It has been reported that gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has the ability to modulate the function of certain ATP-binding cassette (ABC) transporters and to reverse ABC subfamily B member 1 (ABCB1; P-glycoprotein)-and ABC subfamily G member 2 (ABCG2; breast cancer resistance protein/mitoxantrone resistance protein)-mediated multidrug resistance (MDR) in cancer cells. However, it is unknown whether other EGFR TKIs have effects similar to that of gefitinib. In the present study, we have investigated the interaction of another EGFR TKI, erlotinib, with selected ABC drug transporters. Our findings show that erlotinib significantly potentiated the sensitivity of established ABCB1 or ABCG2 substrates and increased the accumulation of paclitaxel or mitoxantrone in ABCB1-or ABCG2-overexpressing cells. Furthermore, erlotinib did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in all cells and was unable to reverse MRP1-mediated MDR and had no effect on the parental cells. However, erlotinib remarkably inhibited the transport of E 2 17BG and methotrexate by ABCG2. In addition, the results of ATPase assays show that erlotinib stimulated the ATPase activity of both ABCB1 and ABCG2. Interestingly, erlotinib slightly inhibited the photolabeling of ABCB1 with [125 I] iodoarylazidoprazosin (IAAP) at high concentration, but it did not inhibit the photolabeling of ABCG2 with IAAP. Overall, we conclude that erlotinib reverses ABCB1-and ABCG2-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1 and ABCG2. These findings may be useful for cancer combinational therapy with erlotinib in the clinic. [Cancer Res 2007;67(22):11012-20]

Interindividual variability in protein expression of organic aniontransporting polypeptides (OATPs) OATP1B1, OATP1B3, OATP2B1, and multidrug resistance-linked P-glycoprotein (P-gp) or ABCB1 was quantified in frozen human livers (n = 64) and cryopreserved human hepatocytes (n = 12) by a validated liquid chromatography tandem mass spectroscopy (LC-MS/MS) method. Membrane isolation, sample workup, and LC-MS/MS analyses were as described before by our laboratory. Briefly, total native membrane proteins, isolated from the liver tissue and cryopreserved hepatocytes, were trypsin digested and quantified by LC-MS/MS using signature peptide(s) unique to each transporter. The mean 6 S.D. (maximum/minimum range in parentheses) protein expression (

The multifactorial nature of MDR indicates that it may be important to develop modulators that can simultaneously inhibit both the function of the drug transporters and key signaling pathways, which are responsible for development of this phenomenon.

In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ⌬F774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (⌬F774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ⌬F774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).Candida albicans is an opportunistic diploid fungus that causes infections in immunocompromised and debilitated patients (34). Widespread and prolonged usage of azoles in recent years has led to the rapid development of the phenomenon of multidrug resistance (MDR), which poses a major hurdle in antifungal therapy. Various mechanisms which contribute towards the development of MDR have been implicated in Candida, and some of these include overexpression of or mutations in the target enzyme of azoles, lanosterol 14␣-demethylase, and overexpression of drug efflux pumps (1, 33) belonging to the ATP-binding cassette (ABC) transporter channel superfamily (18) and to the major facilitator superfamily of transporters (MFS) (22, 35). Among ABC transporters, CDR1 has been shown to play a key role in azole resistance in C. albicans as deduced from its high level of expression found in several azole resistance clinical isolates recovered from patients receiving long-term antifungal therapy (41, 39). Additionally, high-level expression of CDR1 invariably contributes to an increased efflux of fluconazole, thus corroborating its direct involvement in drug efflux (24, 38). Cdr1p has not only acquired significant clinical importance but is considered an important player in any design of strategies to combat antifungal resistance.The CDR1 gene encodes an integral plasma membrane (PM) protein of 1,501 amino acids, with a predicted molecu...

Multidrug resistance due to reduced drug accumulation is a phenomenon predominantly caused by the overexpression of members of the ATP-binding cassette (ABC) transporters, including ABCB1 (P-glycoprotein), ABCG2, and several ABCC family members [multidrug resistanceassociated protein (MRP)]. We previously reported that a thiosemicarbazone derivative, NSC73306, is cytotoxic to carcinoma cells that overexpress functional P-glycoprotein, and it resensitizes these cells to chemotherapeutics. In this study, we investigated the effect of NSC73306 on cells overexpressing other ABC drug transporters, including ABCG2, MRP1, MRP4, and MRP5. Our findings showed that NSC73306 is not more toxic to cells that overexpress these transporters compared with their respective parental cells, and these transporters do not confer resistance to NSC73306 either. In spite of this, we observed that NSC73306 is a transport substrate for ABCG2 that can effectively inhibit ABCG2-mediated drug transport and reverse resistance to both mitoxantrone and topotecan in ABCG2-expressing cells. Interactions between NSC73306 and the ABCG2 drug-binding site(s) were confirmed by its stimulatory effect on ATPase activity (140 -150 nmol/L concentration required for 50% stimulation) and by inhibition of [ 125 I]iodoarylazidoprazosin photolabeling (50% inhibition at 250 -400 nmol/L) of the substrate-binding site(s). Overall, NSC73306 seems to be a potent modulator of ABCG2 that does not interact with MRP1, MRP4, or MRP5. Collectively, these data suggest that NSC73306 can potentially be used, due to its dual mode of action, as an effective agent to overcome drug resistance by eliminating P-glycoprotein -overexpressing cells and by acting as a potent modulator that resensitizes ABCG2-expressing cancer cells to chemotherapeutics.