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Dubin-Johnson syndrome (DJS) is a rare benign autosomal recessive disorder characterized by cholestasis in neonates. The aim of the present study was to describe the clinical characteristics, hepatic profiles, histopathology, gene mutations, and treatment outcomes of neonatal DJS.A multicenter retrospective study was undertaken with patients who had DJS. The authors identified DJS in neonates and reviewed medical records for details. The diagnosis of DJS was based on the presence of unexplained prolonged conjugated hyperbilirubinemia and presence of a mutation in the ATP Binding Cassette Subfamily C Member 2 (ABCC2) gene detected in genomic DNA extracted from circulating blood cells.Eleven children with DJS were identified in the study. The study population comprised eight males and three females. The median age at presentation was 21 days. Dysmorphic features were not recorded in any of the patients. Cholestasis, high serum bile acids, and normal transaminase levels were found in all patients (100%). Serum alkaline phosphatase and gamma glutamyl transferase were elevated in four patients (36%). Hypoalbuminemia and coagulopathy were not noted in these patients. Consanguinity was present in nine patients (82%). All patients had normal abdominal ultrasound findings. Genetic molecular testing showed that 82% of the patients reported a pathogenic variant of the ABCC2 gene defect with the same variant c.2273GT (Gly 758 val) chromosome 10. All patients were alive without liver transplantation.This is the largest study worldwide describing that neonatal DJS is a benign cholestatic disease with favorable outcomes. Low-grade direct hyperbilirubinemia, normal transaminases, and elevated serum bile acids are the main characteristic findings of DJS.

Abstract: ATP-binding cassette (ABC) transporters play a critical role in protecting vital organs such as the brain and placenta against xenobiotics, as well as in modulating the pharmacological and toxicological profile of several drug candidates by restricting their penetration through cellular and tissue barriers. This review paper describes the structure and function of ABC transporters as well as the role of P-glycoprotein, multidrug resistance-associated protein 2 and breast cancer resistance protein in the disposition of drugs. Furthermore, a review of the in vitro and in vivo techniques for evaluating the interaction between drugs and ABC transporters is provided.

Vanillin is used as a flavor ingredient in a diverse range of food categories and is known to inhibit microbial fermentation. In this study, we found that vanillin improved the hydrophobic organic solvent tolerance of Escherichia coli and showed that the AcrAB-TolC efflux pump is implicated in that tolerance. The expression level of the pump was enhanced by the addition of vanillin. AcrAB-TolC efflux pump expression is known to be regulated by transcription activators such as MarA, SoxS, and Rob. Among these three transcription factors, marA transcription was significantly elevated by the addition of vanillin. We found that the AcrAB-TolC efflux pump is involved also in vanillin tolerance. The ΔacrB mutant was more sensitive to vanillin than the parent strain. A complementation test revealed that the introduction of the acrB gene recovered the vanillin tolerance of the ΔacrB mutant.

Methicillin-resistant

Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3',5'-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina.

Background Chemotherapy resistance is a main reason for treatment failure in extranodal NK/T-cell lymphoma (ENKTL). Interleukin-10 (IL-10) is closely related to the occurrence and prognosis of ENKTL. We intended to study the role and molecular mechanism of IL-10 in ENKTL resistance. Methods Fifty serum samples were collected from patients with a histologically proven diagnosis of ENKTL. Fifty healthy volunteers were enrolled as a control group. The level of serum IL-10 was detected by ELISA. The NK/T-cell lymphoma cell lines YT and NK-92 were divided into the control group (untreated), IL-10 group (treated with IL-10), IL-10 + GEM group (treated with IL-10 and gemcitabine simultaneously) and GEM group (treated with gemcitabine). A CCK8 assay and flow cytometry were employed to detect the effects of IL-10 on each group. Western blotting was applied to detect the expression of ABC membrane transporter family proteins and signaling pathway proteins in each group. Results Serum IL-10 levels were higher in ENKTL patients as well asin patients with ineffective treatment. The IC50 value for gemcitabine in YT and NK-92 cells increased significantly in the presence of IL-10. The effects of gemcitabine resulting in cell killing, cell cycle arrest, and apoptosis promotion were also weakened by IL-10. The expression of ABCC4, STAT1, p-STAT1, Tyk2 and p-Tyk2 was significantly increased by IL-10. Conclusion Our results indicate that IL-10 contributes to the resistance of ENKTL cells via ABCC4 and that IL-10 regulates the JAK/STAT signaling pathway in YT and NK-92 cells.

Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes from the brain. Several organic cation and anion transporters are expressed in the CPE cells, but how they functionally mediate transepithelial transport of organic cations and anions remain unclear. In this study, we visualized the transcellular transport of fluorescent organic cation and organic anion probes using live tissue imaging in freshly isolated mouse choroid plexuses (CPs). The cationic probe, 4-[4-(dimethylamino)phenyl]-1-methylpyridinium iodide (IDT307) was transported into CPE cells at the apical membrane and highly accumulated in mitochondria. Consistent with the lack of expression of organic cation efflux transporters, there was little efflux of IDT307 into the blood capillary space. Furthermore, IDT307 uptake and intracellular accumulation was attenuated by approximately 70% in CP tissues from mice with targeted deletion of the plasma membrane monoamine transporter (Pmat). In contrast, the anionic probe fluorescein-methotrexate (FL-MTX) was rapidly transported across the CPE cells into the capillary space with little intracellular accumulation. Rifampicin, an inhibitor of organic anion transporting polypeptides (OATPs), completely blocked FL-MTX uptake into the CPE cells whereas MK-571, a pan-inhibitor of multidrug resistance associated proteins (MRPs), abolished basolateral efflux of FL-MTX. In summary, our results suggest distinct transcellular transport pathways for organic cations and anions at the BCSFB and reveal a pivotal role of PMAT, OATP and MRP transporters in organic cation and anion transport at the blood-cerebrospinal fluid interface. SIGNIFICANCE STATEMENT: Live tissue imaging revealed that while organic cations are transported from the cerebrospinal fluid (CSF) into the choroid plexus epithelial cells by plasma membrane monoamine transporter without efflux into the blood, amphipathic anions in the CSF are efficiently transported across the BCSFB through the collaborated function of apical organic anion transporting polypeptides and basolateral multidrug resistance associated proteins. These findings contribute to a mechanistic understanding of the molecular and cellular pathways for choroid plexus clearance of solutes from the brain.

Drug efflux transporters were highly related to the clinical drug resistance issues, such as cancer multi-drug resistance (MDR) and ocular drug resistance. In the present study, with the focus on human multi-drug resistance protein 1 (MRP1) and P-glycoprotein (P-gp), the inhibitory kinetics of polyoxyethylene (20) sorbitan monooleate (Tween 80) on both drug binding sites and ATPase were in-depth evaluated. We used the stable-cloned ABCB1/Flp-In™-293 and ABCC1/Flp-In™-293 cell lines, and inside-out membrane vesicles for underlying mechanisms investigation while used the drug induced cancer MDR cell line KB/VIN and human retinal pigmented epithelium cell line ARPE-19 for efficacy evaluation. Results showed that Tween 80 exhibited non-competitive inhibition on the doxorubicin efflux of P-gp and MRP1, with the inhibitory affinity 0.00195% (14.89 μM) and 0.00245% (18.7 μM), respectively. Tween 80 inhibited the basal ATPase activity of P-gp and MRP1 in a dose-dependent manner (0.0002-0.02%) and demonstrated significant reversing effects on the doxorubicin, paclitaxel, and vincristine resistance at the concentration of 0.001% (7.63 μM). This was the first thorough study revealing the interactions between Tween 80 and P-gp or MRP1 at a molecular level and these findings suggested that Tween 80 was a potential candidate for future combinatorial regimens applied in the "drug resistance" issue.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae. It consists of the AcrB transporter, which is embedded in the inner membrane, the AcrA adapter located in the periplasm, and the channel protein TolC responsible for the transport of substrates towards the extracellular environment. Besides conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here we report that the AcrAB pump heavily affects the infection process of the LF82 strain, the prototype of Adherent-Invasive Escherichia coli (AIEC) which are highly abundant in the ileal mucosa of Chron disease patients. We found that the deletion of genes encoding AcrA and/or AcrB leads to decreased survival of LF82 within macrophages. Ectopic AcrAB expression in a acrAB defective mutant restores the wild type condition. Furthermore, we demonstrate that inhibition of AcrB and replacement of the transporter with an unfunctional AcrB also interfere with bacterial viability inside macrophages. Overall, these data suggest a pivotal role of the AcrAB efflux pump in bacteria-host cell interactions also in AIEC.

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

Objectives The NorA antiporter of Staphylococcus aureus belongs to the major facilitator superfamily (MFS) and extrudes various kinds of drugs. With no structure available for this drug efflux pump, the aim of this study was to explore its important structural elements that contribute to substrate binding and drug transport. Methods Evolutionary conservative analyses were conducted on different compilations of NorA homologues to identify conservative motifs and residues. Site-directed mutations were constructed to verify the functional changes in NorA efflux capacities and the conformational changes were further measured by fluorescence resonance energy transfer (FRET) and microscale thermophoresis (MST) analysis. Results Besides Motif-A, Motif-B and Motif-C that were reported previously in MFS proteins, two other motifs, Motif-1 and Motif-2, were identified in NorA. Site-directed mutations of Motif-1 and Motif-2 as well as 11 predicted binding sites all caused remarkable reductions in drug resistance and efflux activity. Among these, mutant F16A/E222A/F303A/D307A showed an altered binding affinity for tetraphenylphosphonium chloride when measured by MST and Motif-1 mutant G114D/A117E/D118G/V119I and Motif-2 mutant Q325E/G326E/A328E/G330E displayed obvious conformational alterations when compared with the wild-type NorA in the FRET signal spectra. Conclusions The NorA structure agrees well with the typical structures of MFS proteins, with two newly identified motifs (Motif-1 and Motif-2) that are critical to the structural stability of NorA, and sites F16, E222, F303 and D307 are involved in substrate binding.

Multidrug-resistant plasmid-carrying bacteria are of particular clinical concern as they could transfer antibiotic resistance genes to other bacterial species. However, little is known whether evolutionary adaptation of plasmid-carrying bacteria after long-term antibiotic exposure could affect their subsequent colonization of the human gut. Herein, we combined a long-term evolutionary model based on Escherichia coli K-12 MG1655 and the multidrug-resistant plasmid RP4 with in vivo colonization experiments in mice. We found that the evolutionary adaptation of plasmid-carrying bacteria to antibiotic exposure facilitated colonization of the murine gut and subsequent plasmid transfer to gut bacteria. The evolved plasmid-carrying bacteria exhibited phenotypic alterations, including multidrug resistance, enhanced bacterial growth and biofilm formation capability and decreased plasmid fitness cost, which might be jointly caused by chromosomal mutations (SNPs in rpoC, proQ, and hcaT) and transcriptional modifications. The upregulated transcriptional genes, e.g., type 1 fimbrial-protein pilus (fimA and fimH) and the surface adhesin gene (flu) were likely responsible for the enhanced biofilm-forming capacity. The gene tnaA that encodes a tryptophanase-catalyzing indole formation was transcriptionally upregulated, and increased indole products participated in facilitating the maximum population density of the evolved strains. Furthermore, several chromosomal genes encoding efflux pumps (acriflavine resistance proteins A and B (acrA, acrB), outer-membrane protein (tolC), multidrug-resistance protein (mdtM), and macrolide export proteins A and B (macA, macB)) were transcriptionally upregulated, while most plasmid-harboring genes (conjugal transfer protein (traF) and (trbB), replication protein gene (trfA), beta-lactamase TEM precursor (blaTEM), aminoglycoside 3'-phosphotransferase (aphA) and tetracycline resistance protein A (tetA)) were downregulated. Collectively, these findings demonstrated that evolutionary adaptation of plasmid-carrying bacteria in an antibiotic-influenced environment facilitated colonization of the murine gut by the bacteria and plasmids.

Objectives To determine whether expression of efflux pumps and antibiotic susceptibility are altered in Escherichia coli in response to efflux inhibition. Methods The promoter regions of nine efflux pump genes (acrAB, acrD, acrEF, emrAB, macAB, cusCFBA, mdtK, mdtABC, mdfA) were fused to gfp in pMW82 and fluorescence from each reporter construct was used as a measure of the transcriptional response to conditions in which AcrB was inhibited, absent or made non-functional. Expression was also determined by RT-qPCR. Drug susceptibility of efflux pump mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was investigated. Results Data from the GFP reporter constructs revealed that no increased expression of the tested efflux pump genes was observed when AcrB was absent, made non-functional, or inhibited by an efflux pump inhibitor/competitive substrate, such as PAβN or chlorpromazine. This was confirmed by RT-qPCR for PAβN and chlorpromazine; however, a small but significant increase in macB gene expression was seen when acrB is deleted. Efflux inhibitors only synergized with antibiotics in the presence of a functional AcrB. When AcrB was absent or non-functional, there was no impact on MICs when other efflux pumps were also made non-functional. Conclusions Absence, loss-of-function, or inhibition of E. coli AcrB did not significantly increase expression of other efflux pump genes, which suggests there is no compensatory mechanism to overcome efflux inhibition and supports the discovery of inhibitors of AcrB as antibiotic adjuvants.

Parenteral nutrition (PN)-associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1β derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model.To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium [DSS] for 4 days) followed by central venous catheterization and 14-day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP-binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP-binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP-binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll-1b, C-C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1β exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver-specific effect of GW4064 in this model.GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.

Excretion of [14C]HR011303-derived radioactivity showed significant species difference. Urine (81.50% of dose) was the main excretion route in healthy male subjects, whereas feces (87.16% of dose) was the main excretion route in rats. To further elucidate the underlying cause for excretion species differences of HR011303, studies were conducted to uncover its metabolism and excretion mechanism. M5, a glucuronide metabolite of HR011303, is the main metabolite in humans and rats. Results of rat microsomes incubation study suggested that HR011303 was metabolized to M5 in the rat liver. According to previous studies, M5 is produced in both human liver and kidney microsomes. We found M5 in human liver can be transported to the blood by multidrug resistance-associated protein (MRP) 3 and then the majority of M5 can be hydrolyzed to HR011303. HR011303 enters the human kidney or liver through passive diffusion, whereas M5 is taken up through organic anion transporter (OAT) 3, organic anion-transporting polypeptide (OATP) 1B1, and OATP1B3. When HR011303 alone was present, it can be metabolized to M5 in both sandwich-cultured rat hepatocytes (SCRH) and sandwich-cultured human hepatocytes (SCHH) and excreted into bile as M5 in SCRH. Using transporter inhibitors in sandwich-cultured model and membrane vesicles that expressing MRP2 or Mrp2, we found M5 was substance of MRP2/Mrp2 and the bile efflux of M5 mainly mediated by MRP2/Mrp2. Considering the significant role of MRP3/Mrp3 and MRP2/Mrp2 in the excretion of glucuronides, the competition between them for M5 was possibly the determinant for the different excretion routes in humans and rats. Significance Statement Animal experiments are necessary to predict dosage and safety of candidate drugs prior to clinical trials. However, extrapolation results often differ from actual situation. For HR011303, excretory pathways exhibited a complete reversal, through urine in humans and feces in rats. Such phenomena have been observed in several drugs, but no in-depth studies have been conducted to date. In the present study, the excretion species differences of HR011303 can be explained by the competition for M5 between MRP2/Mrp2 and MRP3/Mrp3.

Sorafenib is a first-line molecular-target drug for advanced hepatocellular carcinoma (HCC), and reducing sorafenib resistance is an important issue to be resolved for the clinical treatment of HCC. In the current study, we identified that ABCC5 is a critical regulator and a promising therapeutic target of acquired sorafenib resistance in human hepatocellular carcinoma cells. The expression of ABCC5 was dramatically induced in sorafenib-resistant HCC cells and was remarkably associated with poor clinical prognoses. The down-regulation of ABCC5 expression could significantly reduce the resistance of sorafenib to HCC cells. Importantly, activation of PI3K/AKT/NRF2 axis was essential for sorafenib to induce ABCC5 expression. ABCC5 increased intracellular glutathione (GSH) and attenuated lipid peroxidation accumulation by stabilizing SLC7A11 protein, which inhibited ferroptosis. Additionally, the inhibition of ABCC5 enhanced the anti-cancer activity of sorafenib in vitro and in vivo. These findings demonstrate a novel molecular mechanism of acquired sorafenib resistance and also suggest that ABCC5 is a new regulator of ferroptosis in HCC cells.

Glioblastoma multiform (GBM) is the most frequent type of malignant brain tumor with a poor prognosis. After optimal surgery, radiotherapy plus temozolomide (TMZ) is the standard treatment for GBM patients. However, the development of TMZ resistance limits its efficacy in GBM management. Runt Related Transcription Factor 1 (RUNX1) and microRNAs have been implicated in drug resistance of TMZ in GBM. In this study, we revealed the underlying mechanism of TMZ resistance and identified miR-128-3p/RUNX1 axis as a novel target for TMZ resistance in GBM. RUNX1 expression was significantly upregulated in GBM tissues as compared to normal tissues, and its expression was even higher in recurrent GBM tissues and TMZ-resistant GBM cells. RUNX1 depletion inhibited the viability, proliferation, migration, invasion and TMZ resistance of GBM cells, which could be rescued by RUNX1 overexpression. We further identified miR-128-3p as a tumor-suppressor whose overexpression restored the sensitivity of TMZ in GBM cells. miR-128-3p negatively regulated RUNX1 and subsequently downregulated multidrug resistance-associated protein 1 (MRP1). Together, the present study indicates that RUNX1 confers TMZ resistance in GBM by upregulating MRP1, which is negatively regulated by miR-128-3p. Targeting miR-128-3p/RUNX1/MRP1 axis provides a potential strategy to overcome TMZ resistance in GBM.

Since single nucleotide polymorphisms (SNPs) have attracted attention, there have been many explorations and improvements in screening and detection methods for SNPs. Traditional methods are complex and time-consuming and rely on expensive instruments. Therefore, there is an urgent need for a low-cost, simple, and accurate method that is convenient for use in resource-poor areas. Thus, a platform based on allele-specific PCR (AS-PCR) and a gold nanoparticle-based lateral flow assay (LFA) was developed, optimized, and used to detect the SNPs of the drug resistance gene

Circular RNAs (circRNAs) exert pivotal functions in many malignancies. However, the roles of circ-ABCC4 in prostate cancer (PCa) radioresistance and progression remain largely unclear. Cell viability, proliferation, apoptosis, invasion, and radioresistance were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell invasion, and colony formation assays. Tumor xenograft experiment was conducted to assess circ-ABCC4 role in xenograft growth in vivo. Dual-luciferase reporter assay was implemented to test the target relation of microRNA-1253 (miR-1253) and circ-ABCC4 or SRY-box transcription factor 4 (SOX4). Circ-ABCC4 enrichment was prominently raised in PCa tissue specimens and cells. Circ-ABCC4 depletion blocked PCa cell viability, proliferation, invasion, and radioresistance and triggered apoptosis. Circ-ABCC4 silencing aggravated irradiation-induced inhibitory effect on xenografts growth. miR-1253 was a downstream molecule of circ-ABCC4, and circ-ABCC4 depletion-mediated anti-cancer impacts in PCa cells were partly counteracted by decreasing miR-1253 abundance. miR-1253 targeted SOX4 mRNA, and miR-1253 blocked PCa cell malignant phenotypes partly by targeting SOX4. Circ-ABCC4 could enhance SOX4 abundance by absorbing miR-1253. Circ-ABCC4 exerted a pro-tumor activity by facilitating PCa cell viability, proliferation, invasion, and radioresistance and suppressing apoptosis.

Background Drug resistance is a major clinical drawback behind the failure of chemotherapy in non‐small cell lung cancer (NSCLC). In this study, we undertook to identify the precise role of circular RNA (circRNA) circ_0058357 in the functional properties of DDP‐resistant NSCLC cells. Methods Circ_0058357, miR‐361‐3p and ATP‐binding cassette (ABC) subfamily C member 1 (ABCC1) were quantified by qRT‐PCR and western blot. Cell survival and viability were gauged by MTT assay. Cell proliferation, apoptosis, invasion and migration were measured by EdU, flow cytometry, transwell and wound‐healing assays, respectively. The direct relationship between miR‐361‐3p and circ_0058357 or ABCC1 was validated by dual‐luciferase reporter assay. Results Our data showed that circ_0058357 was highly expressed in DDP‐resistant NSCLC tissues and cells. Inhibition of circ_0058357 repressed cell growth, invasion, migration, and promoted DDP sensitivity and cell apoptosis of H1299/DDP and A549/DDP cells in vitro. Moreover, inhibition of circ_0058357 diminished the growth of A549/DDP cells and sensitized them to the cytotoxic effect of DDP in vivo. Mechanistically, circ_0058357 contained a miR‐361‐3p binding site and miR‐361‐3p was identified as a molecular mediator of circ_0058357 regulation. MiR‐361‐3p suppressed ABCC1 expression by binding to ABCC1 3′UTR, and miR‐361‐3p‐mediated inhibition of ABCC1 affected the growth, invasion, migration, apoptosis and DDP sensitivity of H1299/DDP and A549/DDP cells. Furthermore, circ_0058357 regulated ABCC1 expression by competitively binding to shared miR‐361‐3p. Conclusions Our findings identified that inhibition of circ_0058357 suppresses the growth and metastasis of H1299/DDP and A549/DDP cells and sensitizes them to DDP therapy partially by targeting the miR‐361‐3p/ABCC1 axis., Circ_0058357 affects ABCC1 expression through miR‐361‐3p. H1299/DDP (a) and A549/DDP (b) cells were transfected with si‐circ_0058357 + anti‐miR‐361‐3p, si‐circ_0058357 + anti‐miR‐NC, si‐circ_0058357 or si‐NC and checked for ABCC1 protein level by western blot.

MRP4 (gene ABCC4) is a polymorphic efflux transporter that has been implicated in drug-induced toxicity. We selected ten commonly observed MRP4 coding variants among Europeans for experimental characterization including nine variants predicted to be deleterious or functional (combined annotation-dependent depletion score >15). We assessed protein localization and activity by quantifying intracellular accumulation of two prototypic substrates, taurocholic acid (TCA) and estradiol 17-β-glucuronide (E217βG), in HEK293T over-expressing MRP4 wildtype or variant where cellular substrate loading was optimized through co-transfection with an uptake transporter. V458M, a novel variant not previously studied, and T1142M, showed reduced activity compared to MRP4 wildtype for E217βG and TCA (P

Objectives This study was performed to identify and characterize circulating Plasmodium species in three provinces of Mindanao approaching malaria elimination. Methods Rapid diagnostic tests (RDT), microscopic examination, and PCR were used to detect malaria parasites. PCR-positive isolates were genotyped for polymorphisms in loci of interest. Results A total of 2639 participants were surveyed in Mindanao between 2010 and 2013. Malaria prevalence by PCR was 3.8% (95% confidence interval (CI): 2.7–5.2%) in Sarangani, 10% (95% CI: 7.7–12.7%) in South Cotabato, and 4.2% (95% CI: 3.2–5.6%) in Tawi-Tawi. P. falciparum and P. vivax were identified in all three provinces, and there was one case of P. malariae in South Cotabato. RDT was inferior to PCR for detecting asymptomatic P. falciparum and P. vivax. In Tawi-Tawi, microscopy failed to identify 46 PCR-positive malaria infections. The presence of pfcrt haplotypes CVMNK, CVIET, and SMNT (codons 72–76), pfmdr1 haplotype NFSND (codons 86, 184, 1034, 1042, 1246), and pvmdr1 haplotype NFL (codons 91, 976, 1076) was confirmed in Mindanao. Conclusions Asymptomatic Plasmodium infections persisted in local communities between 2010 and 2013. PCR successfully identified subpatent malaria infections, and can better characterize malaria epidemiology in communities seeking malaria control and elimination at the local level.

Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer. Many patients with hepatocellular carcinoma are diagnosed at an advanced stage because the early symptoms are not obvious. For advanced hepatocellular carcinoma, immunotherapy and targeted therapy seem to be a promising direction. Finding a new prognostic marker for hepatocellular carcinoma and exploring its role in the immune microenvironment is of great value. ABCC transporters have previously been associated with drug resistance in hepatocellular tumors, but the exact mechanism remains unclear. Here, we conducted a study on ABCC5 in HCC and found that the expression of ABCC5 was up-regulated in HCC and was associated with poor prognosis. Further exploration revealed that ABCC5 was associated with immune infiltration of hepatocellular carcinoma. Single-cell analysis revealed a potential relationship between ABCC5 and immune cell differentiation. Therefore, it is significant to continue to explore the role of ABCC5 in hepatocellular carcinoma.

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun's lipoprotein (Lpp), is still largely unknown. In this paper, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane (IM), OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

Moyamoya disease (MMD) is an occlusive, chronic cerebrovascular disease affected by genetic mutation and the immune response. Furthermore, vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) participate in the neointima of MMD, but the etiology and pathophysiological changes in MMD vessels remain largely unknown. Therefore, we established the circZXDC (ZXD family zinc finger C)–miR-125a-3p–ABCC6 (ATP-binding cassette subfamily C member 6) axis from public datasets and online tools based on “sponge-like” interaction mechanisms to investigate its possible role in VSMCs. The results from a series of in vitro experiments, such as dual luciferase reporter assays, cell transfection, CCK-8 assays, Transwell assays, and Western blotting, indicate a higher level of circZXDC in the MMD plasma, especially in those MMD patients with the RNF213 mutation. Moreover, circZXDC overexpression results in a VSMC phenotype switching toward a synthetic status, with increased proliferation and migration activity. CircZXDC sponges miR-125a-3p to increase ABCC6 expression, which induces ERS (endoplasmic reticulum stress), and subsequently regulates VSMC transdifferentiation from the contractive phenotype to the synthetic phenotype, contributing to the intima thickness of MMD vessels. Our findings provide insight into the pathophysiological mechanisms of MMD and indicate that the circZXDC–miR-125a-3p–ABCC6 axis plays a pivotal role in the progression of MMD. Furthermore, circZXDC might be a diagnostic biomarker and an ABCC6-specific inhibitor and has the potential to become a promising therapeutic option for MMD.

Pseudoxanthoma elasticum (PXE) is a multisystem, genetic, ectopic mineralization disorder with no effective treatment. Inhibition of tissue-nonspecific alkaline phosphatase (TNAP) may prevent ectopic soft tissue calcification by increasing endogenous pyrophosphate (PPi). This study evaluated the anticalcification effects of DS-1211, an orally administered, potent, and highly selective small molecule TNAP inhibitor, in mouse models of PXE. Calcium content in vibrissae was measured in KK/HlJ and ABCC6-/- mice after DS-1211 administration for 13–14 weeks. Pharmacokinetic and pharmacodynamic effects of DS-1211 were evaluated, including plasma alkaline phosphatase (ALP) activity and biomarker changes in PPi and pyridoxal-phosphate (PLP). Anticalcification effects of DS-1211 through TNAP inhibition were further evaluated in ABCC6-/- mice with genetically reduced TNAP activity, ABCC6-/-/TNAP+/+ and ABCC6-/-/TNAP+/-. In KK/HlJ and ABCC6-/- mouse models, DS-1211 inhibited plasma ALP activity in a dose-dependent manner and prevented progression of ectopic calcification compared with vehicle-treated mice. Plasma PPi and PLP increased dose-dependently with DS-1211 in ABCC6-/- mice. Mice with ABCC6-/-/TNAP+/- phenotype had significantly less calcification and higher plasma PPi and PLP than ABCC6-/-/TNAP+/+ mice. TNAP plays an active role in pathomechanistic pathways of dysregulated calcification, demonstrated by reduced ectopic calcification in mice with lower TNAP activity. DS-1211 may be a potential therapeutic drug for PXE.

NorA is one of the main native MDR efflux pumps of

The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.

To explore the genetic etiology in four patients with hyperbilirubinemia, and discuss the correlation between clinical characteristics and molecular basis.The data of clinical manifestation and auxiliary examinations were collected. Genomic DNA of the four patients was extracted and analyzed by next-generation sequencing using the panel including genes involved in hereditary metabolic liver diseases. Suspected variants were verified by Sanger sequencing.All of the four patients were males with normal liver enzymes. It was revealed that all the patients had heterozygous variants, among which c.3011CT, c.2443CT and c.2556del were the variants which have not been reported previously.All of the patients were diagnosed as Dubin-Johnson syndrome (DJS) caused by ABCC2 gene variants. The novel variants add to the spectrum of genetic variants of the disease. Because of the favorite prognosis, precise diagnosis can greatly reduce the psychological pressure of patients and avoid excessive treatments. At the same time, it could provide pertinent genetic counseling for the families.

The small multidrug resistance (SMR) family is composed of widespread microbial membrane proteins that fulfill different transport functions. Four functional SMR subtypes have been identified, which variously transport the small, charged metabolite guanidinium, bulky hydrophobic drugs and antiseptics, polyamines, and glycolipids across the membrane bilayer. The transporters possess a minimalist architecture, with ∼100-residue subunits that require assembly into homodimers or heterodimers for transport. In part because of their simple construction, the SMRs are a tractable system for biochemical and biophysical analysis. Studies of SMR transporters over the last 25years have yielded deep insights for diverse fields, including membrane protein topology and evolution, mechanisms of membrane transport, and bacterial multidrug resistance. Here, we review recent advances in understanding the structures and functions of SMR transporters. New molecular structures of SMRs representing two of the four functional subtypes reveal the conserved structural features that have permitted the emergence of disparate substrate transport functions in the SMR family and illuminate structural similarities with a distantly related membrane transporter family, SLC35/DMT.

Antibiotic combinations remain the frontline therapy for severe infections to reduce mortality. However, conventional antibiotic combinations have some limitations such as the low bioavailability and the rise of resistant strains. Nanoparticles are increasingly used as antibiotic delivery systems to promote bioavailability and hence improve efficacy of antibiotics. In this work, we hypothesize that the simultaneous delivery of two antibiotic-loaded nanoparticles will improve the intracellular bioavailability and thus inhibit emergence of resistance. Accordingly, Chitosan-pluronic nanoparticles were used to construct nanosized ciprofloxacin and meropenem and the antibacterial activity of nanosized combined antibiotics were compared versus unloaded single, unloaded combined, and nanosized single antibiotics. Thirty-six stepwise mutants were selected by exposing two

To explore the genetic etiology and differential diagnosis for a patient with jaundice.Clinical data of the patient and his parents were collected. Genes associated with metabolic liver diseases were subjected to high-throughput sequencing. The pathogenicity of the candidate variants was predicted by using bioinformatics software.High-throughput sequencing revealed that the proband has harbored two variants of the ABCC2 gene (NM_000392) including c.3011CT (p.T1004I) and c.3541CT (p.R1181X), which were respectively inherited from his father and mother. Both variants have been previously unreported and predicted to be pathogenic by bioinformatics analysis.The proband was diagnosed with Dubin-Johnson syndrome due to the compound heterozygous variants of the ABCC2 gene. Genetic testing has enabled accurate differential diagnosis of Dubin-Johnson syndrome in this patient.

Generalized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial calcification, and high mortality during the first year of life, but the natural course and possible differences between the causative genes remain unknown. In all, 247 individual records for patients with GACI (from birth to 58.3 years of age) across 19 countries were reviewed. Overall mortality was 54.7% (13.4% in utero or stillborn), with a 50.4% probability of death before the age of 6 months (critical period). Contrary to previous publications, we found that bisphosphonate treatment had no survival benefit based on a start-time matched analysis and inconclusive results when initiated within 2 weeks of birth. Despite a similar prevalence of GACI phenotypes between ENPP1 and ABCC6 deficiencies, including arterial calcification (77.2% and 89.5%, respectively), organ calcification (65.8% and 84.2%, respectively), and cardiovascular complications (58.4% and 78.9%, respectively), mortality was higher for ENPP1 versus ABCC6 variants (40.5% versus 10.5%, respectively; p = 0.0157). Higher prevalence of rickets was reported in 70.8% of surviving affected individuals with ENPP1 compared with that of ABCC6 (11.8%; p = 0.0001). Eleven affected individuals presenting with rickets and without a GACI diagnosis, termed autosomal recessive hypophosphatemic rickets type 2 (ARHR2), all had confirmed ENPP1 variants. Approximately 70% of these patients demonstrated evidence of ectopic calcification or complications similar to those seen in individuals with GACI, which shows that ARHR2 is not a distinct condition from GACI but represents part of the spectrum of ENPP1 deficiency. Overall, this study identified an early mortality risk in GACI patients despite attempts to treat with bisphosphonates, high prevalence of rickets almost exclusive to ENPP1 deficiency, and a spectrum of heterogenous calcification and multiple organ complications with both ENPP1 and ABCC6 variants, which suggests an overlapping pathology. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

The ATP binding cassette (ABC) transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. By labeling the Cry1Aa protein, we have found that virtually all of the binding is in an oligomeric state, a conformation that allowed higher levels of specific binding that could not be achieved by the monomeric protein on its own. Furthermore, we have observed that Cry1A proteins can hetero-oligomerize in the presence of the transporter, which is reflected in an increase in binding and toxicity to SeABCC2-expressing cells. This synergism can be one of the reasons why B. thuringiensis co-expresses different Cry1 proteins that can apparently have similar binding preferences. The results from in vitro competition and ex vivo competition showed that Cry1Aa, Cry1Ab and Cry1Ac share functional binding sites. By using Cry1Ab–Cry1Ac chimeras, the presence of domain I from Cry1A proteins was revealed to be critical for oligomer formation.

Undue exposure to antimicrobials has led to the acquisition and development of sophisticated bacterial resistance mechanisms, such as efflux pumps, which are able to expel or reduce the intracellular concentration of various antibiotics, making them ineffective. Therefore, inhibiting this mechanism is a promising way to minimize the phenomenon of resistance in bacteria. In this sense, the present study sought to evaluate the activity of the Carvacrol (CAR) and Thymol (THY) terpenes as possible Efflux Pump Inhibitors (EPIs), by determining the Minimum Inhibitory Concentration (MIC) and the association of these compounds in subinhibitory concentrations with the antibiotic Norfloxacin and with Ethidium Bromide (EtBr) against strains SA-1199 (wild-type) and SA-1199B (overexpresses NorA) of Staphylococcus aureus. In order to verify the interaction of the terpenes with the NorA efflux protein, an in silico molecular modeling study was carried out. The assays used to obtain the MIC of CAR and THY were performed by broth microdilution, while the Efflux Pump inhibitory test was performed by the MIC modification method of the antibiotic Norfloxacin and EtBr. docking was performed using the Molegro Virtual Docker (MVD) program. The results of the study revealed that CAR and THY have moderate bacterial activity and are capable of reducing the MIC of Norfloxacin antibiotic and EtBr in strains of S. aureus carrying the NorA efflux pump. The docking results showed that these terpenes act as possible competitive NorA inhibitors and can be investigated as adjuvants in combined therapies aimed at reducing antibiotic resistance.

Dynamic conformational changes play a major role in the function of proteins, including the ATP-Binding Cassette (ABC) transporters. Multidrug Resistance Protein 1 (MRP1) is an ABC exporter that protects tissues from toxic molecules. Overexpression of MRP1 has been shown to confer Multidrug Resistance (MDR), a phenomenon in which cancer cells are capable to defend themselves against a broad variety of drugs. Despite an increasing number of structures of MRP1, including a relatively new bovine cryo-EM structure, the accurate molecular details for its drug extrusion mechanism remain vague. In this study, we used varied computational techniques to explore the unique F583A mutation that is known to essentially lock the transporter in a low-affinity solute binding state. We demonstrate how macro-scale conformational changes affect MRP1’s stability and dynamics, and how these changes correspond to micro-scale structural perturbations in helices 10–11 and the nucleotide-binding domains (NBDs) of the protein in regions known to be crucial for its ATPase activity. We demonstrate how a single substitution of an outward-facing aromatic amino acid causes a long-range allosteric effect that propagates across the membrane, ranging from the extracellular ECL5 loop to the cytoplasmic NBD2 over a distance of nearly 75 Å, leaving the protein in a non-functional state, and provide the putative allosteric pathway. The identified allosteric structural pathway is not only in agreement with experimental data but enhances our mechanical understanding of MRP1, thereby facilitating the rational design of chemosensitizers toward the success of chemotherapy treatments.

Cholesterol gallstone (CGS) disease is characterized by an imbalance in bile acid (BA) metabolism and is closely associated with gut microbiota disorders. However, the role and mechanism by which probiotics targeting the gut microbiota attenuate cholesterol gallstones are still unknown. In this study, Limosilactobacillus reuteri strain CGMCC 17942 and Lactiplantibacillus plantarum strain CGMCC 14407 were individually administered to lithogenic-diet (LD)-fed mice for 8 weeks. Both

Mutations in ABCC6, an ATP-binding cassette transporter with a so far unknown substrate mainly expressed in the liver and kidney, cause pseudoxanthoma elasticum (PXE). Symptoms of PXE in patients originate from the calcification of elastic fibers in the skin, eye, and vessels. Previous studies suggested an involvement of ABCC6 in cholesterol and lipid homeostasis. The intention of this study was to examine the influence of ABCC6 deficiency during adipogenic differentiation of human bone marrow-derived stem cells (hMSCs). Induction of adipogenic differentiation goes along with significantly elevated ABCC6 gene expression in mature adipocytes. We generated an ABCC6-deficient cell culture model using clustered regulatory interspaced short palindromic repeat Cas9 (CRISPR–Cas9) system to clarify the role of ABCC6 in lipid homeostasis. The lack of ABCC6 in hMSCs does not influence gene expression of differentiation markers in adipogenesis but results in a decreased triglyceride content in cell culture medium. Protein and gene expression analysis of mature ABCC6-deficient adipocytes showed diminished intra- and extra-cellular lipolysis, release of lipids, and fatty acid neogenesis. Therefore, our results demonstrate impaired lipid trafficking in adipocytes due to ABCC6 deficiency, highlighting adipose tissue and peripheral lipid metabolism as a relevant target for uncovering systemic PXE pathogenesis.

Background Attenuated Oxaliplatin efficacy is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy and the risks in relapse and metastasis. However, the mechanism of Oxaliplatin de-efficacy during CRC treatment has not been completely elucidated. Methods Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10 transporter. The Cancer Genome Atlas data was analyzed to figure out the correlation between the clinical manifestation and ABCC10 expression. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 assay was conducted to identify the CRC cell viability and Oxaliplatin IC50. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Results CRC patients with higher ABCC10 were prone to relapse and metastasis. Differential ABCC10 expression in multiple CRC cell lines revealed a strong positive correlation between ABCC10 expression level and decreased Oxaliplatin response. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was evidently elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. To explore a strategy to block ABCC10 in CRC cells, we paid a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that neither the inhibition of ERS nor the induction of mild ERS had anti-CRC effect. However, the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the induction of intense ERS. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS down-regulated ABCC10 expression via regulated IRE1-dependent decay activity. Conclusion Oxaliplatin was a substrate of ABCC10 efflux transporter. The intense ERS/IRE1α enhanced Oxaliplatin efficacy through down-regulating ABCC10 in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Dextran is a promising candidate as a nanocarrier of chemotherapeutic drugs due to its biocompatibility, biodegradability, and ability to accumulate in tumors. Furthermore, dextran derivatives interact with P-glycoprotein (P-gp), so we hypothesized that they may be available as tumor-specific drug delivery systems with the ability to reverse multidrug resistance. Here, to test this idea, we investigated whether dextran and its derivatives inhibit breast cancer resistance protein (BCRP), multidrug resistance associated protein 1 (MRP1), and P-gp in vitro. First, we examined their effect on the uptake of specific fluorescent substrates by inside-out Sf-9 membrane vesicles overexpressing BCRP, MRP1, and P-gp. BCRP and MRP1 were significantly inhibited by 2-hydroxypropyl-trimethylammonium-dextran of 4 and 70 kDa (Q-D4 and Q-D70) at a concentration near the clinically used concentration of dextran; however, P-gp was not inhibited. A structure-activity study showed that Q-D4, Q-D70, and 40 kDa diethylaminoethyl-dextran (DEAE-D40) significantly inhibited BCRP, while 4, 40, and 70 kDa dextrans (D4, D40, and D70), dextran sulfate (Sul-D40), and the individual saccharide components of dextran did not. These results suggest that the cationic side chains, but not the saccharides, are important for BCRP inhibition. Finally, cell-based efflux assay was conducted. Q-D4, Q-D70, and DEAE-D40 did not specifically increase the retention of Hoechst33342 in BCRP-overexpressing KB cells. Similarly, Q-D4 and Q-D70 did not affect the intracellular retention of specific fluorescent substrates in MRP1- and P-gp-overexpressing KB cells. The ineffectiveness in cellular systems is presumably due to inability of the dextran derivatives to access transporters located on the cytoplasmic side of the cell membrane.

Normal function of placental extravillous trophoblasts (EVTs), which are responsible for uteroplacental vascular remodeling, is critical for adequate delivery of oxygen and nutrients to the developing fetus and normal fetal programming. Proliferation and invasion of spiral arteries by EVTs depends upon adequate levels of folate. Multidrug resistance-associated protein 1 (MRP1), which is an efflux transporter, is known to remove folate from these cells. We hypothesized that palmitic acid increases MRP1-mediated folate removal from EVTs, thereby interfering with EVTs’ role in early placental vascular remodeling. HTR-8/SVneo and Swan-71 cells, first trimester human EVTs, were grown in the absence or presence of 0.5 mM and 0.7 mM palmitic acid, respectively, for 72 h. Palmitic acid increased ABCC1 gene expression and MRP1 protein expression in both cell lines. The rate of folate efflux from the cells into the media increased with a decrease in migration and invasion functions in the cultured cells. Treatment with N-acetyl cysteine (NAC) prevented the palmitic acid mediated upregulation of MRP1 and restored invasion and migration in the EVTs. Finally, in an ABCC1 knockout subline of Swan-71 cells, there was a significant increase in invasion and migration functions. The novel finding in this study that palmitic acid increases MRP1-mediated folate efflux provides a missing link that helps to explain how maternal consumption of saturated fatty acids compromises the in-utero environment.

The continuous emergence of multidrug-resistant (MDR) bacteria has posed an increasingly serious public health threat which urges people to develop some alternatives. Gallic acid (GA) is a natural ingredient in many traditional Chinese medicines, which has many biological activities, such as antibacterial, and antiseptic. Here, clinical isolates of MDR Escherichia coli (E. coli) were used to evaluate the antibacterial effect of GA and the underlying mechanism. The results revealed that GA exerted bactericidal activity and inhibited the formation of bacterial biofilm. GA enhanced the activities of ceftiofur sodium or tetracycline against E. coli, and facilitated antibiotic accumulation in bacteria. Further analysis of morphological alterations and efflux pump gene expressions confirmed that GA damaged outer and inner membranes, and suppressed the mRNA expressions of acrA, acrB, tolC, acrD and acrF involved in membrane permeability. In addition, GA showed protective effects against bacterial infection and improved the survival rates of Galleria mellonella and BALB/c mice. These data highlight a better understanding of GA against bacteria and provide an alternative strategy for MDR bacterial infection.

Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component (mlaA) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions (ompCF), and over-expressed efflux pumps (acrB, qacE, aceI), are lacking and may be distinguishable using antiseptic rapid fluorescent dye testing assays. Using E. coli K-12 CHX adapted isolates (CHXR1), gene deletion mutants, and over-expressed transformants the phenotypes of these CHX resistance genes were compared using antimicrobial susceptibility tests (AST), rapid fluorescent propidium iodide dye-based membrane integrity assays (RFDMIA), and scanning electron microscopy (SEM). AST findings showed CHXR1, ΔacrB, ΔompCF, and transformants pCA24N-aceI and pCA24N-mlaA conferred greater (two to fourfold) MIC changes when compared to matched controls. Examination of these mutants/transformants using CHX RFDMIA showed that porin dual-deletions (ΔompCF) and mlaA alterations (ΔmlaA; pCA24N-mlaA, CHXR1) were distinguishable from controls. Results for over-expressed (pMS119EH-aceI) and deleted (ΔacrB) efflux pump RFDMIA could not be distinguished with propidium iodide, only with ethidium bromide, suggesting propidium iodide is better suited for detecting porin and mlaA associated CHX resistance mechanisms. SEM of CHXR1 and unadapted E. coli cells exposed to increasing CHX concentrations revealed that CHX does not visibly damage cell envelope integrity at any tested concentration but did identify elongated CHXR1 cells. ΔmlaA confers similar levels of CHX resistance as efflux overexpression and porin deletions, however, only outer membrane-altering porin and mlaA deletions can be reliably distinguished using RFDMIA.

Liver injury is often associated with hepatic retinopathy, resulting from accumulation of retinal toxins due to blood–retinal barrier (BRB) dysfunction. Retinal pigment epithelium highly expresses MRP1/Mrp1. We aimed to investigate whether liver injury affects the function and expression of retinal Mrp1 using bile duct ligation (BDL) rats. Retinal distributions of fluorescein and 2,4-dinitrophenyl-S-glutathione were used for assessing Mrp1 function. BDL significantly increased distributions of the two substrates and bilirubin, downregulated Mrp1 protein, and upregulated phosphorylation of p38 and MK2 in the retina. BDL neither affected the retinal distribution of FITC-dextran nor expressions of ZO-1 and claudin-5, demonstrating intact BRB integrity. In ARPE-19 cells, BDL rat serum or bilirubin decreased MRP1 expression and enhanced p38 and MK2 phosphorylation. Both inhibiting and silencing p38 significantly reversed the bilirubin- and anisomycin-induced decreases in MRP1 protein. Apparent permeability coefficients of fluorescein in the A-to-B direction (Papp, A-to-B) across the ARPE-19 monolayer were greater than Papp, B-to-A. MK571 or bilirubin significantly decreased Papp, A-to-B of fluorescein. Bilirubin treatment significantly downregulated Mrp1 function and expression without affecting integrity of BRB and increased bilirubin levels and phosphorylation of p38 and MK2 in rat retina. In conclusion, BDL downregulates the expression and function of retina Mrp1 by activating the p38 MAPK pathway due to increased bilirubin levels.

The bioavailability of most flavonoids is low but effective in vivo; however, the mechanism of the efficacy of flavonoids has not been elucidated. Kaempferol is typical flavonoids., preliminary research indicates that kaempferol has a significant anti-colon cancer and anti-inflammatory effect. We reported previously that the triple recycling pathways significantly increase the local bioavailability of flavonoids and prolong the residence time of flavonoids in the liver and intestines, which is likely the mode by which flavonoids exert local efficacy. Notably, Efflux transporters (ETs), such as Breast cancer resistance protein (BCRP) and Multi drug resistance-associated protein 2 (MRP2), are the main regulatory molecules of the enterohepatic triple recycling pathways. Thus, our current study explored the regulation of kaempferol by BCRP and MRP2 and the role of BCRP and MRP2 in the suppression of Dextran sulfate sodium (DSS)-induced colitis by kaempferol. Herein, four mouse model was constructed, and the Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established to simultaneously quantify kaempferol and its 3 metabolites and investigate the oral pharmacokinetic characteristics and tissue distribution of these compounds. In Bcrp

The drug/proton antiporter MexB is the engine of the major efflux pump MexAB-OprM in

ATP-binding cassette (ABC) transporters are membrane-spanning proteins involved in cholesterol homeostasis, transport of various molecules in and out of cells and organelles, oxidative stress, immune recognition, and drug efflux. They are long implicated in the development of multidrug resistance in cancer chemotherapy. Existing clinical and molecular evidence has also linked ABC transporters with cancer pathogenesis, prognostics, and therapy. In this review, we aim to provide a comprehensive update on all ABC transporters and their roles in drug resistance in breast cancer (BC). For solid tumours such as BC, various ABC transporters are highly expressed in less differentiated subtypes and metastases. ABCA1, ABCB1 and ABCG2 are key players in BC chemoresistance. Restraining these transporters has evolved as a possible mechanism to reverse this phenomenon. Further, ABCB1 and ABCC1 are important in BC prognosis. Newer therapeutic approaches have been developed to target all these molecules to dysregulate their effect, reduce cell viability, induce apoptosis, and increase drug sensitivity. In the future, targeted therapy for specific genetic variations and upstream or downstream molecules can help improve patient prognosis.

Multidrug resistance-associated protein 1 (MRP1, encoded by the ABCC1 gene) may contribute to the clearance of amyloid-beta (Aβ) peptides from the brain into the blood and stimulation of MRP1 transport activity may be a therapeutic approach to enhance brain Aβ clearance. In this study, we assessed the effect of thiethylperazine, an antiemetic drug which was shown to stimulate MRP1 activity in vitro and to decrease Aβ load in a rapid β-amyloidosis mouse model (APP/PS1-21), on MRP1 transport activity by means of positron emission tomography (PET) imaging with the MRP1 tracer 6-bromo-7-[11C]methylpurine. Groups of wild-type, APP/PS1-21 and Abcc1(−/−) mice underwent PET scans before and after a 5-day oral treatment period with thiethylperazine (15 mg/kg, once daily). The elimination rate constant of radioactivity (kelim) was calculated from time–activity curves in the brain and the lungs as a measure of tissue MRP1 activity. Treatment with thiethylperazine had no significant effect on MRP1 activity in the brain and the lungs of wild-type and APP/PS1-21 mice. This may either be related to a lack of an MRP1-stimulating effect of thiethylperazine in vivo or to other factors, such as substrate-dependent MRP1 stimulation, insufficient target tissue exposure to thiethylperazine or limited sensitivity of the PET tracer to measure MRP1 stimulation.