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.     

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.     

Design,synthesis and biological evaluation of benzamide and phenyltetrazole derivatives with amide and urea linkers as BCRP inhibitors

Breast cancer resistant protein (BCRP/ABCG2), a 72 kDa plasma membrane transporter protein is a member of ABC transporter superfamily. Increased expression of BCRP causes increased efflux and therefore, reduced intracellular accumulation of many unrelated chemotherapeutic agents leading to multidrug resistance (MDR). A series of 31 benzamide and phenyltetrazole derivatives with amide and urea linkers has been synthesized to serve as potential BCRP inhibitors in order to overcome BCRP-mediated MDR. The target derivatives were tested for their cytotoxicity and reversal effects in human non-small cell lung cancer cell line H460 and mitoxantrone resistant cell line H460/MX20 using the MTT assay. In the benzamide series, compounds 6 and 7 exhibited a fold resistance of 1.51 and 1.62, respectively at 10 µM concentration which is similar to that of FTC, a known BCRP inhibitor. Compounds 27 and 31 were the most potent analogues in the phenyltetrazole series with amide linker with a fold resistance of 1.39 and 1.32, respectively at 10 µM concentration. For the phenyltetrazole series with urea linker, 38 exhibited a fold resistance of 1.51 which is similar than that of FTC and is the most potent compound in this series. The target compounds did not exhibit reversal effect in P-gp overexpressing resistant cell line SW620/Ad300 suggesting that they are selective BCRP inhibitors.     

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.     

Discovery of alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as potential dual inhibitors of P-glycoprotein and breast cancer resistance protein

We recently reported alkoxyl biphenyl derivatives bearing dibenzo[c,e]azepine scaffold as novel P-glycoprotein (P-gp, ABCB1) inhibitors. In this study, their ability to reverse breast cancer resistance protein (BCRP, ABCG2)-mediated multidrug resistance was tested in HEK293/BCRP cells which was BCRP-transfected stable HEK293 cells. It was observed that compounds 4d, 4h, 4i increased mitoxantrone accumulation in HEK293/BCRP cells via inhibiting BCRP efflux function. Notably, the inhibitory activity of 4i was comparable to that of the classical BCRP inhibitor Ko143 at an equimolar concentration. Interestingly, 4i had little inhibitory effect on multidrug resistance-associated protein 1 (MRP1, ABCC1), another drug efflux transporter. These results, together with the previous findings, suggest that 4i may be a dual inhibitor of P-gp and BCRP to warrant further investigation.     

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.     

Imatinib and Nilotinib Reverse Multidrug Resistance in Cancer Cells by Inhibiting the Efflux Activity of the MRP7 (ABCC10)

Background

One of the major mechanisms that could produce resistance to antineoplastic drugs in cancer cells is the ATP binding cassette (ABC) transporters. The ABC transporters can significantly decrease the intracellular concentration of antineoplastic drugs by increasing their efflux, thereby lowering the cytotoxic activity of antineoplastic drugs. One of these transporters, the multiple resistant protein 7 (MRP7, ABCC10), has recently been shown to produce resistance to antineoplastic drugs by increasing the efflux of paclitaxel. In this study, we examined the effects of BCR-Abl tyrosine kinase inhibitors imatinib, nilotinib and dasatinib on the activity and expression of MRP7 in HEK293 cells transfected with MRP7, designated HEK-MRP7-2.

Methodology and/or Principal Findings

We report for the first time that imatinib and nilotinib reversed MRP7-mediated multidrug resistance. Our MTT assay results indicated that MRP7 expression in HEK-MRP7-2 cells was not significantly altered by incubation with 5 µM of imatinib or nilotinib for up to 72 hours. In addition, imatinib and nilotinib (1-5 µM) produced a significant concentration-dependent reversal of MRP7-mediated multidrug resistance by enhancing the sensitivity of HEK-MRP7-2 cells to paclitaxel and vincristine. Imatinib and nilotinib, at 5 µM, significantly increased the accumulation of [³H]-paclitaxel in HEK-MRP7-2 cells. The incubation of the HEK-MRP7-2 cells with imatinib or nilotinib (5 µM) also significantly inhibited the efflux of paclitaxel.

Conclusions

Imatinib and nilotinib reverse MRP7-mediated paclitaxel resistance, most likely due to their inhibition of the efflux of paclitaxel via MRP7. These findings suggest that imatinib or nilotinib, in combination with other antineoplastic drugs, may be useful in the treatment of certain resistant cancers.     

Localization and Substrate Selectivity of Sea Urchin Multidrug (MDR) Efflux Transporters

In this study, we cloned, expressed and functionally characterized Stronglycentrotus purpuratus (Sp) ATP-binding cassette (ABC) transporters. This screen identified three multidrug resistance (MDR) transporters with functional homology to the major types of MDR transporters found in humans. When overexpressed in embryos, the apical transporters Sp-ABCB1a, ABCB4a, and ABCG2a can account for as much as 87% of the observed efflux activity, providing a robust assay for their substrate selectivity. Using this assay, we found that sea urchin MDR transporters export canonical MDR susbtrates such as calcein-AM, bodipy-verapamil, bodipy-vinblastine, and mitoxantrone. In addition, we characterized the impact of nonconservative substitutions in the primary sequences of drug binding domains of sea urchin versus murine ABCB1 by mutation of Sp-ABCB1a and treatment of embryos with stereoisomeric cyclic peptide inhibitors (QZ59 compounds). The results indicated that two substitutions in transmembrane helix 6 reverse stereoselectivity of Sp-ABCB1a for QZ59 enantiomers compared with mouse ABCB1a. This suggests that subtle changes in the primary sequence of transporter drug binding domains could fine-tune substrate specificity through evolution.     

