| 名稱 | Alisertib |
| 描述 | Alisertib (MLN 8237) is an Aurora A kinase inhibitor (IC50=1.2 nM) with oral activity and selectivity. Alisertib has antitumor activity, induces apoptosis and autophagy, and induces cell cycle arrest. |
| 細(xì)胞實(shí)驗(yàn) | HCT-116 colorectal carcinoma cells were plated on 6-well dishes (2 × 10^5 per well) and propagated in McCoy's 5A media supplemented with 10% FBS. After 18 hours, alisertib at a final concentration of 0.050, 0.250, or 1.000 μmol/L was added, and the cells were grown for an additional 24 hours. Cells treated with dimethyl sulfoxide (DMSO; 0.2%) served as the untreated vehicle control. The cells were harvested with trypsin EDTA 1×, washed once with PBS, fixed in 70% ethanol, and stored at 4°C for 1 hour. The cells were resuspended in propidium iodide (1:40) and RNAse A (1:5,000) in PBS for 30 minutes at 4°C. Cell-cycle distributions were determined by measuring DNA content using flow cytometry, and samples were analyzed using Winlist 5.0 software [2]. |
| 激酶實(shí)驗(yàn) | Recombinant murine Aurora A and Aurora B protein were expressed in Sf9 cells and purified with GST affinity chromatography. The peptide substrate for Aurora A was conjugated with biotin (Biotin-GLRRASLG). Aurora A kinase (5 nM) was assayed in 50 mM Hepes (pH 7.5)/10 mM MgCl2/5 mM DTT/0.05% Tween 20/2 μM peptide substrate/3.3 μCi/ml [γ-33P]ATP at 2 μM by using Image FlashPlates. Aurora B kinase (2 nM) was assayed with 10 μM biotinylated peptide Biotin-TKQTARKSTGGKAPR in 50 mM Tricine (pH 8.0)/2.5 mM MgCl2/5 mM DTT/10% glycerol/2% BSA/40 μCi/ml [γ-33P]ATP at 250 μM. The conditions for all other in vitro kinase assays are available upon request. MLN8054 was run in a 226 kinase screen at a 1 μM compound concentration with an ATP concentration of 10 μM for all assays [2]. |
| 動(dòng)物實(shí)驗(yàn) | Mice were irradiated (200 cGy), and then 5 × 106 MM1.S cells were inoculated subcutaneously in the right flank. When tumor growth was measurable (～ 2 weeks after the injection), mice were assigned into 4 groups (10 mice each) receiving vehicle orally (100 μL of 10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate) or MLN8237 (7.5 mg/kg, 15 mg/kg, and 30 mg/kg in a final formulation in 10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate) for 21 consecutive days. The maximal tolerated dose of MLN8237 in most mouse strains (continuous dosing for 21 days) is approximately 20 mg/kg twice a day (40 mg/kg per day). Tumor volumes were measured by a Vernier caliper every alternate day and calculated using the following formula: length × width2 × 0.5. Tumor growth inhibition (TGI) was calculated using the formula (Δcontrol average volume ? Δtreated average volume) × 100/(Δcontrol average volume). Mice were killed at the end of the treatment, 2 hours after the last treatment, or when tumor reached 2 cm^3; tumors were immediately collected from mice and evaluated for induction of apoptosis and cell death by TdT-mediated dUTP nick end labeling (TUNEL) assay [1]. |
| 體外活性 | 方法：從 MM 患者獲得的一組 MM 細(xì)胞系和腫瘤細(xì)胞用 Alisertib (0.0001-4 μM) 處理 48-72 h，使用 MTT assay 檢測(cè)細(xì)胞活力。
結(jié)果：盡管 Alisertib 的細(xì)胞毒性活性早在暴露于低于 0.1 μM/L 的 24-48 h 就在幾種細(xì)胞系中檢測(cè)到了，但在所有細(xì)胞系中，在 72 h 時(shí)都發(fā)生了更強(qiáng)烈的細(xì)胞毒性。[1]
方法：人結(jié)直腸癌細(xì)胞 HCT-116 用 Alisertib (0.05-1 μmol/L) 處理 24-48 h，使用 Flow cytometry 檢測(cè)細(xì)胞周期。
結(jié)果：在濃度為 0.050 μmol/L 時(shí)，在 24 和 48 h 時(shí)，G2/M 期的細(xì)胞增加，表型與 Aurora A 抑制一致。在 0.250 和 1.000 μmol/L 的較高濃度下，Alisertib 處理的細(xì)胞顯示出與 Aurora B 抑制一致的表型，顯示 8N DNA 含量的細(xì)胞數(shù)量增加。[2] |
| 體內(nèi)活性 | 方法：為研究抗腫瘤活性，將 Alisertib (7.5-30 mg/kg，10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate) 口服給藥給攜帶 MM1.S 異種移植物的 SCID 小鼠，每天一次，持續(xù)二十一天。
結(jié)果：用 30 mg/kg Alisertib 治療的動(dòng)物的腫瘤負(fù)荷顯著降低。與對(duì)照相比，15 mg/kg (TGI=42%) 和 30 mg/kg (TGI=80%) 處理的動(dòng)物的 TGI 顯著。[1] |
| 存儲(chǔ)條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | 10% DMSO+40% PEG300+5% Tween 80+45% Saline : 5 mg/mL (9.64 mM), Suspension. Please add co-solvents sequentially, clarifying the solution as much as possible before adding the next one. Dissolve by heating and/or sonication if necessary. Working solution is recommended to be prepared and used immediately.
DMSO : 50 mg/mL (96.35 mM)
H2O : < 1 mg/mL (insoluble or slightly soluble)
Ethanol : < 1 mg/mL (insoluble or slightly soluble)
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| 關(guān)鍵字 | arrest | myeloma | MLN8237 | cytotoxicity | spindle | inhibit | mitotic | Autophagy | Aurora Kinase | cancer | multiple | Inhibitor | MLN-8237 | cell-cycle | Apoptosis | Alisertib |
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