Human Multidrug Resistance Associated Protein 4 Confers Resistance to Camptothecins

Purpose: The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included. Methods: HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods. Results: Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) \u3e SN-38 carboxylate (9.70) \u3e rubitecan (9.06) \u3e SN-38 lactone (8.91) \u3e CPT lactone (7.33) \u3e CPT-11 lactone (5.64) \u3e CPT carboxylate (4.30) \u3e CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a γ-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine. Conclusions: Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide