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The overexpression of ATP-binding cassette (ABC) transporters is the main cause of cancer multidrug resistance (MDR), which leads to chemotherapy failure. Uncaria alkaloids are the major active components isolated from uncaria, which is a common Chinese herbal medicine. In this study, the MDR-reversal activities of uncaria alkaloids, including rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine (Icory), hirsutine and hirsuteine, were screened; they all exhibited potent reversal efficacy when combined with doxorubicin. Among them, Icory significantly sensitized ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells to vincristine, doxorubicin and paclitaxel, but not to the non-ABCB1 substrate cisplatin. Noteworthy, Icory selectively reversed ABCB1-overexpressing MDR cancer cells but not ABCC1- or ABCG2-mediated MDR. Further mechanistic study revealed that Icory increased the intracellular accumulation of doxorubicin in ABCB1-overexpressing cells by blocking the efflux function of ABCB1. Instead of inhibiting ABCB1 expression and localization, Icory acts as a substrate of the ABCB1 transporter by competitively binding to substrate binding sites. Collectively, these results indicated that Icory reversed ABCB1-mediated MDR by suppressing its efflux function, and it would be beneficial to increase the efficacy of these types of uncaria alkaloids and develop them to be selective ABCB1-mediated MDR-reversal agents.

Multidrug resistance (MDR) is the main cause of failed chemotherapy treatments. Therefore, preventing MDR is pivotal in treating colorectal cancer (CRC). In a previous study miR-133b was shown to be a tumor suppressor. Additionally, in CRC cells transfected with miR-133b, ATP-binding cassette (ABC) subfamily C member 1(ABCC1) was shown to be significantly down regulated. Whether miR-133b also enhances the chemosensitivity of drugs used to treat CRC by targeting ABCC1 is still unclear. Here, we utilized flow cytometry and high-performance liquid chromatography (HPLC) analysis to identify the ability of miR-133b to reserve MDR in CRC. We then used a dual-luciferase reporter assay to validate that miR-133b targets ABCC1. Further in vivo experiments were designed to validate the method in which miR-133b reversed MDR in CRC cells. The results demonstrated that the level of miR-133b was down-regulated and the expression of ABCC1 was up-regulated in drug-resistant CRC cells compared to non-drug-resistant CRC cells. The restoration of miR-133b expression in CRC drug-resistant cells in vitro resulted in reduced IC50s to chemotherapeutic drugs, significantly induced G1 accumulation, inhibited growth and promoted necrosis in combination with either 5-fluorouracil (5-FU) or vincristine (VCR), and decreased the expression of ABCC1. The dual-luciferase assay demonstrated that miR-133b directly targets ABCC1. The combination of agomiRNA-133b with chemotherapeutic drugs in vivo inhibited tumor growth induced by CRC drug-resistant cells. A xenograft from the in vivo model resulted in up-regulated levels of miR-133b and down-regulated levels of ABCC1. Therefore, miR-133b enhances the chemosensitivity of CRC cells to anti-tumor drugs by directly down-regulating ABCC1. This discovery provides a therapeutic strategy in which miR-133b is used as a potential sensitizer for drug-resistant CRC.

Drug treatments for hepatocellular carcinoma (HCC) often fail because of multidrug resistance (MDR). The mechanisms of MDR are complex but cancer stem cells (CSCs), which are able to self-renew and differentiate, have recently been shown to be involved. The deubiquitinating enzyme ubiquitin-specific protease 22 (USP22) is a marker for CSCs. This study aimed to elucidate the role of USP22 in MDR of HCC and the underlying mechanisms. Using in vitro and in vivo assays, we found that modified USP22 levels were responsible for the altered drug-resistant phenotype of BEL7402 and BEL/FU cells. Downregulation of USP22 dramatically inhibited the expression of ABCC1 (encoding MRP1) but weakly influenced ABCB1 (encoding P-glycoprotein). Sirtuin 1 (SIRT1) was reported previously as a functional mediator of USP22 that could promote HCC cell proliferation and enhance resistance to chemotherapy. In this study, USP22 directly interacted with SIRT1 and positively regulated SIRT1 protein expression. Regulation of the expression of both USP22 and SIRT1 markedly affected the AKT pathway and MRP1 expression. Inhibition of the AKT pathway by its specific inhibitor LY294002 resulted in downregulation of MRP1. USP22 and MRP1 expression was detected in 168 clinical HCC samples by immunohistochemical staining, and a firm relationship between USP22 and MRP1 was identified. Together, these results indicate that USP22 could promote the MDR in HCC cells by activating the SIRT1/AKT/MRP1 pathway. USP22 might be a potential target, through which the MDR of HCC in clinical setting could be reversed.

We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transports NO as dinitrosyl-dithiol-iron complexes (DNICs) composed of iron, NO and glutathione (GSH). Hence, this gas with contrasting anti- and pro-tumor effects, which has been assumed to be freely diffusible, is a tightly-regulated species in M1-MØs. These control systems prevent NO cytotoxicity and may be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets.

Background Multidrug resistance (MDR) is one of the reasons for treatment failure in oral squamous cancer patients; however, the MDR mechanisms remain elusive. Methods Two human oral squamous cell carcinoma (OSCC) cell lines, CAL27 and SCC9, were analyzed by stepwise selection upon exposure to 5-fluorouracil (5-FU) and cisplatin (CDDP) for MDR cell line establishment, and cell viability was analyzed by CCK8 assays. Transcriptomes of the CAL27 and CAL27-MDR cells were analyzed by RNA-seq assay. The molecules in Sonic hedgehog (Shh) signal pathway and MDR related pathway were selected for validation by qRT-PCR and Western blot. Human OSCC tissues were applied for immunohistochemical and clinicopathological analysis. Results The OSCC-MDR cell lines with resistance to 5-FU and CDDP were successfully established. Protein expression levels of ATP-binding cassette (ABC) transporter ABCC1 and ABCG2 were increased 1.6-fold and 2.1-flod, respectively, in OSCC specimen from the patients with chemotherapy. The RNA-seq assay speculated the activation of Hedgehog (Hh) signaling with the upregulation of SHH, PTCH1, and SMO in OSCC-MDR cells as compared with OSCC cells. Furthermore, immunohistochemical studies confirmed that the high expression of SHH, PTCH1, and SMO in OSCC patients was significantly associated with chemotherapy. Overexpression of SHH increased the chemoresistance in OSCC cells, while the mRNA expression levels of PTCH1 and ABCG2 were increased in OSCC cells upon stimulation with recombinant SHH protein. Conclusions These results revealed that the activation of Hh signaling pathway regulating ABC transporters is associated MDR in OSCC.

Background Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. Methods The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. Results Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. Conclusions Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

To disclose how phosphorus deficiency influence phytoremediation of Cd contamination using poplars, root architecture, Cd absorption, Cd translocation and antioxidant defense in poplar roots were investigated using a clone of Populus × euramericana. Root growth was unaltered by Cd exposure regardless of P conditions, while the degree of root proliferation upon P deficiency was changed by high level of Cd exposure. The concentration and content of Cd accumulation in roots were increased by P deficiency. This can be partially explained by the increased expression of genes encoding PM H + -ATPase under the combined conditions of P deficiency and high Cd exposure, which enhanced Cd2+-H+ exchanges and led to an increment of Cd uptake under P deficiency. Despite of the increasing Cd accumulation in roots, the translocation of Cd from roots to aerial tissues sharply decreased upon P deficiency. The relative expression of genes responsible for Cd translocation (HMA4) decreased upon P deficiency and thus inhibited Cd translocation via xylem. GR activity was decreased by P deficiency, which can inhibit the form of GSH and GSH–Cd complexes and decrease Cd translocation via GSH–Cd complexes. The transportation of PC–Cd complexes into vacuole decreased under P deficiency as a result of the low expression of PCS and ABCC1, and thus suppressed Cd tolerance and Cd detoxification in roots. Moreover, P deficiency decreased the levels of antioxidase (GR and CAT) and phytohormones including JA, ABA and GA3, which synchronously reduced antioxidant capacity in roots.

Background 5-Fluorouracil (5-FU) is an antimetabolite that interferes with DNA synthesis and has been widely used as a chemotherapeutic drug in various types of cancers. However, the development of drug resistance greatly limits its application. Overexpression of ATP-binding cassette (ABC) transporters in many types of cancer is responsible for the reduction of the cellular uptake of various anticancer drugs causing multidrug resistance (MDR), the major obstacle in cancer chemotherapy. Recently, we have obtained a novel synthetic 5-FU analog, U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil], combining a uracil skeleton with an exo-cyclic methylidene group. U-332 was highly cytotoxic for HL-60 cells and showed similar cytotoxicity in the 5-FU resistant subclone (HL-60/5FU), in which this analog almost completely abolished expression of the ATP-binding cassette (ABC) transporter, multidrug resistance associate protein 1 (ABCC1). The expression of ABC transporters is usually correlated with NF-κB activation. The aim of this study was to determine the level of NF-κB subunits in the resistant HL-60/5-FU cells and to evaluate the potential of U-332 to inhibit activation of NF-κB family members in this cell line. Methods Anti-proliferative activity of compound U-332 was assessed by the MTT assay. In order to disclose the mechanism of U-332 cytotoxicity, quantitative real-time PCR analysis of the NF-κB family genes, c-Rel, RelA, RelB, NF-κB1, and NF-κB2, was investigated. The ability of U-332 to reduce the activity of NF-κB members was studied by ELISA test. Results In this report it was demonstrated, using RT-PCR and ELISA assay, that members of the NF-κB family c-Rel, RelA, RelB, NF-κB1, and NF-κB2 were all overexpressed in the 5-FU-resistant HL-60/5FU cells and that U-332 potently reduced the activity of c-Rel, RelA and NF-κB1 subunits in this cell line. Conclusions This finding indicates that c-Rel, RelA and NF-κB1 subunits are responsible for the resistance of HL-60/5FU cells to 5-FU and that U-332 is able to reverse this resistance. U-332 can be viewed as an important lead compound in the search for novel drug candidates that would not cause multidrug resistance in cancer cells.

The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). 77 cell lines displayed YM155 IC50s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However,TP53-mutant cells were not generally less sensitive to YM155 thanTP53wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.

Multidrug resistance–associated protein 1 (adenosine triphosphate–binding cassette subfamily C member 1 [ABCC1]) is abundantly expressed at the lung epithelial barrier, where it may influence the pulmonary disposition of inhaled drugs and contribute to variability in therapeutic response. The aim of this study was to assess the impact of ABCC1 on the pulmonary disposition of 6-bromo-7-11C-methylpurine (11C-BMP), a prodrug radiotracer that is intracellularly conjugated with glutathione to form the ABCC1 substrate S-(6-(7-11C-methylpurinyl))glutathione (11C-MPG). Methods: Groups of Abcc1(−/−) rats, wild-type rats pretreated with the ABCC1 inhibitor MK571, and wild-type control rats underwent dynamic PET scans after administration of 11C-BMP intravenously or by intratracheal aerosolization. In vitro transport experiments were performed with unlabeled BMP on the human distal lung epithelial cell line NCI-H441. Results: The pulmonary kinetics of radioactivity significantly differed between wild-type and Abcc1(−/−) rats, but differences were more pronounced after intratracheal than after intravenous administration. After intravenous administration, lung exposure (area under the lung time–activity curve from 0 to 80 min after radiotracer administration [AUClung]) was 77% higher and the elimination slope of radioactivity washout from the lungs (kE,lung) was 70% lower in Abcc1(−/−) rats, whereas after intratracheal administration, AUClung was 352% higher and kE,lung was 86% lower in Abcc1(−/−) rats. Pretreatment with MK571 decreased kE,lung by 20% after intratracheal radiotracer administration. Intracellular accumulation of MPG in NCI-H441 cells was significantly higher and extracellular efflux was lower in the presence than in the absence of MK571. Conclusion: PET with pulmonary administered 11C-BMP can measure ABCC1 activity at the lung epithelial barrier and may be applicable in humans to assess the effects of disease, genetic polymorphisms, or concomitant drug intake on pulmonary ABCC1 activity.

The resistance of tumors against anticancer drugs is a major impediment for chemotherapy. Tumors often develop multidrug resistance as a result of the cellular efflux of chemotherapeutic agents by ABC transporters such as P-glycoprotein (ABCB1/P-gp), Multidrug Resistance Protein 1 (ABCC1/MRP1), or Breast Cancer Resistance Protein (ABCG2/BCRP). By screening a chemolibrary comprising 140 compounds, we identified a set of naturally occurring aurones inducing higher cytotoxicity against P-gp-overexpressing multidrug-resistant (MDR) cells versus sensitive (parental, non-P-gp-overexpressing) cells. Follow-up studies conducted with the P-gp inhibitor tariquidar indicated that the MDR-selective toxicity of azaaurones is not mediated by P-gp. Azaaurone analogs possessing pronounced effects were then designed and synthesized. The knowledge gained from structure&ndash, activity relationships will pave the way for the design of a new class of anticancer drugs selectively targeting multidrug-resistant cancer cells.

Acute myeloid leukemia (AML) is one of the most common hematological neoplasia causing death worldwide. The long-term overall survival is unsatisfactory due to many factors including older age, genetic heterogeneity and molecular characteristics comprising additional mutations, and resistance to chemotherapeutic drugs. The expression of ABCB1/P-glycoprotein, ABCC1/MRP1, ABCG2/BCRP and LRP transporter proteins is considered the major reason for multidrug resistance (MDR) in AML, however conflicting data have been reported. Here, we review the main issues about drug transporter proteins in AML clinical scenario, and highlight the clinicopathological significance of MDR phenotype associated with ABCB1 polymorphisms and FLT3 mutation.

Background: Human ABCC3 (hABCC3) or multidrug resistance protein 3 (MRP3), third member of the subfamily C of the ABC proteins, is associated with multidrug resistance and treatment failure in acute leukemia. Hence, targeting this protein might be a useful approach to provide more effective drugs to overcome cancer drug resistance. Objectives: The present study aimed at predicting the possible ligand binding sites (PBSs) on hABCC3 and examining the possible binding of different chemotherapeutic drugs to this protein. Methods: To predict the PBSs on the hABCC3 transporter, a three-dimensional homology model of hABCC3 was generated in the current study based on bovine ABCC1 (bABCC1) using SWISS-MODEL and MODELLER programs, and then a molecular docking was qualified. Finally, binding affinities of 14 ligands including chemotherapeutic and immunosuppressive drugs, in addition to hABCC3 inhibitors, for hABCC3 were evaluated using binding free energies and the corresponding scores. Molecular docking was performed using AutoDock. Furthermore, Chimera, LigPlot, and Swiss PDB viewer (SPDBV) were used for interactive visualization and analysis of molecular structures. Results: The two PBSs on hABCC3 were predicted using the blind docking method. Docking results in both PBSs showed that vincristine, doxorubicin, and daunorubicin had the highest binding affinities, respectively, with vincristine having the highest docking score. Conclusions: In the current study, three drugs with the highest affinity for hABCC3 were introduced in order to take a step toward the possible hABCC3 targeting in drug-resistant acute leukemia. Furthermore, the application of in-silico methods in targeted cancer therapy, especially leukemia treatment, was highlighted.

Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Chemotherapy resistance is one of the most challenging problems for NSCLC and other cancer treatment. Recent study suggested that miRNAs are involved in therapeutic functions of chemotherapy during cancer treatment. Our present study established doxorubicin (Dox) resistant NSCLC A549 and H460 cells (named A549Dox/R and H460 Dox/R). We found that miR-199a-5p was significantly down regulated in Dox resistant cells. Over expression of miR-199a-5p can increase the Dox sensitivity of resistant cells. Among various targets of miR-199a-5p, chemoresistance can increase the expression of ABCC1 and HIF-1α. Gain and loss of function studies confirmed that both ABCC1 and HIF-1α were involved in the chemoresistance of NSCLC cells. Collectively, our data showed that miR-199a-5p regulated expression of ABCC1 and HIF-1α were involved in Dox resistance of NSCLC.

Methotrexate (MTX) is an efficient chemotherapeutic drug used in the treatment of several disorders in which there is a rapid cellular growth like cancer. MTX and folic acid have a chemically alike structure. MTX shows its chemotherapeutic effects by competing with folic acid in cancerous cells, which results in the reduction of folic acid in cells, and ultimately it causes cell death. But a drawback of MTX is that it causes different side effects by competing with folic acid in normal cells. The action of MTX on folate metabolism involves a complex pattern that includes several metabolizing enzymes and several transporters whose expression and/or function have been suggested to be changed by genetic polymorphisms. The main genes involved in showing important polymorphism are dihydrofolate reductase (DHFR), methotrexate polyglutamates (MTX-PG), thymidylate synthase (TS), solute carrier 19A1(SLC19A1), ATP-binding cassette C1 and two transporters (ABCC1, ABCC2), breast cancer resistance protein (BCRP), methylenetetrahydrofolate reductase (MTHFR), and many others too.

Despite considerable efforts in reversing clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) based on small molecule inhibitors has been hindered. This may be due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mechanisms that underlie this successful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, we report the development of two erythroleukemia variants, RVC and RDC, which were derived by step-wise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC, which retained P-gp expression, showed altered MDR phenotypes that were resistant to cyclosporine modulation. The ABCB1 coding regions were genetically stable even under long-term stringent drug selection. Genomically, ABCB1 is likely the most stable ABC transporter gene when comparing with several ABC superfamily members (such as ABCA1, ABCC1, CFTR, and ABCG2). Our findings suggested that non-P-gp mechanisms were likely responsible for the resistance to CsA modulation in both RVC and RDC cells. Moreover, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low level and unstable drug resistance, thus shedding some light on the benefits of CsA in treating certain types of AML patients.

Brivanib, a promising tyrosine kinase inhibitor, is currently undergoing advanced stages of clinical evaluation for solid tumor therapy. In this work, we investigated possible interactions of this novel drug candidate with ABC drug efflux transporters and cytochrome P450 (CYP450) drug-metabolizing enzymes that participate in cancer multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). First, in accumulation experiments with various model substrates, we identified brivanib as an inhibitor of the ABCB1, ABCG2, and ABCC1 transporters. However, in subsequent combination studies employing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide proliferation assays in both Madin-Darby canine kidney II (MDCKII) and A431 cellular models, only ABCG2 inhibition was revealed to be able to synergistically potentiate mitoxantrone effects. Advantageous to its possible use as MDR antagonist, brivanib's chemosensitizing properties were not impaired by activity of any of the MDR-associated ABC transporters, as observed in comparative viability assay in the MDCKII cell sublines. In incubation experiments with eight recombinant CYP450s, we found that brivanib potently inhibited CYP2C subfamily members and the CYP2B6 isoform. Finally, in induction studies, we demonstrated that brivanib upregulated ABCB1 and CYP1A2 messenger RNA levels in systemic cell models, although this interaction was not significantly manifested at a functional level. In conclusion, brivanib exhibits potential to cause clinically relevant pharmacokinetic DDIs and act as a modulator of ABCG2-mediated MDR. Our findings might be used as an important background for subsequent in vivo investigations and pave the way for the safe and effective use of brivanib in oncological patients.

Drug resistance is a big problem in cancer treatment and one of the most prominent mechanisms underlain is overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1, ABCC1 and ABCG2. Inhibition of ABC transporters is an important approach to overcome drug resistance. The inositol-requiring enzyme 1α (IRE1α), an arm of unfolded protein response (UPR), splices XBP1 mRNA to generate an active transcription factor XBP1s. UPR is implicated in drug resistance. However, the underlying mechanism is unclear. We found that the anticancer drugs such as 5-fluorouracil (5-FU) activated the IRE1α-XBP1 pathway to induce the expression of ABCB1, ABCC1 and ABCG2 in colon cancer cells. Inhibition of IRE1α RNase activity with small molecule 4μ8c suppressed the drug-induced expression of these ABC transporters and sensitized 5-FU-resistant colon cancer cells to drug treatment. In vivo xenograft assay indicates that administration of 4μ8C substantially enhanced the efficacy of 5-FU chemotherapy on 5-FU-resistant colon cancer cells. These results suggest that IRE1α-targeting might be a strategy to cope with drug resistance of colon cancer.

SLC5A1, CCNA1, and ABCC1 have been extensively studied as candidate genes because of their great influence on the reproductive traits of animals. However, little is known about the association between polymorphisms of the SLC5A1, CCNA1, and ABCC1 genes and litter size in Small-Tail Han sheep. In this study, the expression levels of SLC5A1, CCNA1, and ABCC1 in HPG (hypothalamic&ndash, pituitary&ndash, gonadal) axis tissues of polytocous and monotocous Small-Tail Han sheep were analyzed by qPCR. To better understand the effects of four single nucleotide polymorphisms (SNPs) comprising of g.70067210 T &gt, C in SLC5A1, g.25350431 C &gt, T and g.25360220 T &gt, C in CCNA1, and g.14413132 C &gt, T in ABCC1, a population genetic analysis was conducted using data obtained from genotyping in 728 sheep from seven breeds. The results indicated that all genes included in this study were differentially expressed in the pituitary and uterus of polytocous and monotocous Small-Tail Han sheep (p &lt, 0.05). The associations of these four SNPs and the FecB mutation with litter size in 384 Small-Tail Han sheep were analyzed, therefore, and it was found that both g.70067210T &gt, C and the FecB mutation were significantly associated with litter size (p &lt, 0.05). The linear regression analysis of the association of multiple markers (FecB and g.70067210 T &gt, C in SCL5A1) with litter size indicated that homozygous ewes carrying the BB/TT genotype had larger litter size than any ewes with any other genotype. In conclusion, the SLC5A1 SNPs significantly affect litter size in sheep and are useful as genetic marker for litter size.

Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum multidrug resistance and can decrease the bioavailability of many important drugs. Substrates of MRP1 include anti-cancer agents, antibiotics, antivirals, antidepressants and anti-inflammatory drugs. Using calcein as a fluorescent reporter in a high content uptake assay, we recently reported the identification of 12 MRP1 inhibitors after screening an anti-cancer library of 386 compounds. Here, we describe the development of a new high content imaging-based uptake assay using doxorubicin as a fluorescent reporter. Screening the same anti-cancer library of 386 compounds, the new assay identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. Inhibition of MRP1 activity was confirmed using flow cytometry and confocal microscopy-based transport assays. Six drugs (afatinib, celecoxib, doramapimod, mifepristone, MK-2206 and rosiglitazone) were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against vincristine, doxorubicin and etoposide. Mifepristone and doramapimod were most effective in reversal of resistance against vincristine while mifepristone and rosiglitazone were most successful in resensitizing H69AR cells against doxorubicin. Furthermore, resistance towards etoposide was completely reversed in the presence of celecoxib or doramapimod. Selected drugs were also evaluated for resistance reversal in HEK cells that overexpress P-glycoprotein or breast cancer resistance protein. Our results indicate mifepristone and doramapimod as pan inhibitors of these three drug transporters while celecoxib exhibited selective MRP1 inhibition. Together, our findings signify the importance of MRP1 in drug discovery and demonstrate the effectiveness and value of doxorubicin-based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies. On the other hand, anti-cancer drugs that did not interact with MRP1 carry a low risk for developing MRP1-mediated resistance.

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR‐246 (PRIMA‐1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR‐246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR‐246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione‐conjugated MQ (GS‐MQ). Due to the reversibility of MQ conjugation, GS‐MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53‐targeted cancer therapy., Tumor suppressor TP53 is mutated in a large fraction of tumors. APR‐246/Eprenetapopt is the most clinically advanced mutant p53‐targeting drug candidate (Phase III). Besides restoring wild type p53 activity, the active product MQ also disrupts the redox balance, resulting in cancer cell death.

Simple Summary Despite tremendous advances in cancer treatment, chemotherapy remains the first-line choice in many tumor types. The action of numerous chemotherapy drugs is limited by the occurrence of ABC proteins in cancer cell membranes, which remove medicines from cell compartments. In this paper, we show that one of bromodomain inhibitors, namely I-CBP112, was capable of repressing genes that are responsible for multidrug resistance in all three studied cancer cell lines. CBP/p300 bromodomain inhibitor allows for the higher drug accumulation inside cells and considerably potentiated drug effects. At the molecular level, I-CBP112 caused rearrangement of chromatin at the ABC gene promoters by inducing recruitment of LSD1, which removes transcription-promoting histone marks. I-CBP112 emerges as a promising compound to overcome ABC-dependent cancer drug resistance. Abstract The high expression of some ATP-binding cassette (ABC) transporters is linked to multidrug resistance in cancer cells. We aimed to determine if I-CBP112, which is a CBP/p300 bromodomain inhibitor, altered the vulnerability of the MDA-MB-231 cell line to chemotherapy drugs, which are used in neoadjuvant therapy in patients with triple negative breast cancer (TNBC). MDA-MB-231 cells represent TNBC, which is negative for the expression of estrogen and progesterone receptors and HER2 protein. An I-CBP112-induced decrease in the expression of all the studied ABCs in the breast, but also in the lung (A549), and hepatic (HepG2) cancer cell lines was associated with increased accumulation of doxorubicin, daunorubicin, and methotrexate inside the cells as well as with considerable cell sensitization to a wide range of chemotherapeutics. Gene promoters repressed by I-CBP112 in MDA-MB-231 cells, such as ABCC1 and ABCC10, were characterized by enhanced nucleosome acetylation and, simultaneously, by considerably lower trimethylation in the transcription-promoting form of H3K4me3. The CBP/p300 bromodomain inhibitor induced the recruitment of LSD1 to the gene promoters. The inhibition of this demethylase in the presence of I-CBP112 prevented the repression of ABCC1 and ABCC10 and, to a considerable extent, cancer cells’ sensitization to drugs. In conclusion, the CBP/p300 bromodomain inhibitor I-CBP112 can be considered as a potent anti-multidrug-resistance agent, capable of repressing key ABC transporters responsible for drug efflux in various cancer types.

