Whole cells were harvested and washed twice with ice-cold PBS. Cell extracts were collected in cell lysis buffer . Equal amounts of cell lysate from various treatments were resolved by sodium dodecyl sulfate polycrylamide gel electrophoresis . After blocked in TBST with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with appropriately diluted primary antibodies overnight at 4?C. The membranes were then washed thrice with TBST and incubated with HRP-conjugated jak2 inhibitor kinase inhibitor secondary antibody at 1:5000 dilution for 2 h at room temperature. After washed thrice with TBST, the protein-antibody complex were visualized by the enhanced Phototope TM-HRP Detection Kit and exposed to Kodak medical X-ray processor . GAPDH was used as a loading control. 2.7 ABCB1 ATPase activity assay The changes of ATPase activity were estimated by Pgp- Glo? assay systems . The inhibitory effects of BIBF 1120 were examined against a verapamilstimulated ABCB1 ATPase activity. Sodium orthovanadate was used as an ABCB1 ATPase inhibitor. Various concentrations of BIBF 1120 diluted with assay buffer were incubated in 0.1 mM verapamil, 5 mM MgATP and 25 ?g recombinant human ABCB1 membranes at 37?C for 40 min.
Luminescence was initiated by ATP detection buffer. After incubated at room temperature for 20 min to allow luminescent signal to develop, the untreated white opaque 96-well plate was read on luminometer . The changes of relative light units were determined by comparing Na3VO4-treated samples with BIBF 1120 and verapamil combination-treated samples, and hence, the ATP consumed was obtained by comparing to a standard Rocuronium curve. 2.8 Statistics All experiments were repeated at least thrice and the differences were determined by using the Student?s t-test. The significance was determined at P<0.05. 3 Results 3.1 Determination of multidrug resistance ABCB1 is overexpressed in Hep G2/adr and MCF-7/ adr cells, while ABCC1 and ABCG2 were overexpressed in HL60/adr and S1-M1-80 cells, respectively . The basal expressions of ABCB1, ABCC1 and ABCG2 in the parental cell lines were nearly undetectable. MTT assay showed that four MDR cell lines exerted much higher tolerance to multiple anticancer drugs compared with their parental, drug-sensitive cell lines . 3.2 Modulation of multidrug resistance in MDR cell lines by BIBF 1120 The intrinsic in vitro toxicity of BIBF 1120 on different cells was determined using the MTT assay. The IC50 values were 15.28?0.73, 18.94?0.79, 25.16?0.65, 28.54?0.66, 7.64?0.73, 9.12?0.59, 4.35?0.34 and 6.54?0.56 ?M, for Hep G2, Hep G2/adr, MCF-7, MCF-7/adr cells, HL60, HL60/adr S1 and S1-M1-80 respectively .