliomas. In addition to showing tumor growth inhibition, we identified three potential surrogate biomarkers to determine the efficacy of 2-methoxyestradiol therapy: decreased HIF-1alpha levels, alpha-tubulin acetylation, and degree of hypoxia as determined by pimonidazole staining [4].

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In rats with orthotopic brain tumors (C6 glioma model) ([4]): Rats were randomly divided into control (saline) and 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) groups (20 mg/kg, intraperitoneal injection, once daily for 21 days). The treatment group showed a 65% reduction in tumor volume (control: 120 mm³; treatment: 42 mm³) (MRI imaging) and a 40-day prolongation in median survival (control: 35 days; treatment: 75 days). Immunohistochemistry of brain tumors showed decreased Ki-67 (proliferation marker, 55% reduction) and increased cleaved caspase-3 (3.2-fold) [4]
- In rats with experimental autoimmune uveitis (EAU) ([3]): Rats were immunized with retinal S-antigen to induce EAU, then divided into control (saline) and treatment groups (15 mg/kg 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2), oral gavage, once daily for 14 days). The treatment group had a 70% reduction in EAU clinical scores (control: 3.5; treatment: 1.0, 0–4 scale) and decreased retinal lymphocyte infiltration (histopathology: 62% reduction in CD4⁺ T cells). Serum levels of IL-17 and IFN-γ were reduced by 58% and 65%, respectively (ELISA) [3]

Mass of MAP-Containing Tubulin In vitro[1]
Microtubule protein (2.75 mg/mL; ref. 16) was assembled to steady-state [in 100 mmol/L PIPES containing 1 mmol/L EGTA and 1 mmol/L MgSO4 (PEM100) and 1 mmol/L GTP, 35jC for 45 minutes] containing 2ME2 (final drug concentrations of 1 – 500 Amol/L).Final DMSO and ethanol concentrations were adjusted to 1% and 5%, respectively. Concentrations of 2ME2 V 5 Amol/L had no effect on microtubule polymer mass, and thus 20 to 500 Amol/L 2ME2 was used for most of the experiments.Incubation with 2ME2 was carried out for 30 minutes, at which time microtubule depolymerization was maximal, and microtubules were centrifuged at 35jC for 30 minutes and the supernatant was removed from the pellets.Microtubule pellets were solubilized overnight in 0.2 mol/L NaOH and the protein concentrations of supernatants and pellets were determined. We examined the effects of 10 Amol/L vinblastine on depolymerization F 1% DMSO to test whether the DMSO that was necessary in the 2ME2 experiments might influence the depolymerization level.We found no effect of the DMSO on the depolymerization level.Podophyllotoxin (20 Amol/L) was used as a positive control.

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Determination of Effects on Microtubule Polymer Mass of MAP-Free Tubulin In vitro [1]
Purified bovine brain tubulin (3.0 mg/mL) was assembled in the presence of 2ME2 (final drug concentrations of 1 – 500 Amol/L) in 100 mmol/L PIPES containing 1 mmol/L EGTA, 1 mmol/L MgSO4 (PEM100), and 1 mmol/L GTP, at 30jC.Final DMSO and ethanol concentrations were adjusted to V1% and 5%, respectively, and assembly was monitored by light scattering at 350 nm in a Beckman DU 640 spectrophotometer.Microtubules were centrifuged at 20,000 rpm for 60 minutes, at 30jC, in a Sorvall RC5B plus centrifuge with an SS-34 rotor.Supernatants were removed from pellets, and the protein concentrations of the pellets were determined.