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.     

Reversal of P-glycoprotein (P-gp) mediated multidrug resistance in colon cancer cells by cryptotanshinone and dihydrotanshinone of Salvia miltiorrhiza

ObjectiveMultidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer drugs is an obstacle to successful chemotherapy. Overexpression of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) membrane transporter, can mediate the efflux of cytotoxic drugs out of cancer cells, leading to MDR and chemotherapy failure. Thus, development of safe and effective P-gp inhibitors plays an important role in circumvention of MDR. This study investigated the reversal of P-gp mediated multidrug resistance in colon cancer cells by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone isolated from Salvia miltiorrhiza (Danshen), known to be safe in traditional Chinese medicine.MethodsThe inhibitory effects of tanshinones on P-gp function were compared using digoxin bi-directional transport assay in Caco-2 cells. The potentiation of cytotoxicity of anticancer drugs by effective tanshinones were evaluated by MTT assay. Doxorubicin efflux assay by flow cytometry, P-gp protein expression by western blot analysis, immunofluorescence for P-gp by confocal microscopy, quantitative real-time PCR and P-gp ATPase activity assay were used to study the possible underlying mechanisms of action of effective tanshinones.ResultsBi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased digoxin efflux ratio in a concentration-dependent manner, indicating their inhibitory effects on P-gp function; whereas, tanshinone I, tanshinone IIA and miltirone had no inhibitory effects. Moreover, both cryptotanshinone and dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 colon cancer cells. Results from mechanistic studies revealed that these two tanshinones increased intracellular accumulation of the P-gp substrate anticancer drugs, presumably by down-regulating P-gp mRNA and protein levels, and inhibiting P-gp ATPase activity.ConclusionsTaken together, these findings suggest that cryptotanshinone and dihydrotanshinone could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies for colon cancer.     

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.     

Selenoesters and selenoanhydrides as novel multidrug resistance reversing agents: A confirmation study in a colon cancer MDR cell line

Taking into account that multidrug resistance (MDR) is the main cause for chemotherapeutic failure in cancer treatment and as a continuation of our efforts to overcome this problem we report the evaluation of one cyclic selenoanhydride (1) and ten selenoesters (2–11) in MDR human colon adenocarcinoma Colo 320 cell line. The most potent derivatives (1, 9–11) inhibited the ABCB1 efflux pump much stronger than the reference compound verapamil. Particularly, the best one (9) was 4-fold more potent than verapamil at a 10-fold lower concentration. Furthermore, the evaluated derivatives exerted a potent and selective cytotoxic activity. In addition, they were strong apoptosis inducers as the four derivatives triggered apoptotic events in a 64–72% of the examined MDR Colo 320 human adenocarcinoma cells.     

Mitochondrial ABC proteins in health and disease

ABC transporters represent one of the largest families of membrane proteins that are found in all three phyla of life. Mitochondria comprise up to four ABC systems, ABCB7/ATM1, ABCB10/MDL1, ABCB8 and ABCB6. These half-transporters, which assemble into homodimeric complexes, are involved in a number of key cellular processes, e.g. biogenesis of cytosolic iron-sulfur clusters, heme biosynthesis, iron homeostasis, multidrug resistance, and protection against oxidative stress. Here, we summarize recent advances and emerging themes in our understanding of how these ABC systems in the inner and outer mitochondrial membrane fulfill their functions in important (patho) physiological processes, including neurodegenerative and hematological disorders.     

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.     

The phosphodiesterase-5 inhibitor vardenafil is a potent inhibitor of ABCB1/P-glycoprotein transporter

One of the major causes of chemotherapy failure in cancer treatment is multidrug resistance (MDR) which is mediated by the ABCB1/P-glycoprotein. Previously, through the use of an extensive screening process, we found that vardenafil, a phosphodiesterase 5 (PDE-5) inhibitor significantly reverses MDR in ABCB1 overexpressing cancer cells, and its efficacy was greater than that of tadalafil, another PDE-5 inhibitor. The present study was designed to determine the reversal mechanisms of vardenafil and tadalafil on ABC transporters-mediated MDR. Vardenafil or tadalafil alone, at concentrations up to 20 μM, had no significant toxic effects on any of the cell lines used in this study, regardless of their membrane transporter status. However, vardenafil when used in combination with anticancer substrates of ABCB1, significantly potentiated their cytotoxicity in ABCB1 overexpressing cells in a concentration-dependent manner, and this effect was greater than that of tadalafil. The sensitivity of the parenteral cell lines to cytotoxic anticancer drugs was not significantly altered by vardenafil. The differential effects of vardenafil and tadalafil appear to be specific for the ABCB1 transporter as both vardenafil and tadalafil had no significant effect on the reversal of drug resistance conferred by ABCC1 (MRP1) and ABCG2 (BCRP) transporters. Vardenafil significantly increased the intracellular accumulation of [(3)H]-paclitaxel in the ABCB1 overexpressing KB-C2 cells. In addition, vardenafil significantly stimulated the ATPase activity of ABCB1 and inhibited the photolabeling of ABCB1 with [(125)I]-IAAP. Furthermore, Western blot analysis indicated the incubation of cells with either vardenafil or tadalafil for 72 h did not alter ABCB1 protein expression. Overall, our results suggest that vardenafil reverses ABCB1-mediated MDR by directly blocking the drug efflux function of ABCB1.     

