ABC Transporters Associated with Multidrug Resistance

Drug resistance can be a serious obstacle to successful anticancer treatment with tumors often failing to respond either to initial chemotherapy (intrinsic resistance) or to subsequent rounds of treatment (acquired resistance). Studies conducted in the laboratory on tumor cell lines cultured in the presence of cytotoxic drugs reveal that resistance can develop, not only to the selecting drug, but to a number of structurally and functionally dissimilar drugs as well, hence providing multidrug resistance (MDR). This MDR

phenomenon may involve several different types of mechanisms, but a common cause is the presence of multidrug ABC transporters, which prevent access of the drugs to their intracellular targets sites. They accomplish this task by either effluxing the drug out of the cell via the plasma membrane, or by sequestering the drug within intracellular organelles such as the endoplasmic reticulum, lysosome, or peroxisome.

Identification of BCRP as a Multidrug Transporter

P-gp was the first ABC transporter to be associated with MDR (around the mid-1970s) with the MRPs being later discoveries (MRP1 sequenced in 1992). It was not until 1998 that BCRP was revealed as another MDR-associated ABC transporter. It was becoming apparent that there were certain tumor cell lines that showed resistance to several drugs (mainly mitoxantrone, bisantrene, topotecan, and dox-orubicin) yet did not overexpress P-glycoprotein or MRPs. Recognition of this atypical, non-P-gp, non-MRP resistance phenotype initiated a search for another multidrug transporter, leading to the ultimate discovery of BCRP, otherwise termed Mitoxantrone Resistance Protein (MXR) or ABC Transporter in Placenta (ABCP). The different names of this protein derive from the fact that it was characterized and cloned from three different sources in independent laboratories at about the same time: from a multidrug-resistant breast cancer cell line selected in doxorubicin (MCF7/ AdrVp3000, hence BCRP), from a colon cancer line selected in mitoxantrone (S1-M1-80, hence MXR), and from human placenta (hence ABCP) [1].

Though commonly called BCRP by virtue of its initial discovery in a drug-selected breast cancer cell line, it is not clear that the protein is actually often overexpressed in breast cancers in vivo. Indeed only weak BCRP expression has been found in breast tumors. There are, however, many different human tumors in which BCRP expression has been clearly demonstrated, including tumors of the kidney, ovary, stomach, colon, thyroid, brain, endometrium, and testis; squamous tumors (lung, head and neck, and esophagus); soft tissue sarcomas; pheochromocytomas; and hepatocarci-nomas. This may reflect the distribution of BCRP in normal tissues (see later discussion).

Substrate and Inhibitor Profiles of BCRP Compared to Other Multidrug Transporters

The ABC transporters associated with MDR vary somewhat in their mechanisms of action, substrate and inhibitor profiles, and in their tissue locations. Nevertheless there is a significant overlap in these characteristics between the transporters (Figure 2).

Functional studies on both BCRP overexpressing drug-selected and BCRP-transfected cell lines show that the transporter can confer resistance to anthracyclines (dox-orubicin, daunorubicin), anthracenediones (mitoxantrone), camptothecins (topotecan, irinotecan, and its active metabolite, SN-38), and etoposide, but not to vincristine, taxol, or colchicine, which are classical P-gp substrates. Substrates currently recognized to be transported by BCRP are shown in Table I.

Figure 2 Actions of multidrug transporters BCRP, P-gp, and MRP1 in efflux of substances from cells. Drugs can enter and leave cells by passive diffusion along a concentration gradient. Multidrug transporters provide a second route for drug exit and can drive drugs out of the cell against a concentration gradient by exploiting the energy of ATP hydrolysis. This additional efflux reduces drug concentrations inside cells to sublethal levels. P-gp and BCRP (in the form of a dimer) can efflux drugs unmodified (mainly hydrophobic, amphipathic compounds). The MRPs require the presence of reduced glutathione (GSH) to transport unmodified drugs (predominantly organic anions) but can transport drugs following their conjugation (GST, Glutathione transferase). (see color insert)

Figure 2 Actions of multidrug transporters BCRP, P-gp, and MRP1 in efflux of substances from cells. Drugs can enter and leave cells by passive diffusion along a concentration gradient. Multidrug transporters provide a second route for drug exit and can drive drugs out of the cell against a concentration gradient by exploiting the energy of ATP hydrolysis. This additional efflux reduces drug concentrations inside cells to sublethal levels. P-gp and BCRP (in the form of a dimer) can efflux drugs unmodified (mainly hydrophobic, amphipathic compounds). The MRPs require the presence of reduced glutathione (GSH) to transport unmodified drugs (predominantly organic anions) but can transport drugs following their conjugation (GST, Glutathione transferase). (see color insert)

Table I Substrate Profile of BCRP Compared with P-gp and MRP1.

