ATP-binding Cassette Drug Transporters Research Articles

Inhibition of Human Drug Transporter Activities by the Pyrethroid Pesticides Allethrin and Tetramethrin.

Pyrethroids are widely-used chemical insecticides, to which humans are commonly exposed, and known to alter functional expression of drug metabolizing enzymes. Limited data have additionally suggested that drug transporters, that constitute key-actors of the drug detoxification system, may also be targeted by pyrethroids. The present study was therefore designed to analyze the potential regulatory effects of these pesticides towards activities of main ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, using transporter-overexpressing cells. The pyrethroids allethrin and tetramethrin were found to inhibit various ABC and SLC drug transporters, including multidrug resistance-associated protein (MRP) 2, breast cancer resistance protein (BCRP), organic anion transporter polypeptide (OATP) 1B1, organic anion transporter (OAT) 3, multidrug and toxin extrusion transporter (MATE) 1, organic cation transporter (OCT) 1 and OCT2, with IC50 values however ranging from 2.6 μM (OCT1 inhibition by allethrin) to 77.6 μM (OAT3 inhibition by tetramethrin) and thus much higher than pyrethroid concentrations (in the nM range) reached in environmentally pyrethroid-exposed humans. By contrast, allethrin and tetramethrin cis-stimulated OATP2B1 activity and failed to alter activities of OATP1B3, OAT1 and MATE2-K, whereas P-glycoprotein activity was additionally moderately inhibited. Twelve other pyrethoids used at 100 μM did not block activities of the various investigated transporters, or only moderately inhibited some of them (inhibition by less than 50%). In silico analysis of structure-activity relationships next revealed that molecular parameters, including molecular weight and lipophilicity, are associated with transporter inhibition by allethrin/tetramethrin and successfully predicted transporter inhibition by the pyrethroids imiprothrin and prallethrin. Taken together, these data fully demonstrated that two pyrethoids, i.e., allethrin and tetramethrin, can act as regulators of the activity of various ABC and SLC drug transporters, but only when used at high and non-relevant concentrations, making unlikely any contribution of these transporter activity alterations to pyrethroid toxicity in environmentally exposed humans.

Inhibition of SLC drug transporter activities by environmental bisphenols

The plastic component bisphenol A (BPA) is suspected to exert deleterious effects towards human health and targets various cellular and molecular pathways, including activity of ATP-binding cassette drug transporters. The present study was designed to determine whether BPA and some derivatives, like its substitutes bisphenol F (BPF) and bisphenol S (BPS) and the flame retardant tetrabromobisphenol A (TBBPA), may additionally interact with solute carrier (SLC) drug transporters. Activities of the various following SLC transporters were inhibited in a major way (by >60%) by 100μM bisphenols: OCT1 and MATE1 (by BPA and TBBPA), OATP1B1 (by BPA, BPF and TBBPA), OATP1B3 and NTCP (by TBBPA) and OAT3 (by BPA, BPF, BPS and TBBPA); by contrast, activities of other transporters were not impacted (MATE2-K) or were stimulated (notably OCT1 by BPS and OCT2 by BPF). Transporter inhibitions due to bisphenols were concentrations-dependent, with half maximal inhibitory concentrations (IC50) ranging from 0.5μM to 73.5μM. BPA was finally shown to be not transported by OAT3, although inhibiting this transporter in a competitive manner. Taken together, these data indicate that bisphenols interact with SLC transporters, at concentration levels however rather higher than those occurring in humans in response to environmental exposure.

Hernandezine, a Bisbenzylisoquinoline Alkaloid with Selective Inhibitory Activity against Multidrug-Resistance-Linked ATP-Binding Cassette Drug Transporter ABCB1.

The overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (P-glycoprotein, MDR1) is the most studied mechanism of multidrug resistance (MDR), which remains a major obstacle in clinical cancer chemotherapy. Consequently, resensitizing MDR cancer cells by inhibiting the efflux function of ABCB1 has been considered as a potential strategy to overcome ABCB1-mediated MDR in cancer patients. However, the task of developing a suitable modulator of ABCB1 has been hindered mostly by the lack of selectivity and high intrinsic toxicity of candidate compounds. Considering the wide range of diversity and relatively nontoxic nature of natural products, developing a potential modulator of ABCB1 from natural sources is particularly valuable. Through screening of a large collection of purified bioactive natural products, hernandezine was identified as a potent and selective reversing agent for ABCB1-mediated MDR in cancer cells. Experimental data demonstrated that the bisbenzylisoquinoline alkaloid hernandezine is selective for ABCB1, effectively inhibits the transport function of ABCB1, and enhances drug-induced apoptosis in cancer cells. More importantly, hernandezine significantly resensitizes ABCB1-overexpressing cancer cells to multiple chemotherapeutic drugs at nontoxic, nanomolar concentrations. Collectively, these findings reveal that hernandezine has great potential to be further developed into a novel reversal agent for combination therapy in MDR cancer patients.

Molecular Properties of Drugs Interacting with SLC22 Transporters OAT1, OAT3, OCT1, and OCT2: A Machine-Learning Approach.

Statistical analysis was performed on physicochemical descriptors of ∼250 drugs known to interact with one or more SLC22 "drug" transporters (i.e., SLC22A6 or OAT1, SLC22A8 or OAT3, SLC22A1 or OCT1, and SLC22A2 or OCT2), followed by application of machine-learning methods and wet laboratory testing of novel predictions. In addition to molecular charge, organic anion transporters (OATs) were found to prefer interacting with planar structures, whereas organic cation transporters (OCTs) interact with more three-dimensional structures (i.e., greater SP3 character). Moreover, compared with OAT1 ligands, OAT3 ligands possess more acyclic tetravalent bonds and have a more zwitterionic/cationic character. In contrast, OCT1 and OCT2 ligands were not clearly distinquishable form one another by the methods employed. Multiple pharmacophore models were generated on the basis of the drugs and, consistent with the machine-learning analyses, one unique pharmacophore created from ligands of OAT3 possessed cationic properties similar to OCT ligands; this was confirmed by quantitative atomic property field analysis. Virtual screening with this pharmacophore, followed by transport assays, identified several cationic drugs that selectively interact with OAT3 but not OAT1. Although the present analysis may be somewhat limited by the need to rely largely on inhibition data for modeling, wet laboratory/in vitro transport studies, as well as analysis of drug/metabolite handling in Oat and Oct knockout animals, support the general validity of the approach-which can also be applied to other SLC and ATP binding cassette drug transporters. This may make it possible to predict the molecular properties of a drug or metabolite necessary for interaction with the transporter(s), thereby enabling better prediction of drug-drug interactions and drug-metabolite interactions. Furthermore, understanding the overlapping specificities of OATs and OCTs in the context of dynamic transporter tissue expression patterns should help predict net flux in a particular tissue of anionic, cationic, and zwitterionic molecules in normal and pathophysiological states.

Osimertinib (AZD9291) Attenuates the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCB1 in Vitro.

The effectiveness of cancer chemotherapy is often circumvented by multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in human cancer cells. Furthermore, some TKIs are transported by ABCB1, which results in low oral bioavailability, reduced distribution, and the development of acquired resistance to these TKIs. In this study, we investigated the interaction between ABCB1 and osimertinib, a novel selective, irreversible third-generation EGFR TKI that has recently been approved by the U.S. Food and Drug Administration. We also evaluated the potential impact of ABCB1 on the efficacy of osimertinib in cancer cells, which can present a therapeutic challenge to clinicians in the future. We revealed that although osimertinib stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer resistance to osimertinib. Our results suggest that it is unlikely that the overexpression of ABCB1 can be a major contributor to the development of osimertinib resistance in cancer patients. More significantly, we revealed an additional action of osimertinib that directly inhibits the function of ABCB1 without affecting the expression level of ABCB1, enhances drug-induced apoptosis, and reverses the MDR phenotype in ABCB1-overexpressing cancer cells. Considering that osimertinib is a clinically approved third-generation EGFR TKI, our findings suggest that a combination therapy with osimertinib and conventional anticancer drugs may be beneficial to patients with MDR tumors.

