Axitinib (AG-013736) is a multi-targeted tyrosine kinase inhibitor that inhibits VEGFR1, VEGFR2, VEGFR3, and PDGFRβ (IC50=4/20/0.4/2 nM). Axitinib has antitumor activity and is used in the treatment of renal cell carcinoma.

c-Kit:1.7 nM, PDGFRβ:1.6 nM, VEGFR3: 0.1-0.3 nM, VEGFR2/KDR: 0.2 nM, VEGFR2/Flk1:0.18 nM, VEGFR1/FLT1:0.1 nM

方法：胶质瘤细胞 U87、T98 和 U251 用 Axitinib (0.1-100 µM) 处理 72 h，通过 MTT assay 测定细胞活力。
结果：治疗 72 h 后，Axitinib 抑制 U87 和 T98 细胞的生长，IC50 值分别为 12.7 µM 和 8.5 µM。相反，U251 细胞对 Axitinib 介导的细胞毒性作用更具抵抗力。[1]
方法：人上皮卵巢癌细胞 A2780、RMG1、HeyA8 和 HeyA8-MDR 用 Axitinib (1-4 µM) 处理 4 h，通过 Western Blot 检测靶点蛋白表达水平。
结果：不同剂量的 Axitinib 治疗以剂量依赖的方式显著降低了 A2780、RMG1 和 HeyA 8中磷酸化 EGFR2 的表达，但在 HeyA8 MDR 细胞中没有。[2]

方法：为检测体内抗肿瘤活性，将 Axitinib (30 mg/kg，0.5% methyl cellulose) 口服给药给携带 A2780、RMG1 或 HeyA8 MDR 肿瘤的 BALB/c nude 小鼠，每天两次，持续 35-40 天。
结果：在 A2780 和 RMG1 模型中，Axitinib 治疗组的肿瘤重量与对照组相比显著降低了 50%，但在 HeyA8 MDR 模型中差异不显著。[2]

Porcine aorta endothelial (PAE) cells overexpressing full-length VEGFR-2, PDGFR-β, KIT, and NIH-3T3 overexpressing murine VEGFR-2 (Flk-1) or PDGFR-α were generated as described previously. The ELISA capture plates were prepared by coating 96-well ReactiBind plates with 100 μL/well of 2.5 μg/mL anti-VEGFR-2 antibody, 0.75 μg/mL anti-PDGFR-β antibody, 0.25 μg/mL anti-PDGFR-α antibody, 0.5 μg/mL anti-KIT antibody, or 1.20 μg/mL anti-Flk-1 antibody. Measurement of RTK phosphorylation by ELISA was done as described previously [1].

Endothelial or tumor cells were starved for 18 h in the presence of either 1% FBS (HUVEC) or 0.1% FBS (tumor cells). Axitinib was added and cells were incubated for 45 min at 37°C in the presence of 1 mmol/L Na3VO4. The appropriate growth factor was added to the cells, and after 5 min, cells were rinsed with cold PBS and lysed in the lysis buffer and a protease inhibitor cocktail. The lysates were incubated with immunoprecipitation antibodies for the intended proteins overnight at 4°C. Antibody complexes were conjugated to protein A beads and supernatants were separated by SDS-PAGE. The Super Signal West Dura kit was used to detect the chemiluminescent signal [1].

AG-013736, a receptor kinase inhibitor of VEGFRs and, at higher doses, PDGFRs (IC50 = 0.1 nmol/L for VEGFR-1, 0.2 nmol/L for VEGFR-2, 0.1–0.3 nmol/L for VEGFR-3, and 1.6 nmol/L for PDGFRβ; ref. 18), was provided by Pfizer Global Research and given once daily by gavage in a volume of 0.13 mL. Control animals received 0.5% carboxymethylcellulose drug carrier. Irradiations were done on nonanesthetized mice using a 137Cs source operating at 2.4 Gy/min. Mice were confined to plastic jigs with tumor-bearing legs extended through an opening in the side, allowing local irradiations. Fractionated doses were given in five daily 2 Gy fractions per week (omitting weekends). For combination treatments, radiotherapy was delivered first, and AG-013736 was given within ～4 h. Mice were sacrificed, and tumors were excised and then quick frozen (using liquid nitrogen) following 1, 2, or 3 weeks of treatment [3].