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Active efflux of CPT-11 and its metabolites in human KB-derived cell lines
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.
Abstract:
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]carb onylox y camptothecin (CPT-11)] and its metabolites, as well as their contribution to the acquisition of resistance, we studied the uptake of CPT-11, its active metabolite SN-38, and glucuronide conjugate (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1-derived cell lines. These lines included KB-C2, 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 ATP-dependent transport, with a Michaelis constant of 17 microM, 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 KB-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 of 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-fold, respectively, whereas those for KCP-4 were 2.3- and 20-fold, respectively. These results suggest that MRP and P-gp are involved in the active efflux of SN-38 and CPT-11, respectively, from human KB-derived cells. In addition, a difference in substrate specificity among GS-X pump members was demonstrated.
Insights
Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) mediate the efflux of irinotecan (CPT-11) and its metabolite SN-38. This study reveals distinct substrate specificities among glutathione S-conjugate export pump (GS-X pump) family members, impacting drug resistance.
Area of Science:
- Pharmacology
- Molecular Biology
- Cancer Research
Background:
- Chemotherapeutic agents like irinotecan (CPT-11) are subject to efflux by transporter proteins, contributing to drug resistance.
- P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and glutathione S-conjugate export pump (GS-X pump) families are implicated in drug efflux.
- Understanding the specific roles of these pumps in CPT-11 and its metabolite SN-38 transport is crucial for overcoming resistance.
Purpose of the Study:
- To investigate the involvement of P-gp, MRP, and GS-X pump family members in the active efflux of CPT-11 and its metabolites.
- To determine the contribution of these transporters to the acquisition of resistance against CPT-11 and SN-38.
- To elucidate the substrate specificity of different GS-X pump family members.
Main Methods:
- Uptake studies of CPT-11, SN-38, and SN38-glucuronide (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1 derived cell lines overexpressing P-gp (KB-C2), MRP (C-A500), and an unidentified GS-X pump (KCP-4).
- Assessed ATP-dependent transport kinetics (Michaelis constant) for SN-38 and CPT-11.
- Evaluated drug resistance using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Main Results:
- SN-38 carboxylate form showed significant ATP-dependent transport into C-A500 (MRP) vesicles (Km = 17 microM).
- CPT-11 carboxylate form exhibited ATP-dependent uptake primarily in KB-C2 (P-gp) vesicles.
- SN38-Glu uptake was ATP-dependent in C-A500 and KB-C2 vesicles, with higher activity in C-A500; KCP-4 showed minimal uptake.
- Resistance fold-changes compared to KB-3-1 were: KB-C2 (CPT-11: 6.3x, SN-38: 6.8x), C-A500 (CPT-11: 12x, SN-38: 27x), KCP-4 (CPT-11: 2.3x, SN-38: 20x).
Conclusions:
- MRP and P-gp are involved in the active efflux of SN-38 and CPT-11, respectively, from human KB-derived cells.
- The study demonstrates distinct substrate specificities among members of the GS-X pump family.
- These findings highlight the complex role of drug efflux transporters in mediating resistance to irinotecan-based chemotherapy.