Acquisition of drug resistance is a critical issue in chemotherapy of leukemia, and transporters of anti-cancer drugs play important roles in the drug resistance. Comparing expression profiles of transporters at protein level between sensitive and resistance tumor cells could be a rational strategy to identify the transporters responsible for drug resistance. Recently we have developed a method to quantify multiple transporter proteins simultaneously by multi-channel MRM analysis using LC-MS/MS (1). The purpose of this study was to identify transporters involved in vincristine resistance in leukemia cells by comparison of quantitative protein expression profiles of transporters.
Crude membrane fraction was isolated from human myeloid leukemia cell line, K562 and K562/VCR (71-fold resistant to vincristine). Proteins were digested by trypsin and subjected to LC-MS/MS for simultaneous quantification of 34 ABC transporters, 5 SLC transporters and 2 membrane markers. Corresponding stable isotope labeled peptides were used as internal standard.
MDR1 protein, which mediates efflux transport of vincristine, was detected in K562/VCR cells (5.3 pmol/mg protein), but not in K562 cells (determination limit was 0.13 pmol/mg protein), indicating its induction by over 40-fold in K562/VCR cells. Protein expression of MRP1 was not significantly affected in K562/VCR cells, and MRP2, 3, and 7 were not detected in either cells, although these transporters have been reported to transport vincristine. MRP4 was decreased and ENT1 was increased significantly in K562/VCR cells, and these changes could not explain the drug resistance. The present result suggests that MDR1 is the most significant factor to acquire vincristine resistance in K562/VCR cells. A large scale quantitative profiling of transporter proteins using our technique could be a rational strategy to identify transporters responsible for a drug resistance.
(1) Kamiie et al, Pharm Res, E-pub ahead of print (2008)
