Baicalein Inhibits Plasmid-Mediated Horizontal Transmission of the blaKPC Multidrug Resistance Gene from Klebsiella pneumoniae to Escherichia coli.

Carbapenem-resistant bacterial infections pose an urgent threat to public health worldwide. Horizontal transmission of the ß-lacatamase Klebsiella pneumoniae carbapenemase (blaKPC) multidrug resistance gene is a major mechanism for global dissemination of carbapenem resistance. Here, we investigated the effects of baicalein, an active ingredient of a Chinese herbal medicine, on plasmid-mediated horizontal transmission of blaKPC from a meropenem-resistant K. pneumoniae strain (JZ2157) to a meropenem-sensitive Escherichia coli strain (E600). Baicalein showed no direct effects on the growth of JZ2157 or E600. Co-cultivation of JZ2157 and E600 caused the spread of meropenem resistance from JZ2157 to E600. Baicalein at 40 and 400 µg/mL significantly inhibited the spread of meropenem resistance. Co-cultivation also resulted in plasmid-mediated transmission of blaKPC from JZ2157 to E600, which was inhibited by baicalein. Therefore, baicalein may be used in clinical practice to prevent or contain outbreaks of carbapenem-resistant infections by inhibiting the horizontal transfer of resistance genes across bacteria species.

An Efflux Pumps Inhibitor Significantly Improved the Antibacterial Activity of Botanicals from Plectranthus glandulosus towards MDR Phenotypes.

Bacterial multidrug resistance causes many therapeutic failures, making it more difficult to fight against bacterial diseases. This study aimed to investigate the antibacterial activity of extract, fractions, and phytochemicals from Plectranthus glandulosus (Lamiaceae) against multidrug-resistant (MDR) Gram-negative phenotypes expressing efflux pumps. The crude extract after extraction was subjected to column chromatography, and the structures of the isolated compounds were determined using spectrometric and spectroscopic techniques. Antibacterial assays of samples alone and in the presence of an efflux pump inhibitor (phenylalanine-arginine ß-naphthylamide, PAßN) were carried out using the broth microdilution method. The phytochemical study of P. glandulosus plant extract afforded seven major fractions (A-G) which lead to the isolation of seventeen known compounds. The ethanol extract of P. glandulosus was not active at up to 1024 µg/mL, whereas its fractions showed MICs varying from 32 to 512 µg/mL on the studied bacteria. Fraction C of P. glandulosus showed the lowest MIC (32 µg/mL) on E. coli ATCC8739 strain. Fraction D presented the highest activity spectrum by inhibiting the growth of 90% (9/10) of the studied bacteria. The presence of PAßN has improved the activity of extract and all fractions. Overall, the tested phytochemicals showed low activity against the studied bacteria. The overall results obtained in this study show that some fractions from P. glandulosus, mainly fractions C and D, should be investigated more for their possible use to fight against MDR bacteria.

Repurposing of Ribavirin as an Adjunct Therapy against Invasive Candida Strains in an In Vitro Study.

The use of antifungal agents in clinical settings is limited by the appearance of drug resistance and adverse side effects. There is, therefore, an urgent need to develop new drugs to strengthen the treatment of invasive fungal diseases. The aim of this study is to describe the potential repurposing of ribavirin as an adjunct therapy against Candida spp. Primary screening of a Prestwick Chemical library against Candida albicans ATCC 90028 and fluconazole-resistant Candida albicans strains was performed. Subsequently, we evaluated the responses of 100 Candida sp. strains to ribavirin, an antiviral agent, using the broth microdilution method as recommended by CLSI. We checked the involvement of efflux pump activity in the development of ribavirin resistance. We studied time-kill curves and performed a checkerboard assay for a ribavirin-antifungal combination study. Twenty-one nonstandard antifungal compounds were identified, including ribavirin. Ribavirin had antifungal activity in vitro against 63 Candida strains, including strains of C. albicans, C. parapsilosis, and C. tropicalis, with MICs ranging from 0.37 to 3.02 µg/ml, while MICs for C. krusei, C. glabrata, C. lusitaniae, and some C. albicans strains remained high (≥24.16 µg/ml). No relation was observed between efflux pump activity and ribavirin resistance. Ribavirin exhibited fungistatic activity against multidrug-resistant (MDR) C. albicans and fungicidal activity against a C. parapsilosis strain. In addition, ribavirin acted synergistically with azoles against Candida strains for which ribavirin MICs were <24.4 µg/ml. This study highlights the potential clinical application of ribavirin, alone or in association with other antifungal agents, as an adjunct anti-Candida drug.

