Multidrug Resistance Transporter Research Articles

Intranasal administration of carbamazepine-loaded carboxymethyl chitosan nanoparticles for drug delivery to the brain

Epilepsy is considered as a common and diverse set of chronic neurological disorders and its symptoms can be controlled by antiepileptic drugs (AEDs). The presence of p-glycoprotein and multi-drug resistance transporters in the blood-brain barrier could prevent the entry of AEDs into the brain, causing drug resistant epilepsy. To overcome this problem, we propose using carboxymethyl chitosan nanoparticles as a carrier to deliver carbamazepine (CBZ) intra-nasally with the purpose to bypass the blood-brain barrier thus to enhance the brain drug concentration and the treatment efficacy. Results so far indicate that the developed CBZ-NPs have small particle size (218.76 ± 2.41 nm) with high drug loading (around 35%) and high entrapment efficiency (around 80%). The in vitro release profiles of CBZ from the NPs are in accordance with the Korsmeyer-peppas model. The in vivo results show that both encapsulation of CBZ in nanoparticles and the nasal route determined the enhancement of the drug bioavailability and brain targeting characteristics.

In vitro evaluation of antimicrobial agents on Acanthamoeba sp. and evidence of a natural resilience to amphotericin B

The free-living amoeba (FLA) Acanthamoeba sp. is an opportunistic pathogen that can cause amoebic keratitis (AK) or granulomatous amoebic encephalitis (GAE). While current treatments of AK are long with some relapses, no consensus therapy has been developed for GAE remaining lethal in 90% of the cases. In this context, efficient antiacanthamoebal drugs have to be identified. In this work, 15 drugs used in the treatment of AK or GAE or in other parasitic diseases were evaluated for their in vitro activity on A. castellanii. Hexamidine, voriconazole and clotrimazole exhibited the highest activities with IC50 values at 0.05 μM, 0.40 μM and 0.80 μM, respectively, while rifampicin, metronidazole and cotrimoxazole were inactive. Among 15 drug associations evaluated, no synergistic effect was observed, and one antagonism was determined between hexamidine and chlorhexidine. Interestingly, amphotericin B was the only drug presenting an increase of IC50 as a function of treatment duration. The amoebae susceptibility to amphotericin B cultured in the presence of 250 μM of the drug was similar to the one of a naive control, revealing that no resistant strain could be selected. However, the amoebae susceptibility always returned to an initial level at each passage. This natural and non-acquired adaptation to amphotericin B, qualified as resilience, was observed in several strains of A. castellanii and A. polyphaga. Using a pharmacological approach with effectors of different cellular mechanisms or transports, and an ultrastructural analysis of amphotericin B-treated amoebae, the involvement of several mitochondria-dependent pathways as well as multidrug resistant transporters was determined in amphotericin B resilience. Based on the observations from this study, the relevance of using amphotericin B in GAE treatments may be reconsidered, while the use of some other drugs, such as rifampicin or cotrimoxazole, is not relative to intrinsic antiacanthamoebal activity.

CUDC-907, a dual HDAC and PI3K inhibitor, reverses platinum drug resistance.

Platinum (Pt)-based anticancer drugs are the mainstay of treatment for solid cancers. However, resistance to Pt drugs develops rapidly, which can be caused by overexpression of multidrug resistance transporters and activation of DNA repair. CUDC-907 is a potent molecular targeted anticancer agent, rationally designed to simultaneously inhibit histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K). We investigated the potentiation effect of CUDC-907 on Pt drugs in resistant cancer cells. ABCC2 stably-transfected HEK293 cells and two pairs of parental and Pt-resistant cancer cell lines were used to test for the circumvention of resistance by CUDC-907. Chemosensitivity was assessed by the sulphorhodamine B assay. Drug combinations were evaluated by the median effect analysis. ABCC2 transport activity was examined by flow cytometric assay. Cellular Pt drug accumulation and DNA platination were detected by inductively coupled plasma optical emission spectroscopy. ABCC2, ERCC1 and p21 expression were evaluated by quantitative real-time PCR. Cell cycle analysis and apoptosis assay were performed by standard flow cytometric method. The combination of CUDC-907 with cisplatin were found to exhibit synergistic cytotoxic effect in cisplatin-resistant cancer cells. In Pt-resistant cancer cells, CUDC-907 apparently circumvented the resistance through inhibition of ABCC2 and DNA repair but induction of cell cycle arrest. In the presence of CUDC-907, cellular accumulation of Pt drugs and formation of DNA-Pt adducts were found to be increased whereas expression levels of ABCC2 and ERCC1 was inhibited in Pt-resistant cells. The data advocates further development of CUDC-907 as a resistance reversal agent for use in combination cancer chemotherapy.

Sphingolipids facilitate age asymmetry of membrane proteins in dividing yeast cells.