Cardiac fibroblast growth factor‐16 increases efflux drug (ABC) transporter production and resistance to doxorubicin‐induced cardiomyocyte death

Doxorubicin (DOX) is a cytotoxic anthracycline antibiotic that is widely used in cancer treatment. However, its cardiotoxic side effects can be fatal, and have long been an issue for cancer patients both in the short and longer term. Strategies are needed to protect the heart. Fibroblast growth factor (FGF) 16 plays an important role in embryonic mouse heart development. After birth, FGF‐16 is predominantly expressed in the myocardium, and is linked to both anti‐fibrotic and anti‐hypertrophic activities. Supplementation with FGF‐16 increases resistance to DOX‐induced loss of ventricular function in an isolated mouse heart model. However, there is no report on the effects of DOX on endogenous FGF‐16 levels or the mechanism(s) for cardioprotection by FGF‐16. ATP‐binding cassette (ABC) transporters are a family of ATP‐dependent membrane proteins that play an important role in drug distribution and excretion. ABC transporters work as pumps effluxing drugs out of the cell immediately after drug entry thereby reducing cellular drug concentration. Drug concentration positively determines the ABC transporter levels. Previously we showed that FGF‐16 can compete with FGF‐2 binding to the same FGF receptors (FGFRs). Unlike FGF‐16, FGF‐2 is not cardiac‐specific but is cardioprotective and known to induce p‐glycoprotein (MDRs) and multidrug resistance‐associated protein (MRPs) expression in neonatal rat cardiomyocytes. Our hypothesis is that FGF‐16 protects cardiomyocytes from DOX‐induced cell death by increasing the levels of ABC drug transporters. Our results show that 1 μM DOX induces a time‐dependent decrease in endogenous FGF‐16 RNA levels with a rapid 80% reduction by 2 hours in neonatal rat cardiomyocytes. Knockdown of endogenous FGF‐16 levels using siRNA results in a significant increase in apoptotic (Annexin‐V positive) and necrotic (LDH release) cells; this is consistent with endogenous FGF‐16 serving as a cardiac maintenance/survival factor. Overexpression of FGF‐16 using adenoviral delivery significantly and specifically increases efflux drug transporter Mdr1a levels in a dose‐dependent manner. Significant decrease in calcein fluorescence intensity in a functional analysis using calcein AM as a substrate for the efflux drug transporters was consistent with the increased levels of drug transporters RNA post FGF‐16 overexpression. In addition, FGF‐16 adenovirus overexpression in cardiomyocytes treated with 1μM DOX significantly decreased the intracellular DOX concentration as measured by DOX autofluorescence. This was associated with a significant reduction in the apoptotic and necrotic cell population, consistent with a decrease in DOX cytotoxicity. Furthermore, inhibition of FGFR signaling using 20 μM SU5402 blocked the increased efflux effect of the ABC transporters. In conclusion, FGF‐16 increases resistance to DOX‐induced injury by increasing efflux drug transporters via binding to the FGFRs. Understanding the effect of FGF‐16 on drug transporter production and function may offer a further target for intervention in the context of DOX and cardiotoxicity.Support or Funding InformationFunded by the Canadian Institutes of Health Research and Research Manitoba.

Human ATP-Binding Cassette Transporter ABCG2 Confers Resistance to CUDC-907, a Dual Inhibitor of Histone Deacetylase and Phosphatidylinositol 3-Kinase.