The 5-aromatic hydantoin-3-acetate derivatives as inhibitors of the tumour multidrug resistance efflux pump P-glycoprotein (ABCB1): Synthesis,crystallographic and biological studies

A series of arylpiperazine derivatives of hydantoin-3-acetate, including previously obtained 5,5-diphenylhydantoin (1–7) and new-synthesized spirofluorene-hydantoin derivatives (8–12), were investigated in the search for new inhibitors of the tumour multidrug resistance (MDR) efflux pump P-glycoprotein (P-gp, ABCB1) overexpressed in mouse T-lymphoma cells. Synthesis of new compounds (8–12) was performed. Crystal structures of two compounds (8 and 11) were determined by X-ray diffraction method. The conformations of the investigated molecules (8 and 11) in the crystalline samples are different. The bent conformation seems to be more favourable for biological activity than the extended one. The efflux pump inhibitory properties of the compounds 1–12 were evaluated in the fluorescence uptake assay using rhodamine 123 dye in mouse T-lymphoma model in vitro. Their cytotoxic action was examined, too. All compounds with methyl acetate moiety displayed high potency to inhibit the MDR efflux pump. The most active compound, methyl 2-(1-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-5,5-diphenylhydantoin-3-yl)acetate (5), tested at 1/10 of verapamil concentration displayed the 9-fold higher P-gp inhibitory action.     

Sensitivity of apoptosis-resistant colon cancer cells to tanshinones is mediated by autophagic cell death and p53-independent cytotoxicity

BackgroundMultidrug resistance (MDR) develops in nearly all patients with colon cancer. The reversal of MDR plays an important role in the success of colon cancer chemotherapy. One of the commonest mechanisms conferring MDR is the suppression of apoptosis in cancer cells.PurposeThis study investigated the sensitivity of cryptotanshinone (CTS) and dihydrotanshinone (DTS), two lipophilic tanshinones from a traditional Chinese medicine Salvia miltiorrhiza, in apoptosis-resistant colon cancer cells.MethodsCell viability was measured by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein levels were analyzed by western blot analysis. The formation of acidic vesicular organelles was visualized by acridine orange staining.ResultsExperimental results showed that multidrug-resistant colon cancer cells SW620 Ad300 were sensitive to both CTS and DTS in terms of cell death, but with less induction of apoptosis when compared with the parental cells SW620, suggesting that other types of cell death such as autophagy could occur. Indeed, the two tanshinones induced more LC3B-II accumulation in SW620 Ad300 cells with increased autophagic flux. More importantly, cell viability was increased after autophagy inhibition, indicating that autophagy induced by the two tanshinones was pro-cell death. Besides, the cytotoxic actions of the two tanshinones were p53-independent, which could be useful in inhibiting the growth of apoptosis-resistant cancer cells with p53 defects.ConclusionThe current findings strongly indicate that both CTS and DTS could inhibit the growth of apoptosis-resistant colon cancer cells through induction of autophagic cell death and p53-independent cytotoxicity. They are promising candidates to be further developed as therapeutic agents in the adjuvant therapy for colon cancer, especially for the apoptosis-resistant cancer types.     

Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells

Delivery of RNA interference (RNAi)-mediating agents to target cells is one of the major obstacles for the development of RNAi-based therapies. One strategy to overcome this barrier is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. In this study, the tkRNAi approach was used for modulation of the “classical” ABCB1-mediated multidrug resistance (MDR) in human cancer cells. Subsequent to treatment with anti-ABCB1 shRNA expression vector bearing E. coli, MDR cancer cells (EPG85 257RDB) showed 45% less ABCB1 mRNA expression. ABCB1 protein expression levels were reduced to a point at which merely a weak band could be detected. Drug accumulation was enhanced 11-fold, to an extent that it reached 45% of the levels in non-resistant cells and resistance to daunorubicin was decreased by 40%. The data provide the proof-of-concept that tkRNAi is suitable for modulation of “classical” MDR in human cancer cells. Overall, the prototype tkRNAi system tested here did not yet attain the levels of gene silencing seen with conventional siRNAs nor virally delivered shRNAs; but the tkRNAi system for gene-silencing of ABCB1 is still being optimized, and may become a powerful tool for delivery of RNAi effectors for the reversal of cancer MDR in future.