Spatiotemporal expression of the putative MdtABC efflux pump of Phtotorhabdus luminescens occurs in a protease-dependent manner during insect infection.

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.

Increase of zinc resistance in German human derived livestock-associated MRSA between 2000 and 2014.

PROBLEM ADDRESSED: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), particularly of the clonal complex (CC) 398, emerged as zoonotic pathogens predominantly among humans with direct or indirect livestock contact, but also in healthcare settings. The factors contributing to the success of LA-MRSA are only poorly understood. OBJECTIVE: During the past years, the use of heavy metal compounds as feed-supplements was found to influence the co-selection of LA-MRSA in pig herds. This study aimed to determine the prevalence of zinc resistance among MRSA CC398 isolated from patients of a German university hospital located in a pig farming-dense area. METHODS AND APPROACH: In comparison to concurrent healthcare-associated MRSA (HA-MRSA), LA-MRSA CC398 comprising isolates from their first appearance in 2000 to recent isolates from 2014 were included. RESULTS: Among MRSA CC398, the overall resistance rate towards zinc chloride was 57% compared to only 3% among concurrently isolated HA-MRSA. Zinc resistance correlated with the presence of the czrC gene in 100% of the MRSA CC398 and in 67% of the HA-MRSA. CONCLUSIONS: The zinc resistance rate in MRSA CC398 significantly increased from 2009 to 2014 with a maximum in 2014. Alarmingly, zinc resistance has become a frequent phenotype of human LA-MRSA in Germany potentially facilitating co-selection of antibiotic resistance genes.

Analogues of Disulfides from Allium stipitatum Demonstrate Potent Anti-tubercular Activities through Drug Efflux Pump and Biofilm Inhibition.

Disulfides from Allium stipitatum, commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S-methylthiolation of appropriate thiols using S-methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L-1) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32-138 µM (8-32 mg L-1). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M. tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

TanshinoneIIA enhances the chemosensitivity of breast cancer cells to doxorubicin through down-regulating the expression of MDR-related ABC transporters.

As the first-line drug for breast cancer chemotherapy, doxorubicin (Dox) has strong cardiotoxicity. Meanwhile, prolonged Dox treatment of patients with breast cancer may result in resistance of breast cancer cells to Dox and an increased number of Dox-resistant breast cancer stem cells (BCSCs), thereby easily leading to breast cancer relapse. TanshinoneIIA (Tan IIA) has anti-tumor activity in addition to its cardiovascular protective effect. By preparing Dox resistant human breast cancer MCF-7 cells, here, we wanted to assess a new use of Tan IIA in enhancing the chemosensitivity of breast cancer cells to Dox and investigated its possible mechanisms. The results showed that Tan IIA could enhance the anti-tumor effect of Dox on MCF-7 and MCF-7/dox cells in a dose-dependent manner, especially that of on MCF-7/dox cells. Even nontoxic dose of Tan IIA could also promote intracellular Dox accumulation of MCF-7 and MCF-7/dox cells through down-regulating the expression of efflux ABC transporters including P-gp, BCRP and MRP1, which can effectively eliminated cancerous cells including BCSCs, thereby enhancing the chemosensitivity of breast cancer. Therefore, Tan IIA can be used as a new potential chemotherapeutic sensitizer for the combination treatment of breast cancer.

Minocycline and Tigecycline: What Is Their Role in the Treatment of Carbapenem-Resistant Gram-Negative Organisms?

Carbapenem-resistant organisms are increasingly common worldwide, particularly in India and are associated with high mortality rates especially in patients with severe infection such as bacteremia. Existing drugs such as carbapenems and polymyxins have a number of disadvantages, but remain the mainstay of treatment. The tetracycline class of antibiotics was first produced in the 1940s. Minocycline, tetracycline derivative, although licensed for treatment of wide range of infections, has not been considered for treatment of multidrug-resistant organisms until recently and needs further in vivo studies. Tigecycline, a derivative of minocycline, although with certain disadvantages, has been frequently used in the treatment of carbapenem-resistant organisms. In this article, we review the properties of minocycline and tigecycline, the common mechanisms of resistance, and assess their role in the management of carbapenem-resistant organisms.

Inhibition of the multidrug efflux pump LmrS from Staphylococcus aureus by cumin spice Cuminum cyminum.

Staphylococcus aureus is a serious causative agent of infectious disease. Multidrug-resistant strains like methicillin-resistant S. aureus compromise treatment efficacy, causing significant morbidity and mortality. Active efflux represents a major antimicrobial resistance mechanism. The proton-driven multidrug efflux pump, LmrS, actively exports structurally distinct antimicrobials. To circumvent resistance and restore clinical efficacy of antibiotics, efflux pump inhibitors are necessary, and natural edible spices like cumin are potential candidates. The mode of cumin antibacterial action and underlying mechanisms behind drug resistance inhibition, however, are unclear. We tested the hypothesis that cumin inhibits LmrS drug transport. We found that cumin inhibited bacterial growth and LmrS ethidium transport in a dosage-dependent manner. We demonstrate that cumin is antibacterial toward a multidrug-resistant host and that resistance modulation involves multidrug efflux inhibition.

[Identification of multidrug resistance gene MDR1 associated microRNA of salvianolic acid A reversal in lung cancer].

This paper was aimed to investigate the microRNA associated with multidrug resistance gene MDR1 of salvianolic acid A reversal in lung cance. Human lung cancer A549 cells were divided into normal control group and drug group, and the MDR1 expression levels were determined by real-time quantitative PCR. MicroRNA expression profiling of normal control group and drug group were detected by using the latest microRNA microarray. Quantitative RT-PCR was used to validate the differentially expressed miRNA. Forecast of miRNA associated with MDR1 multi-resistant genes of up-regulated miRNA. Experimental results showed that the dosage of MDR1 expression level significantly lowered compared with control group. The miRNA expression spectrum analyses of human lung cancer A549 cells to drug group and the control group were detected by microRNA microarray, 426 differentially expressed miRNA were screened out. Then target prediction were performed for difference up-expression of miRNA and found that there were four obvious increase of miRNA associated with MDR1 multi-resistant genes. Real-time quantitative PCR for 4 microRNA verification, the results were consistent with the chip. So the author considered that salvianolic acid A down lung cancer multidrug resistance gene MDR1 is likely to be affected by the miRNA expression and regulation of target genes, to further clarify the traditional Chinese medicine to reverse multi-drug resistant mechanism provides the experimental basis.

Linezolid-resistant Staphylococcus aureus strain 1128105, the first known clinical isolate possessing the cfr multidrug resistance gene.

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZD(r) clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination.

Assessment of berberine as a multi-target antimicrobial: a multi-omics study for drug discovery and repositioning.

Postgenomics drug development is undergoing major transformation in the age of multi-omics studies and drug repositioning. Rather than applications solely in personalized medicine, omics science thus additionally offers a better understanding of a broader range of drug targets and drug repositioning. Berberine is an isoquinoline alkaloid found in many medicinal plants. We report here a whole genome microarray study in tandem with proteomics techniques for mining the plethora of targets that are putatively involved in the antimicrobial activity of berberine against Escherichia coli. We found DNA replication/repair and transcription to be triggered by berberine, indicating that nucleic acids, in general, are among its targets. Our combined transcriptomics and proteomics multi-omics findings underscore that, in the presence of berberine, cell wall or cell membrane transport and motility-related functions are also specifically regulated. We further report a general decline in metabolism, as seen by repression of genes in carbohydrate and amino acid metabolism, energy production, and conversion. An involvement of multidrug efflux pumps, as well as reduced membrane permeability for developing resistance against berberine in E. coli was noted. Collectively, these findings offer original and significant leads for omics-guided drug discovery and future repositioning approaches in the postgenomics era, using berberine as a multi-omics case study.

Small molecule inhibitors of multidrug resistance gene (MDR1) expression: preclinical evaluation and mechanisms of action.

The resistance of tumors to a number of structurally and functionally unrelated chemotherapeutic drugs has been a major obstacle for successful cancer chemotherapy. An important mechanism leading to multidrug resistance (MDR) is the overexpression of the 170 kDa P-glycoprotein (P-gp), which is a member of the ATP-binding cassette (ABC) superfamily of membrane transporters, encoded by the MDR1 gene. Aiming to overcome MDR and due to the clinical failure of P-gp inhibitors, downregulation of MDR1 expression by small molecules has been studied as a possible cancer adjuvant chemotherapy. Here we review the current knowledge on MDR1 gene expression downregulation by small molecules and the mechanisms underlying those effects observed in cancer cell lines, in an attempt to identify targets for future therapeutic interventions.

Antibiotic susceptibility profile of Aeromonas species isolated from wastewater treatment plant.

This study assessed the prevalence of antibiotic-resistant Aeromonas species isolated from Alice and Fort Beaufort wastewater treatment plant in the Eastern Cape Province of South Africa. Antibiotic susceptibility was determined using the disc diffusion method, and polymerase chain reaction (PCR) assay was employed for the detection of antibiotics resistance genes. Variable susceptibilities were observed against ciprofloxacin, chloramphenicol, nalidixic acid, gentamicin, minocycline, among others. Aeromonas isolates from both locations were 100% resistant to penicillin, oxacillin, ampicillin, and vancomycin. Higher phenotypic resistance was observed in isolates from Fort Beaufort compared to isolates from Alice. Class A pse1 ß-lactamase was detected in 20.8% of the isolates with a lower detection rate of 8.3% for bla(TEM) gene. Class 1 integron was present in 20.8% of Aeromonas isolates while class 2 integron and TetC gene were not detected in any isolate. The antibiotic resistance phenotypes observed in the isolates and the presence of ß-lactamases genes detected in some isolates are of clinical and public health concern as this has consequences for antimicrobial chemotherapy of infections associated with Aeromonas species. This study further supports wastewater as potential reservoirs of antibiotic resistance determinants in the environment.

Different transport activity of human triallelic MDR1 893Ala/Ser/Thr variant and its association with herb extracts.

Individual pharmacokinetic differences for herb-drug interaction have been associated with genetic variations of the multidrug resistance (MDR) gene. A high level expression of MDR protein increases cellular efflux and might decrease drug sensitivity. This study investigated the drug efflux activity difference of human MDR1 triallelic variant 2677G/T/A (rs2032582), as a nonsynonymous 893Ala/Ser/Thr, using Xenopus laevis oocytes and MDR1 overexpressing LLC-PK1 cells. Two MDR1 variants (2667T/893Ser and 2667A/893Thr) were generated using human MDR1 cDNA (2677G/893Ala). No significant difference in the expression of MDR1 893Ala/Ser/Thr was found in X. laevis oocytes. However, the MDR1 2667A/893Thr variant interestingly showed a significant decrease of efflux activity for both digoxin and daunorubicin compared with those of 893Ala and 893Ser variants. In further investigation assessing the inhibitory effects of three herbal extracts on MDR1, 893Ala and 893Ser showed significant decreases of efflux activities in treatments with P. cocos (p = 0.005 for 893Ser) and D. dasycarpus (p = 0.0009 for 893Ala; p = 0.002 for 893Ser) in X. laevis oocytes. The results in this study suggest that herbal medicines could interact with other drugs and change the therapeutic effects depending on the genetic polymorphisms of individuals.

Tetrandrine prevents acquired drug resistance of K562 cells through inhibition of mdr1 gene transcription.

INTRODUCTION: There have been no reports describing successful drug resistance reversal in tumors in the clinic until now. Conversion of drug resistance reversal studies to drug resistance prevention studies may assist in the development of more efficient anti-tumor strategies. The present study demonstrates the prevention of doxorubicin-induced drug resistance by tetrandrine on leukemia cells. MATERIALS AND METHODS: K562 cells were either solely treated with doxorubicin (0.6 mg/ml) or pretreated with tetrandrine of different concentrations (0.5, 1.0 and 2.0 microg/ml) for 24 h followed by combined-treatment with doxorubicin (0.6 microg/ml). RESULTS: The results showed that doxorubicin treatment only resulted in elevated levels of mdr1 mRNA/P-gp expression. Doxorubicin also induced up-regulation of P-gp functional activities, as intracellular retention of rhodamine was decreased; however, 2.0 microg/ml tetrandrine significantly inhibited the overexpression of doxorubicin-induced mdr1 mRNA/P-gp. Consistently, the functional activity of P-gp was also inhibited, which led to increased intracellular drug retention and the recovery of cell sensitivity to chemotherapeutic drugs in combined treatment groups. Both mRNA and protein levels of NF-kappaB were up-regulated in the cells treated with doxorubicin only. Results from an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated the enhanced binding to the promoter region of mdr1 gene compared to the control group. However, tetrandrine could markedly inhibit the doxorubicin-induced expression of NF-kappaB mRNA and protein. In addition, it also attenuated the NF-kappaB DNA-binding activity. CONCLUSION: In summary, tetrandrine can prevent doxorubicin-induced mdr1 mRNA/P-gp expression and P-gp functions in a dose-dependent manner through a mechanism that may involve inhibition of doxorubicin-induced NF-kappaB mRNA expression and protein activity.

Modulation of drug resistance in Staphylococcus aureus by a kaempferol glycoside from Herissantia tiubae (Malvaceae).

In an ongoing project to evaluate natural compounds isolated from plants from the Brazilian biodiversity as modulators of antibiotic resistance, kaempferol-3-O-beta-d-(6''-E-p-coumaroyl) glucopyranoside (tiliroside), isolated from Herissantia tiubae (Malvaceae) was investigated using the strain SA-1199B of Staphylococcus aureus, which overexpresses the norA gene encoding the NorA efflux protein which extrudes hydrophilic fluorquinolones and some biocides, such as benzalkonium chloride, cetrimide, acriflavine and ethidium bromide. The minimum inhibitory concentrations (MICs) of the antibiotics and biocides were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of tiliroside. Although tiliroside did not display relevant antibacterial activity (MIC = 256 microg/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics a reduction in the MIC was observed for norfloxacin (16-fold), ciprofloxacin (16-fold), lomefloxacin (four-fold) and ofloxacin (two-fold), and an impressive reduction in the MICs for the biocides (up to 128-fold). The results presented here represent the first report of a kaempferol glycoside as a putative efflux pump inhibitor in bacteria. The present finding indicates that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial resistance, i.e. a source of potential adjuvants of antibiotics.

Antibiotic-resistance profile in environmental bacteria isolated from penicillin production wastewater treatment plant and the receiving river.

The antibiotic-resistance characteristics of bacterial strains in antibiotic production wastewater treatment plants (WWTP) that contain high concentrations of antibiotics are unknown, as are the environmental effects of the discharge of wastewater from such facilities. In this study, 417 strains were individually isolated from the effluent of a WWTP that treated penicillin G production wastewater, as well as from downstream and upstream areas of the receiving river. The minimum inhibition concentrations (MICs) of 18 antibiotics representing seven classes were then determined for each of these strains. Relatively high similarity in the bacterial composition existed between the wastewater and downstream river samples when compared with the upstream sample. High resistance ratios and MIC values were observed for almost all antibiotics in wastewater isolates, followed by strains from downstream river, of which the resistance ratios and levels were still significantly higher than those of upstream strains. The resistance ratios and levels also significantly differed among strains belonged to different species in the penicillin production wastewater effluent and downstream river. In both samples, the resistances to beta-lactam antibiotics were more frequent, with much higher levels, than the other class antibiotics. Then five clinically important resistant genes mainly coding for extended-spectrum beta-lactamases (ESBLs) were determined for all strains, only bla(TEM-1) which did not belong to ESBL was detected in 17.3% and 11.0% of strains isolated from wastewater and downstream river respectively. Class I integrons were detected in 14% of wastewater isolates and 9.1% of downstream isolates, and primarily contained gene cassettes conferring resistance to aminoglycoside antibiotics. The unexpectedly high levels of multiple antibiotic resistance in strains from wastewater and downstream river were speculated to be mainly due to multidrug efflux systems.

Cellular folate status modulates the expression of BCRP and MRP multidrug transporters in cancer cell lines from different origins.

As cellular folate levels seem to have a different effect on cancer cells from different origins, we extended our initial study to a broader panel of cancer cells. BCRP and MRP1-5 expression was determined in KB, OVCAR-3, IGROV-1, ZR75-1/R/MTX, SCC-11B, SCC-22B, and WiDr either grown in standard RPMI 1640 containing 2.3 micromol/L supraphysiologic concentration of folic acid [high folate (HF)] or adapted to more physiologic concentrations [1-5 nmol/L folic acid or leucovorin; low folate (LF)]. Compared with the HF counterparts, KB LF cells displayed 16.1-fold increased MRP3 and OVCAR-3 LF cells showed 4.8-fold increased MRP4 mRNA levels along with increased MRP3 and MRP4 protein expression, respectively. A marked increase on BCRP protein and mRNA expression was observed in WiDr LF cells. These cells acquired approximately 2-fold resistance to mitoxantrone compared with the HF cell line, a phenotype that could be reverted by the BCRP inhibitor Ko143. Of note, WiDr cells expressed BCRP in the intracellular compartment, similarly to what we have described for Caco-2 cells. Our results provide further evidence for an important role of cellular folate status in the modulation of the expression of multidrug resistance transporters in cancer cells. We show that up-regulation of intracellularly localized BCRP in response to adaptation to LF conditions may be a common feature within a panel of colon cancer cell lines. Under these circumstances, folate supplementation might improve the efficacy of chemotherapeutic drugs by decreasing BCRP expression.