This ability to reverse the P-gp-mediated resistance is comparable to that of another frequently used reversal agent known as verapamil. stimulates ATPase activity in a dose-dependent manner, which is required for the proper function of P-gp. In contrast, ZD6474 does not inhibit the expression level of P-gp. Our results suggest that ZD6474 is usually capable of reversing MDR in cancer cells by directly inhibiting the function of P-gp, a finding that may have clinical implications for ZD6474. EGFR and RET inhibition, as well as tumour angiogenesis VEGFR inhibition (Ryan and Wedge, 2005). ZD6474 is currently in phase III clinical trials for the treatment of follicular, medullary, anaplastic, and locally advanced and metastatic papillary thyroid cancer, as well as for other cancers including non-small cell lung cancer. Multidrug resistance is one of the major causes of failure in cancer chemotherapy, and numerous efforts have been made to overcome MDR. The most employed strategy has been to develop MDR inhibitors including the calcium channel blocker, verapamil (VPL), and the immunosuppressant, cyclosporine A, which reverse MDR by functioning as competitive substrates of P-gp. However, their clinical benefits are limited due to high toxicity at resistance-inhibiting doses. Another important MDR inhibitor, PSC-833, has not been successful in clinical trials either. Recent studies have shown that this tyrosine kinase inhibitors, STI-571 and AG1393, interact TPA 023 with Rabbit polyclonal to AKT1 the human P-gp and human multidrug resistance protein 1 (ABCC1) (Hegedus ZD6474 was synthesised at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China. Docetaxel, ADR, and VNR were obtained from Jiangsu Hengrui Pharmaceutical Co. (Lianyungang, China). Sulforhodamine B, rhodamine-123 (Rho-123), and VPL were purchased from Sigma Chemical Co. (St Louis, MO, USA). The antibody to P-gp was purchased from Alexis Biotechnology Inc. (San Diego, CA, USA). The human breast cancer cells, MCF-7, and their MDR cell subline MCF-7/ADR were maintained in Dulbecco’s modified Eagle medium made up of 10% heat-inactivated fetal bovine serum (FBS), 100?kU?l?1 penicillin, 200?kU?l?1 streptomycin, and 0.01?mg?ml?1 bovine insulin at 37C (5% CO2). The human oral epidermoid carcinoma cell line, KB, and its MDR cell subline, KBV200, were maintained in RPMI 1640 medium made up of 10% heat-inactivatedFBS, 100?kU?l?1 penicillin, and 200?kU?l?1 streptomycin. Cytotoxicity assessments were performed using a sulforhodamine B assay (Schneider The assay was performed according to a previously described method (Ludescher After drug treatment, cells were washed twice with ice-cold phosphate-buffered saline (137?mM NaCl, 2.7?mM KCl, 10?mM Na2HPO4, 1.8?mM KH2PO4, pH 7.4) and total cell lysates were collected in sodium dodecyl sulfate (SDS) sample buffer (50?mM Tris-HCl, pH 6.8, 100?mM dithiothreitol (DTT), 2% SDS, 0.1% bromophenol blue, 10% glycerol). Cell lysates, made up of equal amounts of protein, were separated by SDSCpolyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidine difluoride membranes. After being blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (pH 7.6), membranes were incubated with the TPA 023 appropriate primary antibodies at 4C, overnight, and exposed to the appropriate secondary antibody for 3?h at room temperature. Immunoreactive proteins were visualised using the enhanced chemiluminescence system from Pierce (Rockford, IL, USA). MCF-7 and MCF-7/ADR cells were harvested, and their membranes were isolated and stored at ?80C as described previously (Orlowski P-gp inhibition. Open in a separate window Physique 1 ZD6474 reverses P-gp-mediated resistance. P-gp-negative MCF-7 cells or P-gp-positive MCF-7/adriamycin (ADR) and KBV200 cells were treated with (ADR, docetaxel, or vinorelbine (VNR) in the presence TPA 023 or absence of ZD6474 for different periods of time, and the cell proliferation was determined by the SRB assay. Each point represents the TPA 023 means.d. for three determinations. ZD6474 may not be a substrate of P-gp ZD6474 itself has an equally moderate inhibitory effect on the proliferation of both TPA 023 MCF-7 and MCF-7/ADR cells. As shown in Physique 2A, ZD6474 inhibited the proliferation of MCF-7 and MCF-7/ADR cells in a dose-dependent manner, with IC50s of 17.3 and 19.3?control by Student’s and in the clinic, ZD6474 has not been reported to develop cross-resistance to prior chemotherapeutic brokers, either or (Morelli (2003) treated cells in soft agar for 10C14 days. Finally, the different cells used in these studies may also result in different effects of ZD6474 around the cytotoxicity of chemotherapeutics. These data suggest that the treatment schedule must be considered when ZD6474 is used in combination with other cytotoxic.