CUDC-907 is a novel, dual-acting small molecule compound designed to simultaneously inhibit the activity of histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K). Treatment with CUDC-907 led to sustained inhibition of HDAC and PI3K activity, inhibition of RAF-MEK-MAPK signaling pathway, and inhibition of cancer cell growth. CUDC-907 is currently under evaluation in phase I clinical trials in patients with lymphoma or multiple myeloma, and in patients with advanced solid tumors. However, the risk of developing acquired resistance to CUDC-907 can present a significant therapeutic challenge to clinicians in the future and should be investigated. The overexpression of ATP-binding cassette (ABC) drug transporter ABCB1, ABCC1, or ABCG2 is one of the most common mechanisms of developing multidrug resistance (MDR) in cancers and a major obstacle in chemotherapy. In this study, we reveal that ABCG2 reduces the intracellular accumulation of CUDC-907 and confers significant resistance to CUDC-907, which leads to reduced activity of CUDC-907 to inhibit HDAC and PI3K in human cancer cells. Moreover, although CUDC-907 affects the transport function of ABCG2, it was not potent enough to reverse drug resistance mediated by ABCG2 or affect the expression level of ABCG2 in human cancer cells. Taken together, our findings indicate that ABCG2-mediated CUDC-907 resistance can have serious clinical implications and should be further investigated. More importantly, we demonstrate that the activity of CUDC-907 in ABCG2-overexpressing cancer cells can be restored by inhibiting the function of ABCG2, which provides support for the rationale of combining CUDC-907 with modulators of ABCG2 to improve the pharmacokinetics and efficacy of CUDC-907 in future treatment trials.

Abstract A90: Dichloroacetate can overcome drug resistance via decreased ABC drug transporter expression and PDK2 inhibition

Abstract Introduction: The primary hurdle for effective chemotherapy is the inherent or acquired drug resistance of cancer cells. The glycolytic phenotype (or Warburg effect) is a major metabolic signature of cancer cells. Dichloroacetic acid (DCA) is a non-toxic drug, which can reverse the glycolytic phenotype by inhibiting pyruvate dehydrogenase kinases (PDKs). Recent studies have linked changes in cancer cell metabolism to multi-drug resistance (MDR) phenotypes. We have examined whether reversal of the glycolytic phenotype with DCA can restore sensitivity in drug resistant cancer cells. Methodology: We examined DCA's effects in doxorubicin (DOX)-resistant MDA-MB-231 and MCF7 cells (made by continuous exposure to increasing DOX concentrations for one month) and their DOX-sensitive counterparts by neutral red cell viability assay. Intracellular drug uptake was measured by flow cytometry and expression of PDKs and drug transporters was measured by western blotting. Results: DCA treatment (1mM for 48hrs) significantly (p<0.05) enhanced DOX toxicity in DOX-resistant cells restoring sensitivity to a level similar to parental cells. This is despite DCA having no effect on DOX toxicity in parental cells. PDK2 expression was higher in the drug-selected cells compared to parental cells suggesting the sensitising effect of DCA on drug resistant cells could be an on-target effect. DCA pre-treatment increased the intracellular retention of DOX and mitoxantrone. DCA effectively reduced the expression of ATP-binding cassette (ABC) drug transporters such as P-glycoprotein and ABCG2 in DOX-resistant MDA-MB-231 and MCF7 cells and in vivo in 4T1 mouse mammary tumors. DCA pre-treatment also sensitised the MDR cell line MCF7-FLV1000 towards mitoxantrone induced apoptosis. Conclusion: DCA may be an effective chemo-sensitizing agent in a wide range of MDR cancers, acting by reducing drug transporter expression and inhibiting PDKs. Citation Format: Santhi Achuthan, Richard Callaghan, Anneke C. Blackburn. Dichloroacetate can overcome drug resistance via decreased ABC drug transporter expression and PDK2 inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A90.

Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.

Ro 31–8220 is a potent protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs). Various PKC-independent effects of Ro 31–8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT) 1, involved in uptake of marketed drugs in the liver, in a PKC-independent manner. Ro 31–8220, in contrast to other pan-PKC inhibitors such as staurosporine and chelerythrine, was thus shown to cis-inhibit uptake of the reference OCT1 substrate tetraethylammonium in OCT1-transfected HEK293 cells in a concentration-dependent manner (IC50 = 0.18 μM) and without altering membrane expression of OCT1. This blockage of OCT1 was also observed in human hepatic HepaRG cells that constitutionally express OCT1. It likely occurred through a mixed mechanism of inhibition. Ro 31–8220 additionally trans-inhibited TEA uptake in OCT1-transfected HEK293 cells, which likely discards a transport of Ro 31–8220 by OCT1. Besides Ro 31–8220, 7 additional BIMs, including the PKC inhibitor LY 333531, inhibited OCT1 activity, whereas 4 other BIMs were without effect. In silico analysis of structure-activity relationships next revealed that various molecular descriptors, especially 3D-WHIM descriptors related to total size, correspond to key physico-chemical parameters for inhibition of OCT1 activity by BIMs. In addition to activity of OCT1, Ro 31–8220 inhibited those of other organic cation transporters such as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K, whereas, by contrast, it stimulated that of OCT2. Taken together, these data extend the nature of cellular off-targets of the BIM Ro 31–8220 to OCT1 and other organic cation transporters, which has likely to be kept in mind when using Ro 31–8220 and other BIMs as PKC inhibitors in experimental or clinical studies.

Isoreserpine Reverses Multidrug Resistance Mediated by ABCB1

One of the major obstacles to successful cancer chemotherapy is the development of multidrug resistance (MDR) that is associated with the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (P-glycoprotein/ MDR1). Currently, the most efficient way to overcome ABCB1-mediated MDR in cancer is by direct inhibition of ABCB1 function. Many drugs with known biological activities have been discovered to inhibit the function of ABCB1 and reverse ABCB1-mediated MDR in cancers. However, clinical trial results suggested that many of these clinically active drugs should not be used as ABCB1 modulators due to direct toxicity or undesirable side effects. In this study, we demonstrated that isoreserpine, an indole alkaloid with relatively low toxicity, can significantly inhibit ABCB1-mediated efflux of calcein-AM, a known substrate of ABCB1, in a dose-dependent manner. Moreover, we showed that at non-toxic concentrations, isoreserpine potently reversed ABCB1-mediated resistance to doxorubicin and colchicine in ABCB1-overexpressing human KB-V-1 epidermal cancer cells. Collectively, our findings revealed thatby inhibiting the transport function of ABCB1, isoreserpine can restore drug sensitivity of ABCB1-overexpressing cells to conventional chemotherapeutic drugs. In conclusion, isoreserpine should be further developed into a promising reversal agent for the treatment of MDR in ABCB1-overexpressing cancers.

Inhibitory potential of tuberculosis drugs on ATP-binding cassette drug transporters

Multiple-drug therapy for tuberculosis (TB) and TB-associated co-morbidity increase the likelihood of drug-drug interactions (DDIs). Inhibition of membrane transporters is an important mechanism underlying DDIs. In this study, we assessed the in vitro inhibitory potential of currently used first and second-line TB drugs and of proposed mycobacterial efflux pump inhibitors (EPIs) on the major ABC transporters relevant to drug transport, namely P-gp, BCRP, BSEP and MRP1-5. Membrane vesicles isolated from transporter-overexpressing HEK293 cells were used to study the inhibitory action of TB drugs and EPIs on the transport of model substrates [(3)H]-NMQ (P-gp); [(3)H]-E1S (BCRP); [(3)H]-TCA (BSEP); [(3)H]-E217βG (MRP1, 3 and 4) and [(3)H]-MTX (MRP2 and 5). A strong inhibition (IC50 value <15 μM) was observed for clofazimine (P-gp, BCRP and MRP1), thioridazine (BCRP), timcodar (P-gp, BSEP and MRP1) and SQ109 (P-gp and BCRP). Rifampicin inhibited all transporters, but less potently. Co-administration of clofazimine, thioridazine, timcodar, SQ109 and possibly rifampicin with drugs that are substrates for the inhibited transporters may lead to DDIs. The mycobacterial EPIs potently inhibited a wider range of human ABC transporters than previously reported. These vesicular transport data are especially valuable considering the current emphasis on development of TB drug regimens.

Expression of ABC drug transporters in human placenta from normal and GDM pregnancies and its relation to cytokines

Multidrug resistance (MDR) of cancer is often associated with the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP or ABCG2), in cancer cells, which facilitates the active efflux of a wide variety of chemotherapeutic drugs out of the cells. Marsdenia tenacissima is a traditional Chinese medicinal herb that has long been clinically used for treatment of cancers, particularly in combinational use with anticancer drugs. Polyoxypregnanes (POPs) are identified as main constituents of this herb, and three of them have been reported to exhibit P-gp modulatory effect and thus reverse MDR. Therefore, it is of great necessity to investigate more POPs that have potential to reverse transporters-mediated MDR.We aimed to identify POPs as the chemical basis responsible for circumventing ABC transporters-mediated MDR by M. tenacissima.The MDR reversal effects of M. tenacissima crude extract together with a series of isolated POPs were evaluated on several MDR cancer cell lines that overexpress P-gp, MRP1 or ABCG2. The activities of P-gp, MRP1 and ABCG2 were determined by the flow cytometry-based substrate efflux assay. Molecular docking of POPs to a three-dimensional human P-gp homology structure was also performed.The crude extract of M. tenacissima was firstly found to circumvent P-gp-mediated MDR. Then, 11 polyoxypregnane compounds (POPs) isolated from this herb were found to overcome P-gp-, MRP1- and/or ABCG2-mediated MDR. Further mechanistic study delineated that the reversal of MDR by these POPs was due to significant increase in the intracellular concentrations of the substrate anticancer drugs via their inhibition of different ABC transporter-mediated efflux activities. Furthermore, molecular docking revealed that POPs with P-gp modulatory effect bound to P-gp and fitted well into the cavity between the alpha and beta subunit of P-gp via forming hydrogen bonds. In addition, several key structural determinants for inhibition of P-gp, MRP1 or ABCG2 by POPs were illustrated.Our findings advocated the rational use of M. tenacissima to enhance efficacies of conventional anticancer drugs in tumors with ABC drug transporters-mediated MDR. Furthermore, 11 POPs were found to contribute to MDR reversal effect of M. tenacissima via inhibition of different ABC efflux transporters.

Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60).

Overexpression of ATP-binding cassette (ABC) drug transporters is a major barrier in the success of cancer chemotherapy. One way to overcome overexpression of ABC drug transporter-mediated chemoresistance in acute myeloid leukemia is to suppress ABC drug transporter genes expression by small interference RNA (siRNA). In this study was assessed the involvement of ABCB1 gene in the mechanisms of resistance to etoposide in AML cells. The etoposide-resistant HL-60 cells were generated by stepwise exposure increasing concentrations of etoposide. The etoposide-resistant HL-60 cells were transfected with siRNAs using Transfection Reagent. The ABCB1 mRNA expression were assessed by real-time quantitative PCR. The MDR1/P-gp levels were measured by Western blotting. The sensitivity of resistant HL-60 cells to etoposide after transfection was determined using MTT assay. Apoptosis of resistant HL-60 cells after transfection was detected by flow cytometer. It was found that siRNA effectively inhibited ABCB1 expression at both mRNA and protein levels. Knockdown of the ABCB1 gene correlated with increased sensitivity of the resistant HL-60 cells to etoposide and was observed to lower the cytotoxic index (IC50 etoposide value) after transfection. Our results indicate that product of the ABCB1 gene have effective role in resistance to etoposide in acute myeloid leukemia cells.

Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2.

Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC.

Placental passage of olomoucine II, but not purvalanol A, is affected by p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins (ABCCs)

1. Purine cyclin-dependent kinase inhibitors have recently been recognised as promising candidates for the treatment of various cancers. While pharmacodynamic properties of these compounds are relatively well understood, their pharmacokinetics including possible interactions with placental transport systems have not been characterised to date.2. In this study, we investigated transplacental passage of olomoucine II and purvalanol A in rat focusing on possible role of p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and/or multidrug resistance-associated proteins (ABCCs). Employing the in situ method of dually perfused rat term placenta, we demonstrate transplacental passage of both olomoucine II and purvalanol A against the concentration gradient in foetus-to-mother direction. Using several ATP-binding cassette (ABC) drug transporter inhibitors, we confirm the participation of ABCB1, ABCG2 and ABCCs transporters in the placental passage of olomoucine II, but not purvalanol A.3. Transplacental passage of olomoucine II and purvalanol A from mother to foetus is significantly reduced by active transporters, restricting thereby foetal exposure and providing protection against harmful effects of these xenobiotics. Importantly, we demonstrate that in spite of their considerable structural similarity, the two molecules utilise distinct placental transport systems. These facts should be kept in mind when introducing these prospective anticancer candidates and/or their analogues into the clinical area.

Fallopia japonica, a Natural Modulator, Can Overcome Multidrug Resistance in Cancer Cells.

Resistance of cancer cells to chemotherapy is controlled by the decrease of intracellular drug accumulation, increase of detoxification, and diminished propensity of cancer cells to undergo apoptosis. ATP-binding cassette (ABC) membrane transporters with intracellular metabolic enzymes contribute to the complex and unresolved phenomenon of multidrug resistance (MDR). Natural products as alternative medicine have great potential to discover new MDR inhibitors with diverse modes of action. In this study, we characterized several extracts of traditional Chinese medicine (TCM) plants (N = 16) for their interaction with ABC transporters, cytochrome P3A4 (CYP3A4), and glutathione-S-transferase (GST) activities and their cytotoxic effect on different cancer cell lines. Fallopia japonica (FJ) (Polygonaceae) shows potent inhibitory effect on CYP3A4 P-glycoprotein activity about 1.8-fold when compared to verapamil as positive control. FJ shows significant inhibitory effect (39.81%) compared with the known inhibitor ketoconazole and 100 μg/mL inhibited GST activity to 14 μmol/min/mL. FJ shows moderate cytotoxicity in human Caco-2, HepG-2, and HeLa cell lines; IC50 values were 630.98, 198.80, and 317.37 µg/mL, respectively. LC-ESI-MS were used to identify and quantify the most abundant compounds, emodin, polydatin, and resveratrol, in the most active extract of FJ. Here, we present the prospect of using Fallopia japonica as natural products to modulate the function of ABC drug transporters. We are conducting future study to evaluate the ability of the major active secondary metabolites of Fallopia japonica to modulate MDR and their impact in case of failure of chemotherapy.

Combating and overcoming drug resistance in cancer

Combating and overcoming drug resistance in cancer

Human ATP-Binding Cassette transporters ABCB1 and ABCG2 confer resistance to CUDC-101, a multi-acting inhibitor of histone deacetylase, epidermal growth factor receptor and human epidermal growth factor receptor 2

CUDC-101 is the first small-molecule inhibitor designed to simultaneously inhibit epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and histone deacetylase (HDAC) in cancer cells. Recently, in its first in human phase I study, CUDC-101 showed promising single agent activity against advanced solid tumors and favorable pharmacodynamic profile. However, the risk of developing drug resistance to CUDC-101 can still present a significant therapeutic challenge to clinicians in the future. One of the most common mechanisms of developing multidrug resistance (MDR) in cancer is associated with the overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and ABCG2. Together, they are able to reduce the efficacy and modify the pharmacological properties of anti-cancer agents, including many small molecule tyrosine kinase inhibitors (TKIs). Here, we have investigated the impact of ABCB1 and ABCG2 on the efficacy of CUDC-101 in human cancer cells. We revealed that although CUDC-101 has potent antiproliferative and proapoptotic activities against most cancer cell lines, the overexpression of ABCB1 or ABCG2 in cancer cells significantly reduced the activity of CUDC-101 against HDAC, EGFR and HER2, as well as its cytotoxicity and proapoptotic activity. Moreover, we showed that CUDC-101 modulated the function of both transporters without affecting the protein expression of either ABCB1 or ABCG2. More importantly, our study provides support for the rationale of combining CUDC-101 with modulators of ABC drug transporters to improve drug efficacy and overcome multidrug resistance associated with the overexpression of ABCB1 and ABCG2.

