A dual PI3 kinase/mTOR inhibitor BEZ235 reverses doxorubicin resistance in ABCB1 overexpressing ovarian and pancreatic cancer cell lines

BackgroundMulti-drug resistance (MDR) develops because cancer cells evade toxicity of several structurally unrelated drugs. Besides other mechanisms, MDR is linked to the overexpression of ATP Binding Cassette (ABC), transporters, among which ABCB1 is the best characterized one. Since overactivation of PI3K/Akt/mTOR plays a pivotal role in the growth of human cancers, we hypothesized whether dual PI3K and mTOR inhibitor, BEZ235 (BEZ, dactolisib) reverses resistance to doxorubicin (DOX).MethodsOvarian (A2780) and pancreatic (MiaPaca2) cancer cells were used to generate DOX-resistant clones by overexpressing ABCB1 or stepwise treatment of DOX. Intracellular accumulation of DOX was measured by flow cytometry after treatment with BEZ.ResultsBEZ treatment caused an increase in intracellular levels of DOX which was almost identical to the naïve parental cell lines. BEZ was found to be a weak substrate for ABCB1 as demonstrated by minimal increase in ATPase activity. BEZ treatment caused a dose-dependent decrease in cell viability in combination with DOX, which was associated with an increase in cleaved PARP expression in the drug resistant clones.ConclusionsThese results suggest that BEZ is a non-substrate inhibitor of ABCB1 and is able to effectively re-sensitize cells overexpressing ABCB1 to the effects of DOX.General significanceDual PI3 Kinase/mTOR inhibitor, BEZ, has the potential to reverse MDR in cancer patients.     

Veliparib overcomes multidrug resistance in liver cancer cells

Overexpression of ATP-binding cassette (ABC) transporter is one of the most important factors taking responsibility for the progress of multidrug resistance (MDR) in multiple cancers. In this study, we investigated that veliparib, a PARP inhibitor which is in clinical development, could overcome ABCB1-mediated MDR in liver cancer cells. Veliparib could significantly enhance the cytotoxic effects of a series of conventional chemotherapeutic drugs in ABCB1-overexpression liver cancer cells. Mechanism study showed that veliparib could significantly enhance the accumulation of doxorubicin in ABCB1-overexpression liver cancer cells, without down-regulating the expression level of ABCB1. Finally, veliparib could significantly inhibit the ATPase activity of ABCB1 transporter. This study could provide information that combine veliparib with other chemotherapeutic drugs may benefit liver cancer patients.     

Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2

Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.     

Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance

In this study we investigated the effect of linsitinib on the reversal of multidrug resistance (MDR) mediated by the overexpression of the ATP-binding cassette (ABC) subfamily members ABCB1, ABCG2, ABCC1 and ABCC10. Our results indicate for the first time that linsitinib significantly potentiate the effect of anti-neoplastic drugs mitoxantrone (MX) and SN-38 in ABCG2-overexpressing cells; paclitaxel, docetaxel and vinblastine in ABCC10-overexpressing cells. Linsitinib moderately enhanced the cytotoxicity of vincristine in cell lines overexpressing ABCB1, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, linsitinib significantly increased the intracellular accumulation and decreased the efflux of [³H]-MX in ABCG2-overexpressing cells and [³H]-paclitaxel in ABCC10-overexpressing cells. However, linsitinib, at a concentration that reversed MDR, did not significantly alter the expression levels of either the ABCG2 or ABCC10 transporter proteins. Furthermore, linsitinib did not significantly alter the intracellular localization of ABCG2 or ABCC10. Moreover, linsitinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner. Overall, our study suggests that linsitinib attenuates ABCG2- and ABCC10-mediated MDR by directly inhibiting their function as opposed to altering ABCG2 or ABCC10 protein expression.     

Involvement of ABC transporters in melanogenesis and the development of multidrug resistance of melanoma

Because melanomas are intrinsically resistant to conventional radiotherapy and chemotherapy, many alternative treatment approaches have been developed such as biochemotherapy and immunotherapy. The most common cause of multidrug resistance (MDR) in human cancers is the expression and function of one or more A TP‐b inding c assette (ABC) transporters that efflux anticancer drugs from cells. Melanoma cells express a group of ABC transporters (such as ABCA9, ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, and ABCD1) that may be associated with the resistance of melanoma cells to a broad range of anticancer drugs and/or of melanocytes to toxic melanin intermediates and metabolites. In this review, we propose a model (termed the ABC‐M model) in which the intrinsic MDR of melanoma cells is at least in part because of the transporter systems that may also play a critical role in reducing the cytotoxicity of the melanogenic pathway in melanocytes. The ABC‐M model suggests molecular strategies to reverse MDR function in the context of the melanogenic pathway, which could open therapeutic avenues towards the ultimate goal of circumventing clinical MDR in patients with melanoma.     

