Anti-ABCC5 (aa 2-13) polyclonal antibody

Tumor cells may become resistant to conventional anticancer drugs through the occurrence of transmembrane transporter proteins such as P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or members of the multidrug resistance-associated protein family (MRP1-MRP5; ABCC1-ABCC5). In this report, we studied whether tumor cells that are cytostatic drug resistant because of overexpression of one of the above mentioned proteins are sensitive to a new anticancer agent, interieukin-4 toxin (IL-4 toxin). IL-4 toxin is a fusion protein composed of circularly permuted IL-4 and a truncated form of Pseadomonas exotoxin (PE) [IL-4(38-37)-PE38KDEL]. Ninety-six-h cytotoxicity assays and 10-day clonogenic assays showed that drug-selected multidrug resistant (MDR) tumor cells that overexpress P-glycoprotein or breast cancer resistance proteins are still sensitive to IL-4 toxin. Also, tumor cells transfected with cDNA for MRP2-5 showed no resistance, or marginal resistance, only to the toxin as compared with the parent cells. In contrast, MRP1-overexpressing cells, both drug selected and MRP1 transfected, are clearly resistant to IL-4 toxin with resistance factors of 4.3 to 8.4. MRP1-overexpressing cells were not resistant to PE itself. IL-4 toxin resistance in MRP1-overexpressing cells could be reversed by the MRP1 inhibitors probenecid or MK571 and were not affected by glutathione depletion by DL-buthionine-S,R-sulfoximine. In a transport assay using plasma membrane vesicles prepared from MRP1-overexpressing cells, IL-4 toxin and IL-4, but not PE, inhibited the translocation of the known MRP1 substrate 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). These data suggest that MRP1-overexpressing cells are resistant to IL-4 toxin because of extrusion of this agent by MRP1. Still, the results of this study demonstrate that IL-4 toxin effectively kills most MDR tumor cells and, therefore, represents a promising anticancer drug.

The objective of this paper is to study the effects of poly(ethylene glycol) -block-polylactide (PLA-PEG) nanoparticles on hepatic cells of mouse. Blank PLA-PEG nanoparticles have been successfully prepared and MTT assay suggested that the nanoparticles with HepG2 cell co-culture model did not cause significant changes in membrane integrity in controlled concentration range (0.001-0.1 mg/ml). Immunohistochemical analysis demonstrated that large dose of PLA-PEG nanoparticles injection (42.04 mg/kg, i.v.) did not induce hepatic cell apoptosis. From biochemical assay experiments, although the levels of SOD decreased and those of MDA, NOS increased after treatment with large dose of PLA-PEG nanoparticles injection (42.04 mg/kg, i.v.), they were all not significant (p > 0.05). Then Kunming mice were treated with large dose of PLA-PEG nanoparticles (42.04 mg/kg, i.v.) and after 4 days total RNA was isolated to elucidate patterns of gene expression using a mouse cDNA-microarray (SuperArray). Treatment with nanoparticles resulted in over-exoression of a lot of ATP-binding cassette (ABC) transporters, especially two ABC transporters (ABCA8 and ABCC5/MRP5), and down-regulation of GSTP1, in comparison with the control. ABCA8 could extrude low molecular weight polymers after PLA-PEG nanoparticles hydrolysis outside the cells. We also discovered that ABCC5 expressed multidrug resistance protein 5 (MRP5) to pump out conjugate (GS-X) of PLA-PEG nanoparticles with GSH. The results were confirmed by RT-PCR. Results of in vitro accumulation and efflux experiments indicated that about 51-52% (51.5% and 52.0%) intracellular PLA-PEG nanoparticles was expulsed after mouse primary hepatocytes reached a saturation uptake of nanoparticles during the concentration range of 750-1000 mu g/ml. The results suggested that ABC transporters (especially ABCA8) pump out the polymers after hydrolysis from mouse hepatic cells and large dose of PLA-PEG nanoparticles make mouse hepatic cells gain drug resistance to PLA-PEG nanoparticles. (C) 2006 Elsevier B.V. All rights reserved.