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly392, Tyr404, Arg405), the perpendicular coupling helix (Asn412, Arg415, Lys416), and the ascending α-helix (Glu422, Phe434) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu422 and Arg615 could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.

α-aryl-α-tetralones and α-fluoro-α-aryl-α-tetralones derivatives were synthesized by palladium catalyzed α-arylation reaction of α-tetralones and α-fluoro-α-tetralones, with bromoarenes in moderate to good yields. These compounds were evaluated for their in vitro anti-proliferative effects against human breast cancer and leukemia cell lines with diverse profiles of drug resistance. The most promising compounds, 3b, 3c, 8a and 8c, were effective on both neoplastic models. 3b and 8a induced higher toxicity on multidrug resistant cells and were able to avoid efflux by ABCB1 and ABCC1 transporters. Theoretical calculations of the physicochemical descriptors to predict ADMETox properties were favorable concerning Lipinski’s rule of five, results that reflected on the low effects on non-tumor cells. Therefore, these compounds showed great potential for development of pharmaceutical agents against therapy refractory cancers.

Background and Aim: Hairy enhancer of split-1 (HES1) is a downstream transcriptional factor of Notch signaling pathway, which was found to be related to chemoresistance. This study was aimed to investigate the role of HES1 in chemoresistance of colorectal cancer (CRC). Methods: Tissue microarray was used to analyze the clinical significance of HES1 in radical resected (R0) stage II/III CRC patients that received adjuvant chemotherapy. 5-fluorouracil (5-Fu) chemoresistance was examined in CRC cell lines (RKO and HCT8, LOVO) with stable over-expression and inhibition of HES1 gene by cytotoxicity test. Gene expression microarray was used to investigate the enriched pathways and different expressed of genes in cells with over-expressed HES1. Expression changes of the chemoresistance related genes were confirmed by qPCR and western blot analysis. Results: Stage II CRC patients with higher HES1 expression showed higher recurrence rate after chemotherapy. Colon cancer cell lines which over-expressed HES1 were more resistant to 5-Fu treatment in vitro. Gene expression microarray revealed that HES1 was related to the signaling pathways of epithelial-mesenchymal transition (EMT) and drug metabolism. Immunofluorescence assay showed HES1 over-expression lead to depressed E-cadherin and elevated N-cadherin. QPCR and western blot analysis confirmed that ABCC1, ABCC2 and P-gp1 were induced after HES1 over-expression. Conclusions: HES1 promotes chemoresistance to 5-Fu by prompting EMT and inducing of several ABC transporter genes. HES1 might be a novel therapeutic target in CRC treatment.

Multidrug resistance (MDR) in cancer cells is the development of resistance to a variety of structurally and functionally nonrelated anticancer drugs. This phenomenon has become a major obstacle to cancer chemotherapy seriously affecting the clinical outcome. MDR is associated with increased drug efflux from cells mediated by an energy-dependent mechanism involving the ATP-binding cassette (ABC) transporters, mainly P-glycoprotein (ABCB1), the MDR-associated protein-1 (ABCC1), and the breast cancer resistance protein (ABCG2). The first two transporters have been widely studied already and reviews summarized the results. The ABCG2 protein has been a subject of intense study since its discovery as its overexpression has been detected in resistant cell lines in numerous types of human cancers. To date, a long list of modulators of ABCG2 exists and continues to increase. However, little is known about the clinical consequences of ABCG2 modulation. This makes the design of novel, potent, and nontoxic inhibitors of this efflux protein a major challenge to reverse MDR and thereby increase the success of chemotherapy. The aim of the present review is to describe and highlight specific and nonspecific modulators of ABCG2 reported to date based on the selectivity of the compounds, as many of them are effective against one or more ABC transport proteins.

Neutropenia is a common dose-limiting toxicity associated with irinotecan treatment. Although UGT1A1 variants have been associated with neutropenia, a fraction of neutropenia risk remains unaccounted for. To identify additional genetic markers contributing to variability in irinotecan pharmacokinetics and neutropenia, a regression analysis was performed in 78 irinotecan-treated patients to analyze comprehensively three hepatic efflux transporter genes (ABCB1, ABCC1 and ABCG2). rs6498588 (ABCC1) and rs12720066 (ABCB1) were associated with increased SN-38 exposure, and rs17501331 (ABCC1) and rs12720066 were associated with lower absolute neutrophil count nadir. rs6498588 and a variant in high linkage disequilibrium are located in transcriptionally active regions or are predicted to alter transcription factor binding sites. While enhancer activity was not evident in vitro for genomic regions containing these single-nucleotide polymorphisms, rs6498588 was significantly associated with ABCC1 expression in human liver. These results suggest that genetic variation in ABCC1 and ABCB1 may contribute to irinotecan-induced neutropenia by altering expression of transporters involved in irinotecan metabolite disposition.

// Melanie Spitzwieser 1 , Christine Pirker 2 , Bettina Koblmuller 2 , Georg Pfeiler 3 , Stefan Hacker 4 , Walter Berger 2 , Petra Heffeter 2 , Margit Cichna-Markl 1 1 Department of Analytical Chemistry, University of Vienna, Vienna, Austria 2 Department of Medicine I, Institute of Cancer Research and Comprehensive Cancer Center of the Medical University, Medical University of Vienna, Vienna, Austria 3 Department of Obstetrics and Gynecology, Division of Gynecology and Gynecological Oncology, Medical University of Vienna, Vienna, Austria 4 Department of Plastic and Reconstructive Surgery, Medical University of Vienna, Vienna, Austria Correspondence to: Margit Cichna-Markl, email: margit.cichna@univie.ac.at Keywords: ABC transporter, multidrug resistance, DNA methylation, bisulfite pyrosequencing, breast cancer Received: June 29, 2016 Accepted: September 19, 2016 Published: September 28, 2016 ABSTRACT Overexpression of ABCB1, ABCC1 and ABCG2 in tumor tissues is considered a major cause of limited efficacy of anticancer drugs. Gene expression of ABC transporters is regulated by multiple mechanisms, including changes in the DNA methylation status. Most of the studies published so far only report promoter methylation levels for either ABCB1 or ABCG2 , and data on the methylation status for ABCC1 are scarce. Thus, we determined the promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in 19 human cancer cell lines. In order to contribute to the elucidation of the role of DNA methylation changes in acquisition of a multidrug resistant (MDR) phenotype, we also analyzed the promoter methylation patterns in drug-resistant sublines of the cancer cell lines GLC-4, SW1573, KB-3-1 and HL-60. In addition, we investigated if aberrant promoter methylation levels of ABCB1, ABCC1 and ABCG2 occur in tumor and tumor-surrounding tissues from breast cancer patients. Our data indicates that hypomethylation of the ABCC1 promoter is not cancer type-specific but occurs in cancer cell lines of different origins. Promoter methylation was found to be an important mechanism in gene regulation of ABCB1 in parental cancer cell lines and their drug-resistant sublines. Overexpression of ABCC1 in MDR cell models turned out to be mediated by gene amplification, not by changes in the promoter methylation status of ABCC1 . In contrast to the promoters of ABCC1 and ABCG2 , the promoter of ABCB1 was significantly higher methylated in tumor tissues than in tumor-adjacent and tumor-distant tissues from breast cancer patients.

Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. Fil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. University of Heidelberg; Alemania Fil: Tocchetti, Guillermo Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Arana, Maite Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Catania, Viviana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Theile, Dirk. University of Heidelberg; Alemania Fil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Weiss, Johanna. University of Heidelberg; Alemania

Chemotherapeutic treatment of cancer often fails due to overexpression of the ATP-binding cassette (ABC) transport proteins, like ABCG2, triggering active efflux of various structurally unrelated drugs. This so-called multidrug resistance (MDR) may be reversed by selective, potent, and nontoxic inhibitors of ABCG2. As only a few potent inhibitors are known, new compounds based on a 4-substituted-2-phenylquinazoline scaffold were investigated. Substitution with hydroxy, cyano, nitro, acetamido, and fluoro led to high inhibitory activities toward ABCG2. The ability to reverse MDR of the most active compounds was confirmed in a MTT efficacy assay. Moreover, a negligibly low intrinsic cytotoxicity was found resulting in a high therapeutic ratio. Investigations of the inhibitory activity toward ABCB1 and ABCC1 yielded a high selectivity toward ABCG2 for the quinazoline compounds. Quinoline-based analogues showed lower inhibitory activity and selectivity. The study yielded a variety of promising compounds, some with superior properties compared to those of the standard inhibitor Ko143.

The front-line treatment for adult acute myeloid leukemia (AML) is anthracycline-based combination chemotherapy. However, treatment outcomes remain suboptimal with relapses frequently observed. Among the mechanisms of treatment failure is multidrug resistance (MDR) mediated by the ABCB1, ABCC1, and ABCG2 drug-efflux transporters. Although genetic and phenotypic heterogeneity between leukemic blast cells is a well-recognized phenomenon, there remains minimal data on differences in MDR activity at the individual cell level. Specifically, functional assays that can distinguish the variability in MDR activity between individual leukemic blasts are lacking. Here, we outline a new dielectrophoretic (DEP) chip-based assay. This assay permits measurement of drug accumulation in single cells, termed same-single-cell analysis in the accumulation mode (SASCA-A). Initially, the assay was optimized in pretherapy samples from 20 adults with AML whose leukemic blasts had MDR activity against the anthracyline daunorubicin (DNR) tested using multiple MDR inhibitors. Parameters tested were initial drug accumulation, time to achieve signal saturation, fold-increase of DNR accumulation with MDR inhibition, ease of cell trapping, and ease of maintaining the trapped cells stationary. This enabled categorization into leukemic blast cells with MDR activity (MDR(+)) and leukemic blast cells without MDR activity (MDR(-ve)). Leukemic blasts could also be distinguished from benign white blood cells (notably these also lacked MDR activity). MDR(-ve) blasts were observed to be enriched in samples taken from patients who went on to enter complete remission (CR), whereas MDR(+) blasts were frequently observed in patients who failed to achieve CR following front-line chemotherapy. However, pronounced variability in functional MDR activity between leukemic blasts was observed, with MDR(+) cells not infrequently seen in some patients that went on to achieve CR. Next, we tested MDR activity in two paired AML patient samples. Pretherapy samples taken from patients that achieved CR to front-line chemotherapy were compared with samples taken at time of subsequent relapse. MDR(+) cells were frequently observed in leukemic blast cells in both pretherapy and relapsed samples, consistent with MDR as a mechanism of relapse in these patients. We demonstrate the ability of a new DEP microfluidic chip-based assay to identify heterogeneity in MDR activity in leukemic blasts. The test provides a platform for future studies to characterize the mechanistic basis for heterogeneity in MDR activity at the individual cell level.

GA-13315 is a gibberellin derivative that reveals antitumor and antineoplastic effects both in vitro and in vivo. In the present study, the chemosensitizing effects of GA-13315 in multidrug-resistant cell lines were examined and the underlying mechanisms were investigated. Cytotoxicity and chemosensitizing effects of GA-13315 were determined by MTT assay. Function of ABC transporter was analyzed by measuring intracellular drug accumulation of doxorubicin and rhodamine 123 and by determining the ATPase activity of ABC transporter. Expression levels of apoptosis regulators were analyzed using real-time quantitative PCR and Western blot. GA-13315 selectively killed MCF-7/adr cells that overexpress P-glycoprotein (ABCB1) over the parent MCF-7 cells. In combination with conventional chemotherapeutic agents, GA-13315 at sub-toxic concentrations reversed the multidrug resistance mediated by ABCB1 but exacerbated the resistance conferred by multidrug resistance-associated protein 1 (ABCC1). GA-13315 increased intracellular accumulation of doxorubicin and rhodamine 123 in MCF-7/adr cells and in ABCB1-transfected HEK293 cells but facilitated drug flush-out from cells that overexpress ABCC1. GA-13315 inhibited the ATPase activity of ABCB1 while stimulated that of ABCC1. Moreover, the downregulated expression of Bax in MCF-7/adr cells was restored by GA-13315 markedly. These data suggest that GA-13315 sensitizes multidrug-resistant cells at least partially by impeding the efflux function of ABCB1. The upregulation of Bax by GA-13315 may also contribute to the sensitizing action. The opposite effects of GA-13315 on different ATP-binding cassette transporters and their implications in overcoming drug resistance require further investigation.

The ATP-binding cassette (ABC) transporters are important transmembrane proteins encoded by a supergene family. The majority of ABC proteins are primary active transporters that bind and hydrolyze ATP to mediate the efflux of a diverse range of substrates across lipid membranes. In this study, we cloned and characterized a putative multidrug resistance associated protein 1 (MRP1) from Rhopalosiphum padi encoded by ABCC1. Structural analysis showed that this protein has structural features typical of the ABC transporter family. Phylogenetic analysis indicated that the amino acid sequence was highly similar that of the corresponding protein from Acyrthosiphon pisum. Real-time quantitative polymerase chain reaction (PCR) analysis showed that ABCC1 was expressed throughout all R. padi developmental stages, with the highest level of expression in the fourth larval instar. We also examined ABCC1 expression in four different tissue types and found that it was most highly expressed in the midgut. Exposing R. padi to imidacloprid and chlorpyrifos increased ABCC1 expression. Furthermore, ABCC1 expression was higher in the imidacloprid-resistant (IR) and chlorpyrifos-resistant (CR) strains than in an insecticide-susceptible strain (SS) of R. padi. Exposing R. padi to verapamil in combination with insecticides significantly increased the toxicity of the insecticides. The respective synergy factor of CR and IR R. padi strain was 1.33 and 1.26, which was lower than that (2.72 and 1.64, respectively) of the SS. Our results clarify the biological function of ABCC1 in R. padi, particularly its role in insecticide resistance, and suggest novel strategies for pest management that use ABC transporter inhibitors to increase the effectiveness of insecticides.

Administration of drugs via oral route is the most common and convenient. Three major factors can affect drug absorption from this route: physiologic, physicochemical and formulation. Drug absorption mainly takes place in small intestine after oral administration. Within the small intestine, there are 2 major families of proteins involved in transport that have significant roles on drugs absorption: Solute-carrier and adenosine triphosphate-binding cassette (ABC) superfamilies. There are 48 ABC transporters in humans as well as seven subfamilies: ABCA to ABCG. ABCB1, ABCC1/2, and ABCG2 are widely described for their impacts in the absorption of drugs. Polymorphism on ABC transporters can significantly affect the absorption of drugs from the gastro intestinal (GI) tract. Some polymorphisms can result in over expression of the transporter in the small intestine and hence reduce absorption of drugs and some result in reduction in expression and increase in absorption. These polymorphisms are an important cause of adverse drug reactions and therapeutic failures. Key words: Polymorphism, adenosine triphosphate binding cassette, oral drug absorption, expression, ABC transporters.

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds.