Multiple ATP-binding Cassette Transporters Research Articles

Abstract 2621: Growth hormone receptor antagonist sensitizes melanoma and hepatocarcinoma to drug treatments via downregulation of ABC transporters in vivo

Abstract Knockdown of the growth hormone receptor (GHR) in melanoma cells downregulates ATP-binding cassette (ABC) transporters and sensitizes them to multiple drug treatments in vitro. The goal for this study is to understand whether a GHR antagonist (GHA) could suppress different types of cancers by sensitizing tumors to drug treatments in vivo through the downregulation of ABC transporters. Sera from GHA transgenic mice inhibited the proliferation of mouse melanoma cells (B16F10) in culture and suppressed expression of multiple ABC transporters. When B16-F10 cells were intradermally inoculated into GHA mice, tumor size was markedly reduced, as was STAT5 activation and ABCG1, ABCG2 levels. We then tested the effect of the GHA on the efficacy of cisplatin in vivo. GHA sensitized melanomas to cisplatin, leading to the smallest tumors among all groups. GHKO mice showed the same effect. We further extended this investigation to hepatocellular carcinoma (HCC). GHA mice were subcutaneously inoculated with mouse HCC (Hepa1 6 cells) and subsequently treated with sorafenib. The HCC tumors in GHA mice were markedly sensitive to sorafenib treatment compared to the same in wild-type mice. RNA analysis showed that when HCC was exposed to the combined treatment, relatively decreased ABC transporters expression was found. Immunohistochemical staining showed that phosphorylation of STAT5 and ABCB1 were downregulated in HCC tissues, suggesting that GHA in vivo downregulates ABC transporters in HCC, and therefore sensitizes them to sorafenib. Clinical data derived from HCC patients using the TCGA database showed that multiple ABC transporters in both types of cancer correlate with GHR levels; that is, when GHR levels are relatively high, patient survival is significantly decreased. Additionally, higher ABCC1 levels also lead to significantly decreased survival rate in HCC patients. Collectively, the results indicate that a GHA is effective in sensitizing both melanoma and HCC to available treatments in vivo and may be used as a therapeutic strategy for higher efficacy of tumor clearance. Citation Format: Yanrong Qian, Reetobrata Basu, Samuel Mathes, Prateek Kulkarni, Emily Davis, Darlene Berryman, Edward List, Silvana Duran, John J. Kopchick. Growth hormone receptor antagonist sensitizes melanoma and hepatocarcinoma to drug treatments via downregulation of ABC transporters in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2621.

Mitochondrial Breast Cancer Resistant Protein Sustains the Proliferation and Survival of Drug-Resistant Breast Cancer Cells by Regulating Intracellular Reactive Oxygen Species.

ATP-binding cassette (ABC) transporter family are major contributors to the drug resistance establishment of breast cancer cells. Breast cancer resistant protein (BCRP), one of the ABC transporters, has long been recognized as a pump that effluxes the therapeutic drugs against the concentration gradient. However, recent studies suggest that the biological function of BCRP is not limited in its drug pump activity. Herein, the role of BCRP in the proliferation and survival of drug-resistant breast cancer cells was investigated. We found that BCRP is not the major drug pump to efflux epirubicin in the resistant cells that express multiple ABC transporters. Silencing of BCRP significantly impairs cell proliferation and induces apoptosis of the resistant cells in vitro and in vivo. RNA-sequencing and high-throughput proteomics suggest that BCRP is an inhibitory factor of oxidative phosphorylation (OXPHOS). Further research suggests that BCRP is localized in the mitochondria of the resistant cells. Knockdown of BCRP elevated the intracellular reactive oxygen species level and eventually promotes the cell to undergo apoptosis. This study demonstrated that BCRP exerts important onco-promoting functions in the drug-resistant breast cancer cells independent of its well-recognized drug efflux activity, which shed new light on understanding the complex functional role of ABC transporters in drug-resistant cells.

Borneol: a Promising Monoterpenoid in Enhancing Drug Delivery Across Various Physiological Barriers.

Incorporation of permeation enhancers is one of the most widely employed approaches for delivering drugs across biological membranes. Permeation enhancers aid in delivering drugs across various physiological barriers such as brain capillary endothelium, stratum corneum, corneal epithelium, and mucosal membranes that pose resistance to the entry of a majority of drugs. Borneol is a natural, plant-derived, lipophilic, volatile, bicyclic monoterpenoid belonging to the class of camphene. It has been used under the names "Bing Pian" or "Long Nao" in Traditional Chinese Medicine for more than 1000 years. Borneol has been incorporated predominantly as an adjuvant in the traditional Chinese formulations of centrally acting drugs to improve drug delivery to the brain. This background knowledge and anecdotal evidence have led to extensive research in establishing borneol as a permeation enhancer across the blood-brain barrier. Alteration in cell membrane lipid structures and modulation of multiple ATP binding cassette transporters as well as tight junction proteins are the major contributing factors to blood-brain barrier opening functions of borneol. Owing to these mechanisms of altering membrane properties, borneol has also shown promising potential to improve drug delivery across other physiological barriers as well. The current review focuses on the role of borneol as a permeation enhancer across the blood-brain barrier, mucosal barriers including nasal and gastrointestinal linings, transdermal, transcorneal, and blood optic nerve barrier.

ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species

The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.

Differentially Regulated Transcription Factors and ABC Transporters in a Mitochondrial Dynamics Mutant Can Alter Azole Susceptibility of Aspergillus fumigatus.

Azole resistance of the fungal pathogen Aspergillus fumigatus is an emerging problem. To identify novel mechanisms that could mediate azole resistance in A. fumigatus, we analyzed the transcriptome of a mitochondrial fission/fusion mutant that exhibits increased azole tolerance. Approximately 12% of the annotated genes are differentially regulated in this strain. This comprises upregulation of Cyp51A, the azole target structure, upregulation of ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) transporters and differential regulation of transcription factors. To study their impact on azole tolerance, conditional mutants were constructed of seven ABC transporters and 17 transcription factors. Under repressed conditions, growth rates and azole susceptibility of the mutants were similar to wild type. Under induced conditions, several transcription factor mutants showed growth phenotypes. In addition, four ABC transporter mutants and seven transcription factor mutants exhibited altered azole susceptibility. However, deletion of individual identified ABC transporters and transcription factors did not affect the increased azole tolerance of the fission/fusion mutant. Our results revealed the ability of multiple ABC transporters and transcription factors to modulate the azole susceptibility of A. fumigatus and support a model where mitochondrial dysfunctions trigger a drug resistance network that mediates azole tolerance of this mold.

Multiple ATP-binding cassette transporters genes are involved in thiamethoxam resistance in Aphis gossypii glover

ATP-binding cassette (ABC) transporters represent the largest known group of efflux pumps, utilizing ATP to translocate a broad spectrum of substrates across lipid membranes, which play an important role in phase III of the detoxification process. The presence of ABC transporters and their potential association with insecticide resistance have not been investigated in Aphis gossypii, one of the most economically important agricultural pests worldwide. In this study, the ABC transporter inhibitor-verapamil significantly increased thiamethoxam toxicity against resistant cotton aphids, suggesting that ABCs are involved in thiamethoxam resistance. ABC transporter genes were identified using the A. gossypii genome database and transcriptome data. A total of 69 ABC transporters were identified and grouped into seven subfamilies (A-G), including 4 ABCAs, 5 ABCBs, 25 ABCCs, 2 ABCDs, 1 ABCE, 4 ABCFs and 30 ABCGs. Of these ABC transporters, 53 were predicted to be functional, 19 were full transporters, 30 were half-transporters and 4 had two NBDs. Subfamilies C and G accounted for 77% (32 and 45%, respectively) of the genes. The transcripts of 20 of 26 ABCs based on the transcriptome were upregulated, and ABCA1, ABCA2, ABCB1, ABCB4, ABCB8, ABCD1, ABCD2, ABCE1, ABCF1, ABCF3, ABCG7, ABCG15, ABCG17, ABCG24, ABCG27, ABCG30, MRP1, MRP7, MRP14 and MRP21 transcripts were significantly increased in the thiamethoxan resistant strain compared to the susceptible strain with qRT-PCR. The suppression of overexpressed ABCs (ABCA2, ABCD1, ABCD2, ABCE1 and ABCG15) significantly increased the thiamethoxam sensitivity of resistant aphids. These results suggest that ABC transporters might be involved in thiamethoxam resistance in A. gossypii and will facilitate further work to validate the functional roles of these ABCs in thiamethoxam resistance. These results are useful for understanding the multiple resistance mechanisms of thiamethoxam and the management of insecticide-resistant cotton aphids.

Abstract 1266: Co-expression of P-glycoprotein and ABCG2 in a cell line model reveals both independent and cooperative transporter function

Abstract Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how expression of multiple transporters might confer resistance to chemotherapy drugs. We therefore examined how P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 contribute to drug resistance in a cell line model. HEK-293 cells were transfected with vector encoding full-length ABCB1, ABCG2, or a vector containing both genes under the control of a separate promoter. Cells transfected to express both transporters (B1/G2 cells) expressed high levels of both transporters and were able to transport both the P-gp-specific substrate rhodamine 123 and the ABCG2 specific substrate pheophorbide a when examined by flow cytometry. B1/G2 cells were also cross-resistant to the P-gp substrate doxorubicin, the ABCG2 substrate topotecan as well as mitoxantrone, a substrate of both transporters. The checkpoint inhibitor prexasertib was also found to be a substrate of both P-gp and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that P-gp and ABCG2 can function independently to transport substrates. P-gp and ABCG2 were also found to function cooperatively to transport the common fluorescent substrates mitoxantrone and BODIPY®-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. This was also true in 3-day cytotoxicity assays with mitoxantrone and prexasertib. Thus, P-gp and ABCG2 can independently and cooperatively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed. Citation Format: Andrea Robinson, Robert Robey, Michael Gottesman. Co-expression of P-glycoprotein and ABCG2 in a cell line model reveals both independent and cooperative transporter function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1266.

Coexpression of ABCB1 and ABCG2 in a Cell Line Model Reveals Both Independent and Additive Transporter Function.

Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how coexpression of multiple transporters affected resistance to chemotherapeutic drugs. We therefore examined how coexpression of ABCB1 (P-glycoprotein) and ABCG2 contributes to drug resistance in a cell line model. HEK293 cells were transfected with vector-encoding full-length ABCB1, ABCG2, or a bicistronic vector containing both genes, each under the control of a separate promoter. Cells transfected with both transporters (B1/G2 cells) demonstrated high levels of both transporters, and uptake of both the ABCB1-specific substrate rhodamine 123 and the ABCG2-specific substrate pheophorbide a was reduced when examined by flow cytometry. B1/G2 cells were also cross-resistant to the ABCB1 substrate doxorubicin, the ABCG2 substrate topotecan, as well as mitoxantrone and the cell cycle checkpoint kinase 1 inhibitor prexasertib, both of which were found to be substrates of both ABCB1 and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that ABCB1 and ABCG2, when coexpressed, can function independently to transport substrates. ABCB1 and ABCG2 also functioned additively to transport the common fluorescent substrates mitoxantrone and BODIPY-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. ABCG2 expression was also found to decrease the efficacy of the ABCB1 inhibitor tariquidar in B1/G2 cells. Thus, ABCB1 and ABCG2 can independently and additively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed.

Effect of amyloid beta on ATP-binding cassette transporter expression and activity in porcine brain microvascular endothelial cells

BackgroundDeposition of amyloid-β peptide (Aβ(1–42)) within the brain is characteristic of Alzheimer's disease. Little is known of the effects of Aβ(1–42) on blood-brain barrier (BBB) ATP-binding Cassette (ABC) efflux transporters which influence BBB permeability. The effects of Aβ(1–42) on ABCB1, ABCC5 and ABCG2 activity and expression and pregnane X receptor (PXR) and constitutive androstane receptor (CAR) transcription factors expression were determined in primary porcine brain endothelial cells (PBECs). MethodsThe effect of Aβ(1–42) on transporter activity was determined by measurement of intracellular accumulation of the fluorescent probes calcein (ABCB1), GS–MF (ABCC5) and Hoechst 33342 (ABCG2). Expression of transporters and transcription factors was assessed by Western blotting. ResultsTreatment of PBECs with Aβ(1–42) significantly decreased activity of ABCB1 (Aβ(1–42) at 10 μg/ml, 25 μg/ml and 50 μg/ml), ABCC5 (Aβ(1–42) at 25 μg/ml and 50 μg/ml) and ABCG2 (Aβ(1–42) at 10 μg/ml, 25 μg/ml and 50 μg/ml). Aβ(1–42) also significantly decreased expression of ABCB1 (p < 0.05 at 25 μg/ml and 50 μg/ml), ABCG2 (p < 0.05 at 25 μg/ml and p ≤ 0.001 at 50 μg/ml), ABCC5 (p < 0.05 at 25 μg/ml and 50 μg/ml), PXR (p < 0.05 at 10 μg/ml, 25 μg/ml and 50 μg/ml Aβ(1–42)) and CAR (p < 0.05 at 25 μg/ml and 50 μg/ml Aβ(1–42)). ConclusionAβ(1–42) inhibits multiple ABC transporters and PXR and CAR in PBECs. General significanceAβ(1–42) reduces ABC transporter activity and expression in BBB endothelial cells and has the potential to influence BBB permeability characteristics.

Co-expression of ATP binding cassette transporters is associated with poor prognosis in acute myeloid leukemia

Chemotherapy failure remains a challenge when treating patients with acute myeloid leukemia (AML), who often suffer from persistent or relapsed disease. The multidrug resistance (MDR) mediated by efflux transporters of the ATP binding cassette (ABC) superfamily is a major obstacle for successful chemotherapy. The present study aimed to elucidate whether the expression of ABC transporters was associated with prognostic factors and responses to chemotherapy in patients with AML, with particular focus on whether co-expression of multiple ABC transporters resulted in a worse prognosis. In the present study, the mRNA expression levels of ABC transporters ABCB1, ABCB4, ABCC1, ABCC4 and ABCG2 in bone marrow (BM) mononuclear cell (MNC) samples from 96 de novo patients with AML and in the peripheral blood (PB) MNC samples from 22 normal individuals were investigated using reverse transcription-quantitative polymerase chain reaction analysis. It was revealed that ABCB1, ABCC1, ABCC4 and ABCG2 were expressed at higher levels in patients with AML compared with normal individuals, whereas ABCB4 had a lower expression level. The expression of ABCB4 in patients with AML was significantly lower than in normal individuals (P<0.001). Patients risk status was associated with ABCB1 (P=0.037), ABCC1 (P=0.047), ABCC4 (P=0.015) and ABCG2 (P=0.027). The 4 genes were expressed a significantly higher levels in the poor response group compared with the good response group (ABCB1, P=0.014; ABCC1, P=0.021; ABCC4, P=0.005; ABCG2, P=0.009). The overexpression of the 4 ABC transporters and the complete remission rate were inversely correlated (P<0.001). These results suggest that the co-expression of multiple ABC transporters may contribute to a worse prognosis in AML.

Graphene Quantum Dots Downregulate Multiple Multidrug‐Resistant Genes via Interacting with Their C‐Rich Promoters

Multidrug resistance (MDR) is the major factor in the failure of many forms of chemotherapy, mostly due to the increased efflux of anticancer drugs that mediated by ATP-binding cassette (ABC) transporters. Therefore, inhibiting ABC transporters is one of effective methods of overcoming MDR. However, high enrichment of ABC transporters in cells and their broad substrate spectra made to circumvent MDR are almost insurmountable by a single specific ABC transporter inhibitor. Here, this study demonstrates that graphene quantum dots (GQDs) could downregulate the expressions of P-glycoprotein, multidrug resistance protein MRP1, and breast cancer resistance protein genes via interacting with C-rich regions of their promoters. This is the first example that a single reagent could suppress multiple MDR genes, suggesting that it will be possible to target multiple ABC transporters simultaneously with a single reagent. The inhibitory ability of the GQDs to these drug-resistant genes is validated further by reversing the doxorubicin resistance of MCF-7/ADR cells. Notably, GQDs have superb chemical and physical properties, unique structure, low toxicity, and high biocompatibility; hence, their capability of inhibiting multiple drug-resistant genes holds great potential in cancer therapy.

Multiple ATP-binding cassette transporters are involved in insecticide resistance in the small brown planthopper, Laodelphax striatellus.

ATP-binding cassette (ABC) transporters are membrane-bound proteins involved in the movement of various substrates, including drugs and insecticides, across the lipid membrane. Demonstration of the role of human ABC transporters in multidrug resistance has led to speculation that they might be an important mechanism controlling the fate of insecticides in insects. However, the role of ABC transporters in insects remains largely unknown. The small brown planthopper, Laodelphax striatellus Fallén, has developed resistance to most of the insecticides used for its control. Our goals were to identify the ABC transporters in La. striatellus and to examine their involvement in resistance mechanisms, using related strains resistant to chlorpyrifos, deltamethrin and imidacloprid, compared with the susceptible strain. Based on the transcriptome of La. striatellus, 40 full-length ABC transporters belonging to the ABCA-ABCH subfamilies were identified. Quantitative PCR revealed that over 20% of genes were significantly up-regulated in different resistant strains, and eight genes from the ABCB/C/D/G subfamilies were up-regulated in all three resistant strains, compared with the susceptible strain. Furthermore, synergism studies showed verapamil significantly enhanced insecticide toxicity in various resistant strains but not in the susceptible strain. These results suggest that ABC transporters might be involved in resistance to multiple insecticides in La. striatellus.

Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps.

Melanoma remains one of the most therapy-resistant forms of human cancer despite recent introductions of highly efficacious targeted therapies. The intrinsic therapy resistance of human melanoma is largely due to abundant expression of a repertoire of xenobiotic efflux pumps of the ATP-binding cassette (ABC) transporter family. Here, we report that GH action is a key mediator of chemotherapeutic resistance in human melanoma cells. We investigated multiple ABC efflux pumps (ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1, and ABCG2) reportedly associated with melanoma drug resistance in different human melanoma cells and tested the efficacy of five different anti-cancer compounds (cisplatin, doxorubicin, oridonin, paclitaxel, vemurafenib) with decreased GH action. We found that GH treatment of human melanoma cells upregulates expression of multiple ABC transporters and increases the EC50 of melanoma drug vemurafenib. Also, vemurafenib-resistant melanoma cells had upregulated levels of GH receptor (GHR) expression as well as ABC efflux pumps. GHR knockdown (KD) using siRNA in human melanoma cells treated with sub-EC50 doses of anti-tumor compounds resulted in significantly increased drug retention, decreased cell proliferation and increased drug efficacy, compared to mock-transfected controls. Our set of findings identify an unknown mechanism of GH regulation in mediating melanoma drug resistance and validates GHR as a unique therapeutic target for sensitizing highly therapy-resistant human melanoma cells to lower doses of anti-cancer drugs.

Increased expression of ATP binding cassette transporter genes following exposure of Haemonchus contortus larvae to a high concentration of monepantel in vitro.

BackgroundThere is some evidence that ATP binding cassette (ABC) transporters play a role in resistance to anthelmintics, particularly against macrocyclic lactones. Some anthelmintics, including ivermectin (IVM), have been shown to induce transcription of multiple ABC transporters in nematodes; however, the effects of monepantel (MPL) on transcription of these transporter genes has not been studied.MethodsLarvae of two MPL-susceptible isolates of Haemonchus contortus were exposed to MPL at two concentrations (2.5 and 250 μg/ml) for periods of 3, 6 and 24 h. Transcription levels of sixteen ABC transporter genes were measured at the end of the incubation periods. The consequences of MPL exposure were examined by measuring rhodamine-123 efflux from the larvae, and their sensitivity to subsequent treatment with IVM or levamisole.ResultsMultiple ABC transporter genes showed significantly higher transcription in both worm isolates following exposure to MPL at 250 μg/ml for 3, 6 or 24 h, particularly the P-glycoprotein (P-gp) genes pgp-11, pgp-12 and pgp-14. Of these, only pgp-11 maintained the elevated levels 24 h after the end of the drug exposure period. In contrast, there was only a single instance of low-level upregulation as a result of exposure to MPL at 2.5 μg/ml. Larvae exposed to MPL at 250 μg/ml showed an increased efflux of rhodamine-123 and a proportion of the larval population showed an ability to subsequently tolerate higher concentrations of IVM in migration assays. There was no increased tolerance to IVM following pre-exposure to MPL at 2.5 μg/ml.ConclusionsExposure of H. contortus larvae to 250 μg/ml MPL results in increased transcription of multiple transporter genes and increased R-123 efflux. The subsequent ability of a proportion of the larvae to tolerate IVM suggests a protective role of ABC transporters across different chemical entities. However, these observations were only made at a concentration of MPL well above that experienced by parasitic life stages in vivo, and hence their significance remains unclear.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1806-9) contains supplementary material, which is available to authorized users.

A Selective ATP-Binding Cassette Subfamily G Member 2 Efflux Inhibitor Revealed via High-Throughput Flow Cytometry

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)–driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

Beyond ABC: Another Mechanism of Drug Resistance in Melanoma Side Population

It has been shown that a side population (SP), which is characterized by high chemical efflux capacity, is present in human melanoma cell lines. However, it was not clear whether patients' samples contain the same subpopulation. In this issue, Luo et al. (2012) report that they have isolated SP cells directly from patients' melanomas. SP cells are resistant to paclitaxel because of the upregulation of ABCB1 and ABCB5. Notably, these cells are also resistant to temozolomide, which is not a substrate for ATP-Binding Cassette (ABC) transporters, in an interleukin (IL)-8-dependent manner. This study provides novel clues for understanding how a small, but critical, subpopulation within melanomas is resistant to therapies.

Drug Efflux Transporters and Multidrug Resistance in Acute Leukemia: Therapeutic Impact and Novel Approaches to Mediation

Multidrug resistance (MDR), which is mediated by multiple drug efflux ATP-binding cassette (ABC) transporters, is a critical issue in the treatment of acute leukemia, with permeability glycoprotein (P-gp), multidrug resistance-associated protein 1, and breast cancer resistance protein (i.e., ABCG2) consistently being shown to be key effectors of MDR in cell line studies. Studies have demonstrated that intrinsic MDR can arise as a result of specific gene expression profiles and that drug-induced overexpression of P-gp and other MDR proteins can result in acquired resistance, with multiple ABC transporters having been shown to be overexpressed in cell lines selected for resistance to multiple drugs used to treat acute leukemia. Furthermore, numerous anticancer drugs, including agents commonly used for the treatment of acute leukemia (e.g., doxorubicin, vincristine, mitoxantrone, and methotrexate), have been shown to be P-gp substrates or to be susceptible to efflux mediated by other MDR proteins, and multiple clinical studies have demonstrated associations between P-gp or other MDR protein expression and responses to therapy or survival rates in acute leukemia. Here we review the importance of MDR in cancer, with a focus on acute leukemia, and we highlight the need for rapid accurate assessment of MDR status for optimal treatment selection. We also address the latest research on overcoming MDR, from inhibition of P-gp and other MDR proteins through various approaches (including direct antagonism and gene silencing) to the design of novel agents or novel delivery systems for existing therapeutic agents, to evade cellular efflux.

Multiple ABC transporters are involved in the acquisition of petrobactin in Bacillus anthracis

In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. The petrobactin-binding receptor FpuA is required for these processes. Here additional components of petrobactin reacquisition are described. To identify these proteins, mutants of candidate permease and ATPase genes were generated allowing for characterization of multiple petrobactin ATP-binding cassette (ABC)-import systems. Either of two distinct permeases, FpuB or FatCD, is required for iron acquisition and play redundant roles in petrobactin transport. A mutant strain lacking both permeases, ΔfpuBΔfatCD, was incapable of using petrobactin as an iron source and exhibited attenuated virulence in a murine model of inhalational anthrax infection. ATPase mutants were generated in either of the permease mutant backgrounds to identify the ATPase(s) interacting with each individual permease channel. Mutants lacking the FpuB permease and FatE ATPase (ΔfpuBΔfatE) and a mutant lacking the distinct ATPases FpuC and FpuD generated in the ΔfatCD background (ΔfatCDΔfpuCΔfpuD) displayed phenotypic characteristics of a mutant deficient in petrobactin import. A mutant lacking all three of the identified ATPases (ΔfatEΔfpuCΔfpuD) exhibited the same growth defect in iron-depleted conditions. Taken together, these results provide the first description of the permease and ATPase proteins required for the import of petrobactin in B. anthracis.

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Pharmacokinetic Assessment of Multiple ATP-binding Cassette Transporters: The Power of Combination Knockout Mice

A TP-binding cassette (ABC) multidrug transporters are cellular efflux pumps with broad and often widely overlapping substrate specificities. They can have a major impact on the pharmacokinetics and hence overall pharmacological behavior of many drugs. To study their separate roles and functional overlap, or complementarity, a collection of mice deficient in two or more ABC transporters has been generated. This review discusses recent findings obtained with these models, focusing on pharmacokinetic studies with a number of clinically relevant drugs. In addition, the characterization of these mice and some physiological aspects of ABC multidrug transporters are addressed.