Kun Lee

MRP Subfamily Transporters and Resistance to Anticancer Agents

The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin–Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1–3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.

Evaluation of the Role of Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 in Hepatic Basolateral Excretion of Sulfate and Glucuronide Metabolites of Acetaminophen, 4-Methylumbelliferone, and Harmol in Abcc3–/– and Abcc4–/– Mice

Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3–/– and Abcc4–/– mice using a cassette dosing approach. In the livers of Abcc3–/– and Abcc4–/– mice, the basolateral excretory clearance of acetaminophen sulfate was reduced ∼20 and ∼20%, 4-methylumbelliferyl sulfate was reduced ∼50 and ∼65%, and harmol sulfate was decreased ∼30 and ∼45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by ∼96, ∼85, and ∼40%, respectively, in the livers of Abcc3–/– mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.

Physiological and pharmacological functions of Mrp2, Mrp3 and Mrp4 as determined from recent studies on gene-disrupted mice

The MRP family is composed of nine transporters, at least eight of which are lipophilic anion transporters that are capable of conferring resistance to various anticancer agents. Recently, mice with gene disruptions in Mrp2, Mrp3 and Mrp4 have been developed. This review will discuss insights into the physiological and pharmacological functions of Mrp2, Mrp3 and Mrp4 afforded by investigations of these new mouse models.

Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol a on MRP1 function

We previously isolated agosterol A (AG‐A) from a marine Spongia sp. and found that it completely reversed colchicine resistance in P‐glycoprotein (Pgp)‐over‐expressing KB‐C2 cells and vincristine resistance in multidrug‐resistance protein (MRP)1‐over‐expressing CV60 cells. However, a tri‐deacetylated derivative of AG‐A (IAG‐A) showed almost no activity in reversing Pgp‐ or MRP1‐mediated drug resistance. In this study, we examined the mechanisms by which AG‐A reverses MRP1‐mediated drug resistance by investigating the interaction between agosterols and MRP1 in MRP1‐over‐expressing human KB carcinoma (KB/MRP) cells. [3H]‐Leukotriene C4 (LTC4), [3H]‐2,4‐dinitrophenyl‐S‐glutathione uptake into membrane vesicles prepared from KB/MRP cells and intracellular [3H]‐vincristine accumulation and efflux in KB/MRP cells were measured with or without AG‐A and/or inactive IAG‐A. AG‐A reduced MRP1‐mediated [3H]‐LTC4 transport in a dose‐dependent manner, but IAG‐A did not. Inhibition by AG‐A was competitive, with a Ki value of 31 μM. AG‐A at 10 μM enhanced the accumulation of [3H]‐vincristine in KB/MRP cells to the level of that in control cells in the absence of the agent. Likewise, ATP‐dependent efflux of [3H]‐vincristine from KB/MRP cells was enhanced compared with KB‐3‐1 cells and inhibited by AG‐A. In addition, AG‐A reduced intracellular levels of glutathione, a compound required for MRP1‐mediated transport of some anti‐cancer drugs. These findings suggest that AG‐A reverses MRP1‐mediated drug resistance by directly inhibiting the capacity of MRP1 to transport drugs. In addition, the capacity of AG‐A to reduce cellular glutathione levels may contribute to the modulating activity of MRP1.