Abstract LB-224: Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping

Abstract Nilotinib (Tasigna®) is a novel tyrosine kinase inhibitor (TKI) that has been used to treat chronic phase chronic myeloid leukemia patients who develop resistance due to the emergence of point mutations within the BCR-ABL kinase domain. It is known to be transported by the ATP binding-cassette (ABC) drug efflux transporters P-glycoprotein (P-gp) and ABCG2, which not only alters the efficacy of nilotinib but might also result in lower bioavailability. Identification of a nilotinib binding conformation in the drug-binding pocket of these transporters is important to understand its interactions and can be used to design the next generation of TKIs that do not interact with ABC drug transporters. A structure-activity relationship (SAR)-based approach for nilotinib was used in this study to identify an initial pharmacophore interacting with P-gp. [125I]-Iodoarylazidoprazosin (IAAP) photolabeling and FACS-based transport of fluorescent substrates were carried out with 26 structural derivatives of nilotinib to evaluate their potency for inhibiting the binding of substrate and the transport function of P-gp. IC50 values from these assays were used for 3D pharmacophore modeling and quantitative structure-activity relationships (QSARs) to propose the spatial arrangement of chemical features that are essential for inhibitory activity of nilotinib towards P-gp. Molecular docking of nilotinib in a homology model of human P-gp was performed to identify interactions with the drug-binding pocket. This docked model was evaluated by mutational mapping studies replacing the Y307, M949 and A985 residues with Cysteine in the primary-binding site, which resulted in the loss of nilotinib's activity to inhibit substrate binding to P-gp. The orientation of nilotinib in this binding pocket was further substantiated by assessing the interaction of nilotinib's structural derivatives with P-gp using photoaffinity labeling, ATP hydrolysis and transport assays. These results demonstrate that the pyridine and pyrimidine rings in nilotinib play a key role in its interaction with the drug-binding pocket, while orientation of the imidazole ring determines the affinity of nilotinib for P-gp. These results for the first time identify the primary binding site of nilotinib in the drug-binding pocket of P-gp and can be exploited to design novel tyrosine kinase inhibitors that would not be recognized by ABC drug transporters. Citation Format: Suneet Shukla, Eduardo E. Chufan, Satyakam Singh, Amanda P. Skoumbourdis, Khyati Kapoor, Matthew B. Boxer, Damien Y. Duveau, Craig J. Thomas, Tanaji T. Talele, Suresh V. Ambudkar. Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-224. doi:10.1158/1538-7445.AM2014-LB-224

Abstract 766: Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364

Abstract The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. Plk1 inhibitors BI 2536, volasertib and GSK461364, were designed to selectively inhibit cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to Plk1 inhibitors is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that in human cancer cells, the overexpression of ABCB1 and/or ABCG2 can lead to acquired resistance to three selective Plk1 inhibitors, BI 2536, volasertib and GSK461364. Moreover, these Plk1 inhibitors stimulate the ATPase activity of ABCB1 and ABCG2, as well as competitively inhibit the drug substrate transport mediated by ABCB1 and ABCG2. More significantly, the reduced chemosensitivity and Plk1 inhibitors-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor of ABCB1 and ABCG2. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to Plk1 inhibitors, a combined regimen of Plk1 inhibitors and modulators or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic. Citation Format: Chung-Pu Wu, Hong-May Sim, Suresh V. Ambudkar. Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2014-766