Endoxifen

Cell experiment:

MCF7 and Ishikawa cells are grown in 10% triple charcoal-stripped serum-containing medium for 3 d. The cells are then plated at a density of 2,000 cells per well in 96-well tissue culture plates and treated as indicated every 48 h. Cell proliferation assays are conducted 8 days after the first treatment using a CellTiter-Glo Luminescent Cell Viability kit[1].

Animal experiment:

Mice[2] GLAST::CreERT2 mice are used. Tamoxifen is injected as active metabolite 4-hydroxytamoxifen (4-OH-TAM), or 4-hydroxy-N-desmethyltamoxifen (Endoxifen). 4-OH-TAM is dissolved at 1 mM concentration in 2% ethanol/PBS and 5 μL injected into the ventricles. Endoxifen has not been used before for the induction of the ERT2 system. Endoxifen hydrochloride hydrate has a recommended solubility of over 10 mg/mL (25 mM) in 100% DMSO, corresponding to a maximum concentration of 25 mM or a dose of 125 nmol in a 5 μL volume. At this concentration, a proportion of mice developed neurological signs suggestive of neurotoxicity. Subsequently, concentrations of 2.5, 5, 10, 12.5 and 25 mM, dissolved in 10, 20, 40, 50 and 100% DMSO, respectively, corresponding to a drug dose of 12.5, 25, 50, 62.5 and 125 nmol are used. For testing DMSO toxicity, groups of three mice are injected with 5 uL PBS, 50% DMSO in PBS or 100% DMSO in PBS. Mice are culled 9 days after injection and coronally sectioned brains are stained for cleaved caspase 3, GFAP and Iba1.

References:

[1]. Wu X, et al. The tamoxifen metabolite, Endoxifen, is a potent antiestrogen that targets estrogen receptor alpha fordegradation in breast cancer cells. Cancer Res. 2009 Mar 1;69(5):1722-7.
[2]. Benedykcinska A, et al.Generation of brain tumours in mice by Cre-mediated recombination of neural progenitors in situ with the tamoxifen metabolite Endoxifen. Dis Model Mech. 2016 Feb;9(2):211-20.