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.

PDGFRβ:57 nM (cell free), c-Kit:68 nM (cell free), B-Raf:22 nM (cell free), VEGFR3:20 nM (cell free), B-Raf V599E:38 nM (cell free), Raf-1:6 nM (cell free0

方法：人肝癌细胞 HepG2 和 HuH-7 用 Sorafenib (2-20 µmol/L) 处理 48 h，使用 MTT 方法检测细胞生长抑制情况。
结果：Sorafenib 剂量依赖性地抑制 HepG2 和 HuH-7 细胞生长，IC50 均约为 6 µmol/L。[1]
方法：人急性早幼粒白血病细胞 NB4 用 Sorafenib (1.5-12 µM) 处理 24-48 h，使用 Flow Cytometry 方法检测细胞凋亡情况。
结果：Sorafenib 剂量依赖性 NB4 细胞凋亡，早期和晚期凋亡细胞比例均显著增加。[2]
方法：大鼠肝胆管癌细胞 LCC-2 用 Sorafenib (2.5-5 μM) 处理 12 h，使用 JC-1 染料检测线粒体膜电位。
结果：Sorafenib 使分离的线粒体去极化。[3]

方法：为检测体内抗肿瘤活性，将 Sorafenib (7.5-60 mg/kg) 口服给药给携带人肿瘤 MDA-MB-231、Colo-205、HT-29、DLD-1、NCI-H460 和 A549 的 NCr-nu/nu 小鼠，每天一次，持续二至四天。
结果：Sorafenib 在各种人类肿瘤异种移植物模型中显示出广泛的口服抗肿瘤功效。[4]
方法：为检测体内抗肿瘤活性，将 Sorafenib (30 mg/kg/每周五次) 和 everolimus (10 mg/kg/每周三次) 灌胃给药给携带去势抵抗性前列腺癌肿瘤 CRPC 的 PTEN 突变小鼠，每天一次，持续四周。
结果：Sorafenib 给药增加了 CRPC 中雄激素受体 p-GSK3β 和 p-ERK1/2 的表达水平。Sorafenib 和 everolimus 联合治疗克服了 CRPC 单药的治疗逃逸。[5]

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.

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].

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.