Increased sensitivity to vincristine of MDR cells by the leukotriene D4 receptor antagonist, ONO-1078
Introduction
Resistance to multiple anticancer agents is a major obstacle for treatment of many types of tumors by chemotherapy. Multidrug resistance (MDR) in cultured cell lines is known to be conferred by two different membrane glycoproteins, the 170 kDa P-glycoprotein (P-gp) [9]and the 190 kDa multidrug resistance protein (MRP) [5], and the 110 kDa lung resistance protein (LRP) [17].
P-gp-mediated MDR is reversed by a variety of compounds, including calcium channel blockers and their analogs 1, 7, 16. Almost all agents that reverse P-gp-mediated MDR could not reverse multidrug resistance protein (MRP)-mediated MDR [3], but some agents, such as MK571, a leukotriene D4 (LTD4) receptor antagonist [8], genistein, a protein kinase inhibitor [21], PAK-104P, a pyridine analog [18], and buthionine sulfoximine, an inhibitor of glutathione synthesis [20], did reverse the MRP-mediated MDR. MK571 and PAK-104P completely reversed VCR resistance in MRP-mediated MDR cells 8, 18. MK571 inhibited the photolabeling of MRP by [3H]LTC4, a substrate for MRP, and the transport of [3H]LTC4 into membrane vesicles from MDR cells overexpressing MRP11, 14. PAK-104P competitively inhibited the ATP-dependent [3H]LTC4 transport in membrane vesicles isolated from C-A120 cells [18]. Both MK571 and PAK-104P with different structures appeared to interact with the transporting activity of MRP to inhibit drug efflux from the cells. It will contribute to the elucidation of the mechanism for the reversal to know whether other LTD4 receptor antagonists interact with MRP.
4-Oxo-8-[p-(4-phenylbutyloxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate (ONO-1078) is another LTD4 receptor antagonist that is structurally unrelated to MK571, though like MK571 it is negatively charged. ONO-1078 is used clinically for the treatment of allergic asthma and other immediate hypersensitivity diseases. We examined whether ONO-1078 reverses MRP-mediated MDR and whether it directly interacts with MRP.
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Cell culture and cell lines
Human epidermoid carcinoma KB cells were subcloned twice; a single recloned line, KB-3-1, was used as the parental cell line for the present study [2]. The KB-3-1 cells were cultured in minimal essential medium (MEM) (Nissui Seiyaku, Tokyo, Japan) with 1 mg/ml bactopeptone (Difco, Detroit, MI), 0.292 mg/ml glutamine and 100 units/ml penicillin, supplemented with 10% newborn calf serum (Flow Laboratories, Mclean, VA). The multidrug-resistant CV60 cell line is a subline of the multidrug-resistant
Expression of MRP and P-gp
The 190 kDa MRP was detected in membrane vesicles prepared from CV60 cells but not in those from parental KB-3-1 cells or KB-C2 cells by immunoblotting analysis. The 170 kDa P-gp was detected in KB-C2 cells but not in KB-3-1 cells or CV60 cells (Fig. 2).
The effect of ONO-1078 on sensitivity to VCR
IC50 values for VCR of KB-3-1 cells without or with ONO-1078 at 20 and 100 μM were 41, 18 and 3.1 ng/ml, respectively. IC50 values for VCR of CV60 cells without or with ONO-1078 at 20 and 100 μM were 750, 410 and 50 ng/ml, respectively. CV60
Discussion
Many studies have analyzed the structural features and the mechanism of action of agents that reverse P-gp-mediated MDR, but little is known about agents that reverse MRP-associated MDR [15]. Our results suggested that ONO-1078, an LTD4 receptor antagonist, reversed the resistance to VCR in CV60 cells by inhibiting the active efflux of VCR from the cells and increasing the intracellular concentration of VCR. MRP is an organic anion transporter and LTC4 is one of the best substrates of the
Acknowledgements
This work was supported in part by grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan. We thank Ono Pharmaceutical Co., Ltd. for providing ONO-1078.
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