Epothilone B (EPO 906) is a compound isolated from the myxobacterium Sorangium cellulosum. Similar to paclitaxel, patupilone induces microtubule polymerization and stabilizes microtubules against depolymerization conditions. In addition to promoting tubulin polymerization and stabilization of microtubules, this agent is cytotoxic for cells overexpressing P-glycoprotein, a characteristic that distinguishes it from the taxanes. Patupilone may cause complete cell-cycle arrest.

Tubulin:1.8 μM(EC0.01)

Epothilone B相比Epothilone A展现出更佳的活性。Epothilone B的EC0.01为1.8 μM，在HCT116细胞中强力抑制细胞增殖，IC50为0.8 nM。[1] Epothilone B能够引起KB3-1、KBV-1、Hela及Hs578T细胞的有丝分裂阻滞并表现出细胞毒性，其IC50介于3 nM至92 nM之间。Epothilone B与微管蛋白竞争结合，IC50为3.3 μM。[2] 在过表达GFP-α-tubulin的MCF-7细胞中，Epothilone B（3.5 nM）高效阻断微管动态，并以IC50为3.5 nM引起有丝分裂阻滞。[3] 在包括RPMI 8226、U266、MM.1S、LR5和MR20等多种骨髓瘤（MM）细胞中，Epothilone B直接抑制增殖，IC50在1 nM至10 nM之间，并在10 nM时诱导细胞周期阻滞和凋亡。[4] 最近的一项研究揭示，在卵巢癌Hey细胞中，Epothilone B（5 nM–100 nM）增强表面上皮细胞粘附抗原（EpCAM）的表达，但不影响EpCAM的转录或细胞内总水平。[5]

在RPMI 8226细胞的小鼠异种移植模型中，Epothilone B（2.5 mg/kg–4 mg/kg）能延长生存期并抑制肿瘤生长。[4] 相似地，在包括DU145和PC3的前列腺癌细胞的小鼠异种移植模型中，同样剂量的Epothilone B也能抑制肿瘤生长。[6]

Tubulin polymerization assay: Calf brain microtubule proteins (MTP) are purified, which includes approximately 15%–20% microtubule associated proteins. The buffer (MES buffer) used for the Epothilone B-microtubule studies contains 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 are 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 is monitored spectrophotometrically by using a spectrophotometer equipped with a thermostatically regulated liquid circulator. The temperature is held at 35 °C and changes in turbidity (representative of polymer mass) are monitored at 350 nm. Effective concentration (EC0.01), defined as the interpolated concentration capable of inducing an initial slope of 0.01 OD/min rate, is calculated using the formula EC0.01 = concentration/slope and expressed as the mean with standard deviation obtained from three different concentrations.

For mitotic block and aberrant mitosis, cells are plated either in 48-well plates (for trypan blue and cell counting) or onto coverslips. After 24 hours, cells are treated with Epothilone B and scored at regular intervals. For the cytotoxicity analysis, cells are counted and scored as trypan blue positive or negative. Concurrently, coverslips andaliquots of cells in the culture supernatant are fixed and stained with Hoechst33342 in PBS. These cells are scored for cells blocked at the G2/M transition and aberrant mitosis.(Only for Reference)