Western blot analysis of extracts from HUVE cells, serum-starved overnight and untreated (-) or pretreated with Axitinib (1 nM, 2 hr; +) prior to treatment with Human Vascular Endothelial Growth Factor-165 (hVEGF165) #8065 (50 ng/ml, 5 min; +), using Phospho-VEGF Receptor 2 (Tyr1175) (19A10) Rabbit mAb #2478 (upper) or VEGF Receptor 2 (55B11) Rabbit mAb #2479 (lower).
Western blot analysis of extracts from NIH/3T3 cells, serum-starved overnight and untreated (-) or pretreated with Axitinib (100 nM, 2 hr; +) prior to treatment with Human Platelet-Derived Growth Factor BB (hPDGF-BB) #8912 (100 ng/ml, 5 min; +), using Phospho-PDGF Receptor β (Tyr1009) (42F9) Rabbit mAb #3124 (upper) or PDGF Receptor β (28E1) Rabbit mAb #3169 (lower).
Western blot analysis of extracts from NCI-H526 cells, serum-starved overnight and untreated (-) or pretreated with Axitinib (1000 nM, 2 hr; +) prior to treatment with Human Stem Cell Factor (hSCF) #8925 (100 ng/ml, 5 min; +), using Phospho-c-Kit (Tyr703) (D12E12) Rabbit mAb #3073 (upper) or c-Kit (D13A2) XP® Rabbit mAb #3074 (lower).
Chemical structure of axitinib.
Axitinib is supplied as a lyophilized powder. For a 10 mM stock, reconstitute the 5 mg in 1.29 ml DMSO. Working concentrations and length of treatment can vary depending on the desired effect, but it is typically used as a pretreatment at 1-1000 nM for 0.5-2 hr prior to treating with a stimulator. It can also be used alone, with varying treatment times lasting up to 72 hr.
Store lyophilized or in solution at -20ºC, desiccated. Protect from light. In lyophilized form, the chemical is stable for 24 months. Once in solution, use within 3 months to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.
Axitinib is a selective inhibitor of VEGFR, PDGFR, and c-kit tyrosine kinases. Researchers performing cellular phosphorylation assays have shown that axitinib very potently inhibits VEGF-1, -2, and -3 with IC50 values of approximately 1.2 nM, 0.2 nM, and 0.1-0.3 nM, respectively. It also effectively inhibits PDGF-α (IC50 = 5.0 nM), PDGF-β (IC50 = 1.6 nM), and c-kit (IC50 = 1.7 nM) (1). Axitinib exhibits little activity against a variety of off-target protein kinases when used at 1 µM (1). It inhibits VEGF-induced endothelial cell proliferation, survival, and tube formation, as well as phosphorylation of downstream targets Akt, eNOS, and ERK1/2 in a dose-dependent manner (1). Research studies demonstrate that axitinib suppresses T cell proliferation in a dose dependent manner through G2/M mitotic arrest, while apoptosis is largely prevented though stabilization of Mcl-1 and inactivation of caspase-9 (2). Axitinib has been shown to reduce both hypoxic-induced tissue permeability and overexpression/secretion of VEGF and PDGF in HUVE and RPE cells, as well as alter junction protein expression (3).
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