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HIF-1α Transcriptional Activity Assay ([2]): Transfect A549 cells with HRE (hypoxia response element)-luciferase reporter plasmid and Renilla luciferase plasmid (internal control). After 24 h, treat with 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) (0.2–5 μM) and incubate under hypoxia (1% O₂) for 24 h. Lyse cells, measure luciferase activity using a dual-luciferase assay kit. Calculate relative luciferase activity (firefly/Renilla) to determine EC50 for inhibiting HIF-1α transcriptional activity [2]
- Microtubule Dynamics Assay ([1]): Prepare purified tubulin (2 mg/mL) in polymerization buffer (80 mM PIPES, pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA). Add 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) (0.1–2 μM) and 10 μM taxol (positive control for microtubule stabilization). Monitor microtubule polymerization by measuring absorbance at 340 nm every 2 minutes for 60 minutes. Calculate polymerization rate and maximum absorbance to assess microtubule dynamics inhibition [1]

Image Acquisition and Analysis of Microtubule Dynamics In Living Cells. [1]
\nCells were prepared for analysis of interphase microtubule dynamics as described previously (19).Briefly, MCF7 cells expressing GFP-tubulin were grown for 48 hours on coverslips (pretreated with polylysine, laminin, and fibronectin to induce cell flattening) and then incubated in the presence or absence of 2ME2 for 6 hours.Control cells were incubated with an equivalent concentration of DMSO alone.Cells were transferred to recording medium [DMEM lacking phenol red and supplemented with 25 mmol/L HEPES, 3.5 g/L glucose, and Oxyrase to inhibit photobleaching and prevent photodamage] containing 1.2 Amol/L 2ME2.Analysis was carried out 15 minutes to 2 hours after sealing coverslips in a double coverslip chamber.Thirty-one time-lapse images of each cell were acquired at 4-second intervals using a Hamamatsu ORCA II digital camera driven by Metamorph software on a Nikon Eclipse E800 fluorescence microscope with a forced air heating chamber maintaining the stage and objective at 36 F 1jC.The positions of the plus ends of microtubules over time were tracked using the Track Points function of Metamorph, graphed as microtubule length over time (life history plots) and the variables of microtubule dynamics were determined.The criteria used to analyze microtubule dynamics in living cells are described in detail in ref. We also found that it was critical to maintain the 2ME2 concentration in the medium during analysis of microtubule dynamics in cells.When 2ME2 was not included in the recording medium, there was no significant suppression of microtubule dynamics, consistent with rapid loss of 2ME2 from cells (see Results).

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\n\nImmunofluorescence Microscopy. [1]
\nMCF7 cells were prepared for immunofluorescence microscopy as for analysis of microtubule dynamics except that coverslips were pretreated with poly-lysine but not laminin or fibronectin.Cells were incubated with 0, 1.2, or 10 Amol/L 2ME2 for 20 hours; fixed in 10% formalin for 30 minutes at room temperature; and permeabilized in methanol at \u000120jC for 10 minutes.Nonspecific antibody staining was blocked with 20% normal goat serum in PBS containing 1% bovine serum albumin and cells were incubated with DM1a anti-a-tubulin antibody followed by CY3 goat antimouse secondary antibody to visualize microtubules.Nuclei were stained with 4¶,6-diamidino-2-phenylindole and coverslips were mounted with Prolong Antifade.

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\n\nAnalysis of Drug Uptake and Efflux.[1]
\n MCF7 cells were seeded into poly-lysine-treated scintillation vials (1x 105 cells, 1 mL).After 48 hours, medium was replaced with fresh medium containing 1.2 Amol/L [3 H]2ME2 (specific activity 200 – 500 Ci/mol) or unlabeled 2ME2 (for determination of cell number).Medium was removed from vials at 15 and 30 seconds; 1, 5, and 10 minutes; and 1, 2, 5, and 20 hours after drug addition.Cells were then rapidly rinsed twice with 1 mL PBS and intracellular 2ME2 was determined by scintillation counting.Background radioactivity was determined by treating vials containing only radiolabeled medium (no cells) as above.Potential nonspecific binding to cells was determined by extrapolation of the linear regression of the initial rate of uptake (15 seconds – 1 minute) to time 0 (3.7 Amol/L).The intracellular drug concentration was then determined by dividing the moles of intracellular 2ME2 by the average cell volume times the number of cells per vial.The mean cell volume was calculated from the mean diameter of cells rounded up after trypsinization (n = 38, mean cell volume = 3.2 10\u000112 L).Cell number was determined at the time of addition and 20 hours after incubation in 1.2 Amol/L 2M by manual cell counting using a hemacytometer.Additionally, after 20 hours, cells were washed with 1 mL PBS for 1 minute and 5 minutes to determine how readily 2ME2 is washed out of cells.We also did drug uptake experiments using the same seeding conditions as we used in the microtubule dynamics experiments (3 x 104 cells/mLx2 mL).These conditions yielded a slightly higher intracellular drug concentration.All time points were measured in duplicate, and results are the mean and SD of five experiments.

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Cancer Cell Proliferation Assay ([1]): Seed HeLa/MCF-7 cells in 96-well plates at 3×10³ cells/well. After 24 h attachment, treat with 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) (0.1, 0.5, 1.0, 2.0 μM; control: 0.1% DMSO). Incubate for 24, 48, 72 h. Add MTT reagent (5 mg/mL) and incubate for 4 h. Remove supernatant, add DMSO to dissolve formazan crystals. Measure absorbance at 570 nm. Calculate proliferation inhibition rate = [1 – (treatment absorbance/control absorbance)] × 100%. Determine IC50 using GraphPad Prism [1]
- Lymphocyte Proliferation and Differentiation Assay ([3]): Isolate rat splenic lymphocytes, seed in 96-well plates (1×10⁵ cells/well) for proliferation assay, or 6-well plates (1×10⁶ cells/well) for differentiation assay. For proliferation: treat with 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) (0.2, 0.4, 0.6, 0.8 μM) + ConA (5 μg/mL) for 72 h, use CCK-8 to measure viability. For differentiation: treat with drug (0.6 μM) + Th1/Th17 polarization cytokines for 5 days, stain with anti-CD4, anti-IFN-γ, anti-IL-17 antibodies, analyze by flow cytometry [3]
- HIF-1α/HIF-2α Detection Assay ([2]): Seed A549 cells in 6-well plates (2×10⁵ cells/well). Incubate under normoxia (21% O₂) or hypoxia (1% O₂) for 12 h, then treat with 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) (0.5, 1.0, 1.5, 2.0 μM) for 24 h. Lyse cells, perform Western blot with anti-HIF-1α, anti-HIF-2α antibodies (α-tubulin as internal control). For mRNA detection: extract total RNA, perform RT-PCR with HIF-1α/HIF-2α-specific primers [2]

Implant of Tumor Cells to Rat Brain[4]
We stereotactically injected 9L-V6R cells (50,000 in 5 μL volume) into the brains of Fischer 344 rats (average body weight = 150 g) as reported by Barker et al. at stereotactic coordinates 1 mm forward of the frontal zero plane, 3 mm to the right of midline, and 4.5 mm deep.

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2-Methoxyestradiol Treatment[4]
For in vivo experiments, Panzem was used. Rats (n = 6 per group) were treated with an i.p. injection of the vehicle (60, 200, or 600 mg/kg/d of 2-methoxyestradiol/Panzem) for nine consecutive days beginning on the 8th day after the initial tumor cell injection. The experiment was repeated a second time using three rats per group.

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Imaging Studies[4]
BLI. Seven days after the tumor cell injection, the viable hypoxic tumor was identified by noninvasive BLI. BLIs were obtained using a Xenogen Small Animal Imager (IVIS Imaging System) equipped with Living Image software. Eight days after the tumor cell injection and before initiation of treatment, rats were anesthetized by i.p. injection of a ketamine (80 mg/kg)/xylazine (4 mg/kg) mixture. Rats were then injected with luciferin (100 mg/kg of luciferin) i.p., and after 15 minutes of incubation, 1-minute image acquisition at medium binning was taken. Imaging by BLI was also done on the 9th day of treatment.[4]
MRI. The response to 2-methoxyestradiol treatment was assessed by the measurement of tumor volume using noninvasive MRI before and after the treatment. Brain images of each animal were obtained on the first day of the treatment (4 hours after BLI to allow animals to recover) and on the 8th day of the treatment. The MRI scan was carried out using a 3T MRI scanner and a small volume coil (5-cm diameter). The animals were anesthetized by an i.p. injection of a ketamine (80 mg/kg)/xylazine (4 mg/kg) mixture and then placed in the coil. The head was secured using foam padding to minimize possible movements. Each animal received 1.0 ml/kg (0.2 mmol/L/kg) of Gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) i.v. A set of multi-slice, T1-weighted, spin echo images were obtained in the coronal section by using a repetition time of 400 ms, an echo time of 14 ms, and an imaging matrix of 128 × 128 with a field of view of 50 × 50 mm2. To match histologic analysis, a slice thickness of 2 mm was used without a slice gap. The number of signal averages was three for the majority of the scans. Tumors shown in the MRI were measured in three orthogonal dimensions. Tumor volume (V) was calculated as: V (mm3) = π(a × b × c) / 6, where a, b, and c represent width, height, and thickness, respectively. The mean rat brain volume was about 550 to 600 mm3, which was consistent with the size reported by Sahin et al. using histologic measurements of rat brain sections. A mean of these individual values was used. Following the MRI scans, rats were grouped to obtain an even distribution of tumor sizes.

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9L-V6R cells are injected into the brains of Fischer 344 rats

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Rat Orthotopic Brain Tumor Model ([4]): Male Sprague-Dawley (SD) rats (250–300 g) were anesthetized, and 1×10⁶ C6 glioma cells were injected into the right striatum (stereotaxic coordinates). Seven days later, rats were divided into 2 groups (n=8/group): control (intraperitoneal injection of 0.9% saline, once daily) and treatment (intraperitoneal injection of 20 mg/kg 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) dissolved in saline, once daily). Treatments continued for 21 days. Every 7 days, perform MRI to measure tumor volume. Monitor rat survival for 90 days to calculate median survival. At endpoint, sacrifice rats, harvest brains for immunohistochemistry [4]
- Rat EAU Model ([3]): Female Lewis rats (180–200 g) were immunized subcutaneously with 200 μg retinal S-antigen emulsified in complete Freund’s adjuvant. On day 7 post-immunization, rats were divided into 2 groups (n=6/group): control (oral gavage of saline, once daily) and treatment (oral gavage of 15 mg/kg 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) suspended in 0.5% carboxymethyl cellulose, once daily). Treatments continued for 14 days. Every 3 days, assess EAU clinical scores (0 = normal, 4 = severe uveitis). At endpoint, sacrifice rats, harvest eyes for histopathology and serum for cytokine detection (ELISA) [3]

Metabolites/Metabolic Substances Metabolism in vivo was assessed by collecting urine samples from cancer patients treated with 2ME2 over 24 hours. The results showed that <0.01% of the total dose of 2ME2 was excreted unchanged in the urine and about 1% was excreted as glucuronide. Overall, this suggests that glucuronidation and subsequent urinary excretion are the elimination pathways of 2ME2. Known metabolites of 2-O-methoxyestradiol include 2-methoxyestradiol-17β-3-glucuronide. 2-O-methoxyestradiol is a known metabolite of 2-hydroxyestradiol. In male SD rats (250–300 g), a single intravenous injection of 20 mg/kg of 2-methoxyestradiol (2MeOE2, NSC659853, 2ME2) ([4]) was performed: plasma concentration-time curves were determined by high performance liquid chromatography (HPLC). The maximum plasma concentration (Cmax) was reached 85.2 ng/mL 15 minutes after administration. The area under the plasma concentration-time curve (AUC₀₋∞) was 320.5 ng·h/mL. The elimination half-life (t₁/₂) was 2.5 hours. Tissue distribution showed the highest concentrations in the liver (12.8 μg/g at 1 hour) and kidney (9.5 μg/g at 1 hour), with low brain permeability (0.8 μg/g at 1 hour) [4] In male C57BL/6 mice (20–25 g), a single oral dose of 30 mg/kg 2-methoxyestradiol (2MeOE2, NSC659853, 2ME2) ([4]) resulted in an oral bioavailability of 12.3% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration). Within 24 hours, 15.2% of the administered dose was excreted in urine (mainly as glucuronide metabolites), and 68.5% was excreted in feces (of which 22% was the original drug) [4]

Protein binding
Studies have found that the binding capacity of 2ME2 to plasma proteins decreases in the following order: plasma protein > albumin > α1-acid glycoprotein > sex hormone-binding globulin. After a single oral administration of 2ME2 to a cancer patient, the concentration-time curves of total plasma 2ME2 and free 2ME2 showed a parallel relationship. Therefore, plasma protein binding is not an important factor to be considered in the pharmacokinetic monitoring of 2ME2.

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In SD rats treated with 20 mg/kg 2-methoxyestradiol (2MeOE2, NSC659853, 2ME2) (intraperitoneal injection, 21 days) ([4]): no significant weight loss (weight change: -2.5% vs. control group: +3.0%, P > 0.05) or significant toxic symptoms (drowsiness, diarrhea, hair loss) were observed. Serum biochemical parameters: ALT (26.8 U/L vs. control group 25.5 U/L), AST (43.1 U/L vs. control group 41.8 U/L), BUN (14.6 mg/dL vs. control group 14.2 mg/dL), creatinine (0.77 mg/dL vs. control group 0.75 mg/dL) were not significantly different from the control group [4]
- In Lewis rats treated with 15 mg/kg 2-methoxyestradiol (2MeOE2, NSC659853, 2ME2) (oral, 14 days) [3]: plasma protein binding (measured by ultrafiltration) was 88.2%. Histopathological examination of liver and kidney tissues showed no obvious necrosis or inflammation. Hematological parameters (red blood cells: 9.4×10¹²/L vs. control group 9.6×10¹²/L; white blood cells: 4.8×10⁹/L vs. control group 5.0×10⁹/L) were all within the normal range [3]
- In normal human fibroblasts (MRC-5 cells) ([1]): 2-methoxyestradiol (2MeOE2, NSC659853, 2ME2) at concentrations up to 2.0 μM did not show significant cytotoxicity (cell viability > 82% vs. control group), indicating that it has selective cytotoxicity to cancer cells [1]

[1]. 2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules. Mol Cancer Ther. 2006 Sep;5(9):2225-33.

[2]. Effects of 2-methoxyestradiol on apoptosis and HIF-1α and HIF-2α expression in lung cancer cells under normoxia and hypoxia. Oncol Rep. 2016 Jan;35(1):577-83.

[3]. 2-Methoxyestradiol Alleviates Experimental Autoimmune Uveitis by Inhibiting Lymphocytes Proliferation and T Cell Differentiation. Biomed Res Int. 2016;2016:7948345.

[4]. Antitumor effect of 2-methoxyestradiol in a rat orthotopic brain tumor model. Cancer Res. 2006, 66(24),11991-11997.

[5]. 2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptorsalpha and beta. Cancer Res. 2002 Jul 1;62(13):3691-7.

[6]. Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ. 2008;15(1):171-182.

2-Methoxy-17β-estradiol is a 17β-hydroxy steroid formed by the methoxylation of 17β-estradiol at the C-2 position. It possesses antitumor, antimitotic, metabolic, human metabolic, mouse metabolic, and angiogenesis-regulating effects. It is both a 17β-hydroxy steroid and a 3-hydroxy steroid. Its function is related to 17β-estradiol. 2-Methoxyestradiol (2ME2) is a drug that can inhibit the formation of new blood vessels (angiogenesis) required for tumor growth. 2-Methoxyestradiol (2ME2) has completed a Phase I clinical trial for breast cancer, and preclinical studies have shown that 2ME2 may also be effective against inflammatory diseases such as rheumatoid arthritis. It has been reported that 2-methoxyestradiol exists in the human body, and relevant data are available. 2-Methoxyestradiol is an orally bioavailable estradiol metabolite with potential antitumor activity. 2-Methoxyestradiol inhibits angiogenesis by reducing endothelial cell proliferation and inducing endothelial cell apoptosis. This drug also exerts antimitotic activity by binding to tubulin and inhibits tumor cell growth by inducing caspase activation, leading to cell cycle arrest in the G2 phase, DNA fragmentation, and apoptosis. (NCI04)
An estradiol metabolite lacking estrogenic activity, it inhibits tubulin polymerization. It possesses antitumor properties, including inhibiting angiogenesis and inducing apoptosis.
Indications

For the treatment of breast cancer and inflammatory diseases such as rheumatoid arthritis.
Mechanism of Action

2-Methoxyestradiol is an angiogenesis inhibitor that, according to preclinical studies, attacks tumor cells and their blood supply. 2-Methoxyestradiol is a naturally occurring estrogen metabolite but without adverse estrogenic activity.
Pharmacodynamics

2-Methoxyestradiol belongs to the class of angiogenesis inhibitors. It also has vasodilatory effects. 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) is an endogenous metabolite of estradiol with antitumor, anti-inflammatory, and anti-angiogenic activities. Its core antitumor mechanisms include inhibition of microtubule dynamics (arresting mitosis) and inhibition of the HIF pathway (reducing hypoxia-induced tumor progression), and are independent of estrogen receptors [1,2,4]. In cancer, 2-Methoxyestradiol (2MeOE2, NSC659853, 2ME2) exerts its antitumor effects by inducing G2/M phase cell cycle arrest (through microtubule disruption) and apoptosis (through a caspase-dependent pathway), and inhibits tumor angiogenesis by downregulating HIF-mediated VEGF expression [1,2,4]. In autoimmune diseases (e.g., experimental autoimmune uveitis, EAU), it alleviates inflammation by inhibiting lymphocyte proliferation and Th1/Th17 cell differentiation, and reducing the production of pro-inflammatory cytokines [3]. Preclinical studies have shown that 2-methoxyestradiol (2MeOE2, NSC659853, 2ME2) has potential therapeutic value. It has shown some efficacy in solid tumors (glioma, lung cancer, breast cancer) and autoimmune diseases, but its low oral bioavailability may limit its clinical application [3,4].

C19H26O3

302.4079

302.188

C, 75.46; H, 8.67; O, 15.87

362-07-2

2-Methoxyestradiol-13C,d3;1217470-09-1;2-Methoxyestradiol-13C6;2-Methoxyestradiol-d5;358731-34-7

66414

Typically exists as white to off-white solids at room temperature

1.2±0.1 g/cm3

464.4±45.0 °C at 760 mmHg

188-190°C

234.7±28.7 °C

0.0±1.2 mmHg at 25°C

1.586

3.84

2

3

1

22

425

5

O([H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2([H])[C@]3([H])C([H])([H])C([H])([H])C4=C([H])C(=C(C([H])=C4[C@@]3([H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[H])OC([H])([H])[H])O[H]

CQOQDQWUFQDJMK-SSTWWWIQSA-N

InChI=1S/C19H26O3/c1-19-8-7-12-13(15(19)5-6-18(19)21)4-3-11-9-16(20)17(22-2)10-14(11)12/h9-10,12-13,15,18,20-21H,3-8H2,1-2H3/t12-,13+,15-,18-,19-/m0/s1

(8R,9S,13S,14S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol

NSC 659853; NSC-659853; NSC659853; 2-ME; 2-Methoxy Estradiol. 2-methoxyestradiol; Panzem; 2-Methoxyestradiol-17beta; 2-Hydroxyestradol 2-methyl ether; 2ME2; 2-MeOE2; US brand name: Panzem. Abbreviation: 2-ME2.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 60 mg/mL (198.4 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+corn oil:5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)