Table II Tissue Distribution and Putative Functions of BCRP at These Locations.

Table I Substrate Profile of BCRP Compared with P-gp and MRP1.

Substrate

BCRP

P-gp

MRP1

Paclitaxel

Z

Verapamil

Z

Colchicine

Z

Vinblastine

Z

Z

Etoposide (VP-16)

Z

Z

Z

Daunorubicin

Z

Z

Z

Doxorubicin

Z

Z

Z

Epirubicin

Z

Z

Z

Mitoxantrone

Z

Z

Z

Methotrexate

Za

Z

Z

Prazosin

Z

Z

Topotecan

Z

Z

Bisantrene

Z

Z

Rhodamine-123

Z

Z

Flavopiridol

Z

Lysotracker Green

Z

SN-38

Z

e2-glu

Z

Z

Estrone 3-sulfate

Z

Z

LTC4

Z

GSH

Placenta—syncytiotrophoblast

Apical membrane of epithelium of small intestine and colon

Liver canalicular membrane

Apical membrane of lobules and lactiferous ducts of breast

Endothelium of capillaries and Unknown

"Side" population of hematopoietic stem cells

E2-GLU, 17ß-Estradiol 17-(ß-D-glucuronide); GSH, reduced glutathione; LTC4, leukotriene C4. Rhodamine 123 and Lysotracker Green are fluorescent dyes.

a Methotrexate is effluxed only by the wild-type BCRP

E2-GLU, 17ß-Estradiol 17-(ß-D-glucuronide); GSH, reduced glutathione; LTC4, leukotriene C4. Rhodamine 123 and Lysotracker Green are fluorescent dyes.

a Methotrexate is effluxed only by the wild-type BCRP

A point mutation affecting substrate specificity has recently been reported in BCRP. Though wild-type BCRP has an arginine at the 482nd position (at the start of the third transmembrane segment), BCRP overexpressed in certain drug-selected cell lines was shown to contain either a glycine or threonine at this site. This point mutation causes a paradigm shift in the protein's substrate specificity—wild-type BCRP is incapable of effluxing the fluorescent dye rhodamine 123 or anthracycline drugs such as doxorubicin, but the mutants can handle both these substrates. Conversely, only wild-type BCRP transports the antifolate cytotoxic methotrexate [1]. Polymorphisms of the BCRP gene have also been described in human populations.

Subcellular Location of BCRP

BCRP seems to be predominantly localized to the plasma membrane of both drug-selected and transfected cell lines. This sets it apart from other half-transporters that are localized mainly to intracellular membranes such as the mitochondrion (ABCB7), the peroxisome (ALD subfamily), and the endoplasmic reticulum (Tap1/Tap2). Such a location for

Localization

Putative function

Placenta—syncytiotrophoblast

Apical membrane of epithelium of small intestine and colon

Liver canalicular membrane

Apical membrane of lobules and lactiferous ducts of breast

Protection of fetus, excretion of substrates

Reduced uptake/excretion of substrates into maternal circulation

Excretion of substrates by the liver into bile

Unknown

Endothelium of capillaries and Unknown

"Side" population of hematopoietic stem cells

Unknown

BCRP is consistent with a putative role in the efflux of substrates from the cell. Furthermore, it is apparent in polarized cell lines such as BCRP-transfected MDCK-II Madine-Darby canine kidney cells that the transporter becomes localized primarily to the apical aspect of the plasma membrane, where it mediates the translocation of substrates from basal to apical side. However, a role for BCRP in the intracellular trafficking of molecules cannot be ruled out as some immunocytochemical studies have reported perinuclear staining for BCRP in several topotecan- and mitoxantrone-resistant cell lines [1].

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