2-Methoxyestradiol  (Synonyms: 二甲氧基雌二醇; 2-ME2; NSC-659853)

Microtubule protein (2.75 mg/mL) is assembled to steady-state [in 100 mM PIPES containing 1 mM EGTA and 1 mM MgSO₄ (PEM100) and 1 mM GTP, 35°C for 45 minutes] containing 2-Methoxyestradiol (final drug concentrations of 1-500 μM). Final DMSO and ethanol concentrations are adjusted to 1% and 5%, respectively. Concentrations of 2-Methoxyestradiol ≤ 5 μM have no effect on microtubule polymer mass, and thus 20 to 500 μM 2-Methoxyestradiol is used for most of the experiments. Incubation with 2-Methoxyestradiol is carried out for 30 minutes, at which time microtubule depolymerization is maximal, and microtubules are centrifuged at 35°C for 30 minutes and the supernatant is removed from the pellets. Microtubule pellets are solubilized overnight in 0.2 M NaOH and the protein concentrations of supernatants and pellets are determined^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

MCF7 breast carcinoma cells stably transfected with green fluorescent protein (GFP)-α-tubulin are cultured in DMEM supplemented with nonessential amino acids, 0.1% penicillin/streptomycin, 10% fetal bovine serum, and 0.4 mg/mL G418 at 37°C in 5% CO₂. Transfection of MCF7 cells with GFP-α-tubulin is carried out. To evaluate mitotic indices, cells are plated at a concentration of 6×10⁴/2 mL into six-well plates. After 48 hours, cells are incubated in the absence or presence of 2-Methoxyestradiol at concentrations ranging from 100 nM to 30 μM for 20 hours. To collect both floating and attached cells, medium is collected; attached cells are rinsed with Versene (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, and 0.5 mM EDTA), detached by trypsinization, and added back to the medium. Cells are collected by centrifugation and fixed with 10% formalin for 30 minutes, permeabilized in ice-cold methanol for 10 minutes, and stained with 4′,6-diamidino-2-phenylindole to visualize nuclei. Results are the mean and SE of seven experiments in each of which 500 cells are counted for each concentration. The mitotic IC₅₀ is the drug concentration that induced one half of the maximal mitotic accumulation^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
6~8-week-old C57BL/6 mice are used. C57BL/6 mice are immunized subcutaneously 0.1 mL at tail and 0.05 mL at both thigh sites with IRBP antigen complex. 500 ng Pertussis toxin is injected concurrently. This day is settled as day 0. Then mice are divided into 4 groups, each group containing 5 mice. 15 mg/kg 2-Methoxyestradiol or vehicle is abdominal injected during 0-13 days, 0-6 days, and 7-13 days. At day 14 eyes or lymphoglandula is collected after euthanasia.
Rats^[4]
Fischer 344 rats (average body weight=150 g, n=6 per group) are 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 is repeated a second time using three rats per group.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Kamath K, et al. 2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules. Mol Cancer Ther. 2006 Sep;5(9):2225-33.  [Content Brief]

  [2]. Aquino-Gálvez A, et al. 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.  [Content Brief]

  [3]. Xu L, et al. 2-Methoxyestradiol Alleviates Experimental Autoimmune Uveitis by Inhibiting Lymphocytes Proliferation and T Cell Differentiation. Biomed Res Int. 2016;2016:7948345.  [Content Brief]

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

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

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