Tepoxalin increases chemotherapy efficacy in drug-resistant breast cancer cells overexpressing the multidrug transporter gene ABCB1

Effective cancer chemotherapy treatment requires both therapy delivery and retention by malignant cells. Cancer cell overexpression of the multidrug transmembrane transporter gene ABCB1 (MDR1, multi-drug resistance protein 1) thwarts therapy retention, leading to a drug-resistant phenotype. We explored the phenotypic impact of ABCB1 overexpression in normal human mammary epithelial cells (HMECs) via acute adenoviral delivery and in breast cancer cell lines with stable integration of inducible ABCB1 expression. One hundred sixty-two genes were differentially expressed between ABCB1-expressing and GFP-expressing HMECs, including the gene encoding the cyclooxygenase-2 protein, PTGS2. Several breast cancer cell lines with inducible ABCB1 expression demonstrated sensitivity to the 5-lipoxygenase, cyclooxygenase-1/2 inhibitor tepoxalin in two-dimensional drug response assays, and combination treatment of tepoxalin either with chemotherapies or with histone deacetylase (HDAC) inhibitors improved therapeutic efficacy in these lines. Moreover, selection for the ABCB1-expressing cell population was reduced in three-dimensional co-cultures of ABCB1-expressing and GFP-expressing cells when chemotherapy was given in combination with tepoxalin. Further study is warranted to ascertain the clinical potential of tepoxalin, an FDA-approved therapeutic for use in domesticated mammals, to restore chemosensitivity and improve drug response in patients with ABCB1-overexpressing drug-resistant breast cancers.     

Mitochondria of a human multidrug‐resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P‐glycoprotein (P‐gp), breast cancer resistant protein and multiple resistance protein‐1. The MDR phenotype is associated with the constitutive expression of COX‐2 and iNOS, whereas celecoxib, a specific inhibitor of COX‐2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX‐2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1‐positive cells, whereas COX‐2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P‐gp in mitochondria of MDR cancer cells independently from inhibition of COX‐2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.     

BIRB796, the Inhibitor of p38 Mitogen-Activated Protein Kinase,Enhances the Efficacy of Chemotherapeutic Agents in ABCB1 Overexpression Cells

ATP-binding-cassette family membrane proteins play an important role in multidrug resistance. In this study, we investigated BIRB796, an orally active inhibitor of p38 mitogen-activated protein kinase, reversed MDR induced by ABCB1, ABCG2 and ABCC1. Our results showed that BIRB796 could reverse ABCB1-mediated MDR in both the drug selected and transfected ABCB1-overexpressing cell models, but did not enhance the efficacy of substrate-chemotherapeutical agents in ABCC1 or ABCG2 overexpression cells and their parental sensitive cells. Furthermore, BIRB796 increased the intracellular accumulation of the ABCB1 substrates, such as rhodamine 123 and doxorubicin. Moreover, BIRB796 bidirectionally mediated the ATPase activity of ABCB1, stimulating at low concentration, inhibiting at high concentration. However, BIRB796 did not alter the expression of ABCB1 both at protein and mRNA level. The down-regulation of p38 by siRNA neither affected the expression of ABCB1 nor the cytotoxic effect of paclitaxel on KBV200. The binding model of BIRB796 within the large cavity of the transmembrane region of ABCB1 may form the basis for future lead optimization studies. Importantly, BIRB796 also enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBV200 cell xenografts in nude mice. Overall, we conclude that BIRB796 reverses ABCB1-mediated MDR by directly inhibiting its transport function. These findings may be useful for cancer combinational therapy with BIRB796 in the clinic.     

Purvalanol A,Olomoucine II and Roscovitine Inhibit ABCB1 Transporter and Synergistically Potentiate Cytotoxic Effects of Daunorubicin In Vitro

Cyclin-dependent kinase inhibitors (CDKi) have high potential applicability in anticancer therapy, but various aspects of their pharmacokinetics, especially their interactions with drug efflux transporters, have not yet been evaluated in detail. Thus, we investigated interactions of five CDKi (purvalanol A, olomoucine II, roscovitine, flavopiridol and SNS-032) with the ABCB1 transporter. Four of the compounds inhibited efflux of two ABCB1 substrates, Hoechst 33342 and daunorubicin, in MDCKII-ABCB1 cells: Olomoucine II most strongly, followed by roscovitine, purvalanol A, and flavopiridol. SNS-032 inhibited ABCB1-mediated efflux of Hoechst 33342 but not daunorubicin. In addition, purvalanol A, SNS-032 and flavopiridol lowered the stimulated ATPase activity in ABCB1 membrane preparations, while olomoucine II and roscovitine not only inhibited the stimulated ATPase but also significantly activated the basal ABCB1 ATPase, suggesting that these two CDKi are ABCB1 substrates. We further revealed that the strongest ABCB1 inhibitors (purvalanol A, olomoucine II and roscovitine) synergistically potentiate the antiproliferative effect of daunorubicin, a commonly used anticancer drug and ABCB1 substrate, in MDCKII-ABCB1 cells as well as in human carcinoma HCT-8 and HepG2 cells. We suggest that this pronounced synergism is at least partly caused by (i) CDKi-mediated inhibition of ABCB1 transporter leading to increased intracellular retention of daunorubicin and (ii) native cytotoxic activity of the CDKi. Our results indicate that co-administration of the tested CDKi with anticancer drugs that are ABCB1 substrates may allow significant dose reduction in the treatment of ABCB1-expressing tumors.     

Discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) that modulates ABCB1-mediated multidrug resistance (MDR)

Multidrug resistance (MDR) has been shown to reduce the effectiveness of chemotherapy. Strategies to overcoming MDR have been widely explored in the last decades, leading to a generation of numerous small molecules targeting ABC and MRP transporters. Among the ABC family, ABCB1 plays key roles in the development of drug resistance and is the most well studied. In this work, we report the discovery of non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) from our structurally diverse in-house compound collection that selectively modulates ABCB1-mediated multidrug resistance. WS-10 enhanced the intracellular accumulation of paclitaxel in SW620/Ad300 cells, but did not affect the expression of ABCB1 Protein and ABCB1 localization. The cellular thermal shift assay (CETSA) showed that WS-10 was able to bind to ABCB1, which could be responsible for the reversal effect of WS-10 toward paclitaxel and doxorubicin in SW620/Ad300 cells. Docking simulations were performed to show the possible binding modes of WS-10 within ABCB1 transporter. To conclude, WS-10 could be used as a template for designing new ABCB1 modulators to overcome ABCB1-mediated multidrug resistance.     

Discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) that modulates ABCB1-mediated multidrug resistance (MDR)

Multidrug resistance (MDR) has been shown to reduce the effectiveness of chemotherapy. Strategies to overcoming MDR have been widely explored in the last decades, leading to a generation of numerous small molecules targeting ABC and MRP transporters. Among the ABC family, ABCB1 plays key roles in the development of drug resistance and is the most well studied. In this work, we report the discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) from our structurally diverse in-house compound collection that selectively modulates ABCB1-mediated multidrug resistance. WS-10 enhanced the intracellular accumulation of paclitaxel in SW620/Ad300 cells, but did not affect the expression of ABCB1 Protein and ABCB1 localization. The cellular thermal shift assay (CETSA) showed that WS-10 was able to bind to ABCB1, which could be responsible for the reversal effect of WS-10 toward paclitaxel and doxorubicin in SW620/Ad300 cells. Docking simulations were performed to show the possible binding modes of WS-10 within ABCB1 transporter. To conclude, WS-10 could be used as a template for designing new ABCB1 modulators to overcome ABCB1-mediated multidrug resistance.     

Inhibition of cyclic GMP hydrolysis in human corpus cavernosum smooth muscle cells by vardenafil, a novel, selective phosphodiesterase type 5 inhibitor.

One of the key mediators of penile erectile function is nitric oxide (NO), which activates soluble guanylyl cyclase within the smooth muscle of erectile tissue and stimulates the production of cGMP. In addition to synthesis by cyclases, intracellular cGMP concentrations are tightly regulated by phosphodiesterases, which hydrolyze and inactivate cyclic nucleotides. In this study, we compared the inhibition of cGMP hydrolysis by vardenafil and sildenafil; two inhibitors selective for phosphodiesterase type 5 (PDE5). Vardenafil is a novel, high affinity PDE5 inhibitor currently under clinical development. In soluble extracts of human corpus cavernosum smooth muscle cells, vardenafil and sildenafil effectively inhibited cGMP hydrolysis at substrate concentrations of 1, 5 and 10 microM cGMP. The IC50 values for vardenafil were approximately 5-fold lower than for sildenafil at the substrate concentrations tested. Dixon plot analyses of the inhibition data demonstrated that vardenafil had a smaller inhibition constant (Ki = 4.5 nM) than sildenafil (Ki = 14.7 nM) in the same cellular extracts. In intact cells, 10 microM of the nitric oxide donor sodium nitroprusside resulted in a minimal (17%) increase in cGMP, relative to basal levels (321 +/- 65 fmol/mg prot). Treatment of cells with 10, 50 or 100 nM vardenafil, in the presence of 10 microM sodium nitroprusside, elevated cGMP levels in a dose dependent fashion, from 63% to 137% of basal levels. Equimolar concentrations of sildenafil also caused dose dependent increases in intracellular cGMP, but to a lesser extent (27-60%). These observations suggest that vardenafil is a more potent PDE5 inhibitor, than sildenafil in vitro. The more pronounced increase of cGMP in the presence of NO in intact cells suggests that vardenafil will be effective at lower doses than sildenafil under clinical conditions.     

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.     

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.     

Refinement of safety and efficacy of anti-cancer chemotherapeutics by tailoring their site-specific intracellular bioavailability through transporter modulation

Low intracellular bioavailability, off-site toxicities, and multi drug resistance (MDR) are the major constraints involved in cancer chemotherapy. Many anticancer molecules fail to become a good lead in drug discovery because of their poor site-specific bioavailability. Concentration of a molecule at target sites is largely varied because of the wavering expression of transporters. Recent anticancer drug discovery strategies are paying high attention to enhance target site bioavailability by modulating drug transporters. The level of genetic expression of transporters is an important determinant to understand their ability to facilitate drug transport across the cellular membrane. Solid carrier (SLC) transporters are the major influx transporters involved in the transportation of most anti-cancer drugs. In contrast, ATP-binding cassette (ABC) superfamily is the most studied class of efflux transporters concerning cancer and is significantly involved in efflux of chemotherapeutics resulting in MDR. Balancing SLC and ABC transporters is essential to avoid therapeutic failure and minimize MDR in chemotherapy. Unfortunately, comprehensive literature on the possible approaches of tailoring site-specific bioavailability of anticancer drugs through transporter modulation is not available till date. This review critically discussed the role of different specific transporter proteins in deciding the intracellular bioavailability of anticancer molecules. Different strategies for reversal of MDR in chemotherapy by incorporation of chemosensitizers have been proposed in this review. Targeted strategies for administration of the chemotherapeutics to the intracellular site of action through clinically relevant transporters employing newer nanotechnology-based formulation platforms have been explained. The discussion embedded in this review is timely considering the current need of addressing the ambiguity observed in pharmacokinetic and clinical outcomes of the chemotherapeutics in anti-cancer treatment regimens.     

Tumor necrosis factor alpha induces stronger cytotoxicity in ABCG2-overexpressing resistant breast cancer cells compared with their drug-sensitive parental line

Tumor necrosis factor alpha (TNF-α) has been reported to modulate the multidrug resistance (MDR) phenotype in vitro and in vivo. Multidrug-resistant cells overexpressing the ABCB1 transporter are more susceptible to inhibition of proliferation and induction of apoptosis by TNF-α than their drug-sensitive counterparts. This study was aimed to investigate TNF-α modulatory and antiproliferative effects on drug-resistant cells overexpressing ABCG2. The effects of TNF-α on viability and proliferation rate of MCF-7 breast cancer cells and their ABCG2-overexpressing sublines MCF-7/mitoxantrone (MX) cells were studied using dye exclusion assay, dimethylthiazolyl-2,5-diphenyl tetrazolium bromide technique, and flow cytometric analysis of cell cycle. TNF-α influence on MX accumulation was investigated by flow cytometry. ABCG2-overexpressing cells were more susceptible to antiproliferative and cytotoxic effects of TNF-α than their parental cells. TNF-α increased accumulation of MX in both parental and resistant cells. Higher sensitivity of MDR cells to TNF-α cytotoxicity would help in characterization of its complex modulatory effects on cancer cells and benefit us in designing new approaches to overcome MDR.     

Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance

Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance.