Tubulin polymerization assay

Calf brain microtubule proteins (MTP) were purified, which included approximately 15% ~ 20% microtubule associated proteins. The buffer (MES buffer) used for the Epothilone B-microtubule studies contained 0.1 M 2-morpholinoethanesulfonic acid (MES), 1 mM EGTA, 0.5 mM MgCl2 and 3 M glycerol at pH 6.6. Samples for electron microscopy were placed on carbon-over-Parlodion-coated grids (300 mesh) and negatively stained with 2% uranyl acetate. Microtubule assembly in the presence or absence of Epothilone B was monitored spectrophotometrically by using a spectrophotometer equipped with a thermostatically regulated liquid circulator. The temperature was held at 35 ℃ and changes in turbidity (representative of polymer mass) were monitored at 350 nm.

Cell lines

Hs578T and Hela cells

Preparation method

The solubility of this compound in DMSO is >25.4 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

100 nM; 24 hrs

Applications

In Hs578T and Hela cells, Epothilone B arrested cells at the G2-M phase, causing cytotoxicity. In Hs578T cells treated with Epothilone B, there was a larger percentage (up to 38%) of cells showing multimininucleation.

Animal models

Mouse xenograft models of RPMI 8226 cells

Dosage form

1-time dose at 4 mg/kg or weekly dose at 2.5 mg/kg (given on days 6, 13, 20 and 27); i.v.

Applications

In mouse xenograft models of RPMI 8226 cells, Epothilone B (2.5 ~ 4 mg/kg) prolonged survival and inhibited tumor growth.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Epothilone B (EPO906, Patupilone) is a naturally occurring microtubule-stabilizing macrolide that was first isolated from the myxobacterium Sorangium cellulosum. The EC0.01 value of Epothilone B is 1.8 nM [1].

Epothilone B exhibits the same mechanism of action as paclitaxel. More importantly, in cell culture, Epothilone B is active against paclitaxel-resistant cell lines that express P-glycoprotein. Furthermore, Epothilone B has better solubility in water than paclitaxel [1].

Epothilone B induced an increase in the level of the intracellular free calcium only during the longer times following the treatment in SKOV-3 cells. Besides, the ovarian cancer cells treated with Epothilone B showed a time course dependent increase in the cytosolic fraction of cytochrome c [2].

References:
[1] Regueiro-Ren A1, Borzilleri RM, Zheng X, Kim SH, Johnson JA, Fairchild CR, Lee FY, Long BH, Vite GD. Synthesis and biological activity of novel epothilone aziridines. Org Lett. 2001 Aug 23;3(17):2693-6.
[2] Rogalska A1, Gajek A2, Marczak A2. Epothilone B induces extrinsic pathway of apoptosis in human SKOV-3 ovarian cancer cells. Toxicol In Vitro. 2014 Jun;28(4):675-83.