Active efflux of CPT-11 and its metabolites in human KB-derived cell lines

To investigate the possible involvement of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and/or other glutathione S-conjugate export pump (GS-X pump) family members on the active efflux of irinotecan [ (7-ethyl-10-[4-(1-piperidino)-1-pipertidino)-1-piperidino]carbonyloxy campt othecin (CPT-11)] and its metabolites, as well as their contribution to the acquisition of resistance, we studied the uptake of CPT-11, its active met abolite SN-38, and glucuronide conjugate (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1-derived cell lines. These lines included KB-C 2, C-A500, and KCP-4, which overexpress P-gp, MRP, and the unidentified GS- X pump, respectively. The carboxylate form of SN-38 exhibited significant A TP-dependent transport, with a Michaelis constant of 17 mu M, into membrane vesicles from C-A500 but not from other cell lines. Among these KB-derived cells, significant ATP-dependent uptake of the carboxylate form of CPT-11 was only observed in KB-C2 vesicles. In addition, the uptake of the lactone and carboxylate forms of SN38-Glu into membrane vesicles from C-A500 and K B-C2, but not KCP-4, was ATP dependent, although the transport activity in C-A500 was much higher than that in KB-C2. The 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay revealed that the resistance o f KB-C2 to CPT-11 and SN-38, compared with that of KB-3-1, was 6.3- and 6.8 -fold, respectively; the corresponding figures for C-A500 were 12- and 27-f old, respectively, whereas those for KCP-4 were 2.3- and 20-fold, respectiv ely. These results suggest that MRP and P-gp are involved in the active eff lux of SN-38 and CPT-11, respectively, from human KB-derived cells. In addi tion, a difference in substrate specificity among GS-X pump members was dem onstrated.