| 名稱 | Sorafenib |
| 描述 | Sorafenib (Bay 43-9006) is a multikinase inhibitor that inhibits Raf-1, B-Raf, VEGFR2, VEGFR3, VEGFR4, PDGFRβ, FLT3, c-Kit, and others (IC50=6/22/90/15/20/20/57/58 nM) with oral activity. Sorafenib has antitumor activity and can induce autophagy and apoptosis as well as agonistic ferroptosis. |
| 細(xì)胞實(shí)驗(yàn) | Tumor cell lines were plated at 2 × 105 cells per well in 12-well tissue culture plates in DMEM growth media (10% heat-inactivated FCS) overnight. Cells were washed once with serum-free media and incubated in DMEM supplemented with 0.1% fatty acid-free BSA containing various concentrations of BAY 43-9006 in 0.1% DMSO for 120 minutes to measure changes in basal pMEK 1/2, pERK 1/2, or pPKB. Cells were washed with cold PBS (PBS containing 0.1 mmol/L vanadate) and lysed in a 1% (v/v) Triton X-100 solution containing protease inhibitors. Lysates were clarified by centrifugation, subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked in TBS-BSA, and probed with anti-pMEK 1/2 (Ser217/Ser221; 1:1000), anti-MEK 1/2, anti-pERK 1/2 (Thr202/Tyr204; 1:1000), anti-ERK 1/2, anti-pPKB (Ser473; 1:1000), or anti-PKB primary antibodies. Blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and developed with Amersham ECL reagent on Amersham Hyperfilm [1]. |
| 激酶實(shí)驗(yàn) | Recombinant baculoviruses expressing Raf-1 (residues 305–648) and B-Raf (residues 409–765) are purified as fusion proteins. Full-length human MEK-1 is generated by PCR and purified as a fusion protein from Escherichia coli lysates. Sorafenib tosylate is added to a mixture of Raf-1 (80 ng), or B-Raf (80 ng) with MEK-1 (1 μg) in assay buffer [20 mM Tris (pH 8.2), 100 mM NaCl, 5 mM MgCl2, and 0.15% β-mercaptoethanol] at a final concentration of 1% DMSO. The Raf kinase assay (final volume of 50 μL) is initiated by adding 25 μL of 10 μM γ[33P]ATP (400 Ci/mol) and incubated at 32 °C for 25 minutes. Phosphorylated MEK-1 is harvested by filtration onto a phosphocellulose mat, and 1% phosphoric acid is used to wash away unbound radioactivity. After drying by microwave heating, a β-plate counter is used to quantify filter-bound radioactivity. Human VEGFR2 (KDR) kinase domain is expressed and purified from Sf9 lysates. Time-resolved fluorescence energy transfer assays for VEGFR2 are performed in 96-well opaque plates in the time-resolved fluorescence energy transfer format. Final reaction conditions are as follows: 1 to 10 μM ATP, 25 nM poly GT-biotin, 2 nM Europium-labeled phospho (p)-Tyr antibody (PY20), 10 nM APC, 1 to 7 nM cytoplasmic kinase domain in final concentrations of 1% DMSO, 50 mM HEPES (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 0.015% Brij-35, 0.1 mg/mL BSA, and 0.1% β-mercaptoethanol. Reaction volumes are 100 μL and are initiated by the addition of enzyme. Plates are read at both 615 and 665 nM on a Perkin-Elmer VictorV Multilabel counter at ~1.5 to 2.0 hours after reaction initiation. Signal is calculated as a ratio: (665 nm/615 nM) × 10,000 for each well. For IC50 generation, Sorafenib tosylate is added before the enzyme initiation. A 50-fold stock plate is made with Sorafenib tosylate serially diluted 1:3 in a 50% DMSO/50% distilled water solution. Final Sorafenib tosylate concentrations range from 10 μM to 4.56 nM in 1% DMSO. |
| 動(dòng)物實(shí)驗(yàn) | Female NCr-nu/nu mice (Taconic Farms, Germantown, NY) were used for all studies. Three to five million cells were injected s.c. into the right flank of each mouse. DLD-1 tumors were established and maintained as a serial in vivo passage of s.c. fragments (3 × 3 mm) implanted in the flank using a 12-gauge trocar. A new generation of the passage was initiated every three weeks, and studies were conducted between generations 3 and 12 of this line. Treatment was initiated when tumors in all mice in each experiment ranged in size from 75 to 144 mg for antitumor efficacy studies and from 100 to 250 mg for studies of microvessel density and ERK phosphorylation. All treatment was administered orally once daily for the duration indicated in each experiment. |
| 體外活性 | 方法：人肝癌細(xì)胞 HepG2 和 HuH-7 用 Sorafenib (2-20 μmol/L) 處理 48 h，使用 MTT 方法檢測細(xì)胞生長抑制情況。
結(jié)果：Sorafenib 劑量依賴性地抑制 HepG2 和 HuH-7 細(xì)胞生長，IC50 均約為 6 μmol/L。[1]
方法：人急性早幼粒白血病細(xì)胞 NB4 用 Sorafenib (1.5-12 μM) 處理 24-48 h，使用 Flow Cytometry 方法檢測細(xì)胞凋亡情況。
結(jié)果：Sorafenib 劑量依賴性 NB4 細(xì)胞凋亡，早期和晚期凋亡細(xì)胞比例均顯著增加。[2]
方法：大鼠肝膽管癌細(xì)胞 LCC-2 用 Sorafenib (2.5-5 μM) 處理 12 h，使用 JC-1 染料檢測線粒體膜電位。
結(jié)果：Sorafenib 使分離的線粒體去極化。[3] |
| 體內(nèi)活性 | 方法：為檢測體內(nèi)抗腫瘤活性，將 Sorafenib (7.5-60 mg/kg) 口服給藥給攜帶人腫瘤 MDA-MB-231、Colo-205、HT-29、DLD-1、NCI-H460 和 A549 的 NCr-nu/nu 小鼠，每天一次，持續(xù)二至四天。
結(jié)果：Sorafenib 在各種人類腫瘤異種移植物模型中顯示出廣泛的口服抗腫瘤功效。[4]
方法：為檢測體內(nèi)抗腫瘤活性，將 Sorafenib (30 mg/kg/每周五次) 和 everolimus (10 mg/kg/每周三次) 灌胃給藥給攜帶去勢抵抗性前列腺癌腫瘤 CRPC 的 PTEN 突變小鼠，每天一次，持續(xù)四周。
結(jié)果：Sorafenib 給藥增加了 CRPC 中雄激素受體 p-GSK3β 和 p-ERK1/2 的表達(dá)水平。Sorafenib 和 everolimus 聯(lián)合治療克服了 CRPC 單藥的治療逃逸。[5] |
| 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 | H2O : < 1 mg/mL (insoluble or slightly soluble)
10% DMSO+40% PEG300+5% Tween 80+45% Saline : 5.9 mg/mL (12.69 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.
DMF : 3.33 mg/mL (7.17 mM）
DMSO : 55 mg/mL (118.33 mM)
Ethanol : < 1 mg/mL (insoluble or slightly soluble)
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| 關(guān)鍵字 | inhibit | Inhibitor | CD135 | Vascular endothelial growth factor receptor | Ferroptosis | Fms like tyrosine kinase 3 | Raf | Sorafenib | Raf kinases | Apoptosis | Autophagy | Cluster of differentiation antigen 135 | VEGFR | FLT3 |
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