Though axitinib was successful as an oral agent in earlier phases of clinical development, its interaction with ABC drug transporters hasn’t been characterized. The goal of this operate was to investigate the interaction of axitinib with ABCB1, ABCC1, ABCC4, ABCG2 and lung resistance?associated protein . We display right here that axitinib targets CSCs, increases the efficacy of chemotherapeutic agents and reverses ABCG2-mediated drug resistance by inhibiting the drug efflux perform of ABCG2 and rising the intracellular accumulation of cytotoxic agents in ABCG2-overexpressing cells and SP cells. 3- -2,5- diphenyllapatinibrazolium bromide , vincristine, topotecan, mitoxantrone, Dox, rhodamine 123, verapamil, MK571 and FTC were products of Sigma Chemical Co . Axitinib was purchased from Selleck Chemicals . Dulbecco?s modified Eagle?s medium and RPMI 1640 were solutions of Gibco BRL . Anti-mitogen-activated protein kinase 1/2 , P-ERK , P-AKT and glyceraldehyde-3- phosphate dehydrogenase antibodies were purchased from Kangchen Co.
. AKT antibody was a merchandise of Cell Signaling Technology Inc. . ABCG2 antibody was obtained from Chemicon International, Inc. . Other selleck chemical straight from the source schedule laboratory reagents were of analytical grade and obtained from commercial sources. Cell Culture The following cell lines had been cultured in DMEM or RPMI 1640 containing 10% fetal bovine serum at 37?C in the presence of 5% CO2: the human colon carcinoma cell line S1 and its mitoxantrone- picked derivative ABCG2- overexpressing S1-M1-80 cell line had been kindly supplied by SE Bates ; the human oral epidermoid carcinoma cell line KB and its vincristine-selected derivative ABCB1-overexpressing cell line KBv200 were a gift from Xu-Yi Liu ; the human leukemia cell line HL60 and its Dox-selected derivative ABCC1-overexpressing cell line HL60/ADR were purchased in the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China; the human lung squamous carcinoma cell line SW1573 and its Dox-selected derivative LRP-overexpressing SW1573/2R120 had been provided by EAC Wiemer ; the murine fibroblasts cell line NIH3T3 and the ABCC4-transfected ABCC4 stable expressing NIH3T3/ MRP4-2 cells were kindly presented by Z-S Chen .
The human lung cancer A549 cell line was obtained from ATCC . HEK293/pcDNA3.one, ABCG2-482-G2 and ABCG2-482-T7 cells have been established by selection with G418 after transfecting HEK293 with either an empty pcDNA3.one vector or a pcDNA3.1 vector containing Ubiquinone full-length ABCG2 coding either glycine or threonine at the amino acid 482 position, respectively .
These cells have been obtained from SE Bates and had been cultured in medium with two mg/mL G418. Cell Cytotoxicity Test The MTT assay was used to assess the sensitivity of cells to drugs as previously described . Briefly, cells were distributed evenly into 96-well microtiter plates and then various concentrations of axitinib had been added to the wells.