One proposed mechanism of cellular aging is the gradual loss of certain cellular components that are insufficiently renewed. In an earlier study, multidrug resistance transporters (MDRs) were postulated to be such aging determinants during the yeast replicative life span (RLS). Aged MDR proteins were asymmetrically retained by the aging mother cell and did not diffuse freely into the bud, whereas newly synthesized MDR proteins were thought to be deposited mostly in the bud before cytokinesis. In this study, we further demonstrate the proposed age asymmetry of MDR proteins in dividing yeast cells and investigate the mechanism that controls diffusive properties of MDR proteins to maintain this asymmetry. We found that long-chain sphingolipids, but not the septin/endoplasmic reticulum-based membrane diffusion barrier, are important for restricting MDR diffusion. Depletion of sphingolipids or shortening of their long acyl chains resulted in an increase in the lateral mobility of MDR proteins, causing aged MDR protein in the mother cell to enter the bud. We used a mathematical model to understand the effect of diminished MDR age asymmetry on yeast cell aging, the result of which was qualitatively consistent with the observed RLS shortening in sphingolipid mutants.

A MDR transporter contributes to the different extracellular production of sesquiterpene pyridine alkaloids between adventitious root and hairy root liquid cultures of Tripterygium wilfordii Hook.f.

TwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids. Hairy roots (HRs) exhibit high growth rate and biochemical and genetic stability. However, varying secondary metabolites in HR liquid cultures mainly remain in root tissues, and this condition may affect cell growth and cause inconvenience in downstream extraction. Studies pay less attention to adventitious root (AR) liquid cultures though release ratio of some metabolites in AR liquid cultures is significantly higher than that of HR. In Tripterygium wilfordii Hook.f., release ratio of wilforine in AR liquid cultures reached 92.75 and 13.32% in HR on day 15 of culture. To explore potential roles of transporters in this phenomenon, we cloned and functionally identified a multidrug resistance (MDR) transporter, TwMDR1, which shows high expression levels in HRs and is correlated to transmembrane transportation of alkaloids. Nicotiana tabacum cells with overexpressed TwMDR1 efficiently transported wilforine and wilforgine in an inward direction. To further prove the feasibility of genetically engineered TwMDR1 and improve alkaloid production, we performed a transient RNAi experiment on TwMDR1 in T. wilfordii Hook.f. suspension cells. Results indicated that release ratios of wilforine and wilforgine increased by 1.94- and 1.64-folds compared with that of the control group, respectively. This study provides bases for future studies that aim at increasing secretion ratios of alkaloids in root liquid cultures in vitro.

Draft Genome Sequence of Multidrug-Resistant Cellulosimicrobium sp. Strain KWT-B, Isolated from Feces of Hirundo rustica.

ABSTRACTMigratory birds have been postulated as potential spreaders of antibiotic resistance. Multidrug-resistant Cellulosimicrobium sp. strain KWT-B was isolated from the feces of Hirundo rustica. A draft genome sequence indicated that the strain harbors multidrug-resistant transporters, multidrug efflux pumps, a vancomycin-resistant protein, and metallo-beta-lactamases.

Extreme low dose of 5-fluorouracil reverses MDR in cancer by sensitizing cancer associated fibroblasts and down-regulating P-gp.

We conducted a prospective, meaningful study of extreme low dose of 5-fluorouracil (5FU) as a metronomic agent targeting cancer associated fibroblasts (CAFs) to reverse Multidrug resistance (MDR) by sensitizing cancer associated fibroblasts and down-regulating P-glycoprotein (P-gp). The combination of 5FU and Taxol inhibited resistant KB-8-5 tumor growth by 79% and H460/Tax-R tumor growth by 55%. The inhibition was significant for both tumor types compared with Taxol treatment alone (p<0.001 and p = 0.0067, respectively). Nevertheless, the low-dose 5FU (2.2 mg/kg compared to the therapeutic dose of 50–150 mg/kg) showed negligible tumor inhibitory effect. The tumor growth inhibition study on resistant tumors demonstrated that the continuous administration of low dose 5FU with Taxol significantly inhibited the tumor growth. The treatment overcomes drug resistance in tumors by down-regulating multi-drug resistance transporter protein (P-gp), and more importantly, by eliminating CAFs recruited by resistant tumors. Compared with traditional metronomic chemotherapy, 5FU as metronomic agent targeting CAFs can avoid the disadvantages resulted from the concomitant administration of antiangiogenetic drug. The approach has good translational potential for clinical trials when treating stroma-rich drug resistant tumors.

The multidrug-resistance transporter MdfA from Escherichia coli: crystallization and X-ray diffraction analysis.

The active efflux of antibiotics by multidrug-resistance (MDR) transporters is a major pathway of drug resistance and complicates the clinical treatment of bacterial infections. MdfA is a member of the major facilitator superfamily (MFS) from Escherichia coli and provides resistance to a wide variety of dissimilar toxic compounds, including neutral, cationic and zwitterionic substances. The 12-transmembrane-helix MdfA was expressed as a GFP-octahistidine fusion protein with a TEV protease cleavage site. Following tag removal, MdfA was purified using two chromatographic steps, complexed with a Fab fragment and further purified using size-exclusion chromatography. MdfA and MdfA-Fab complexes were subjected to both vapour-diffusion and lipidic cubic phase (LCP) crystallization techniques. Vapour-diffusion-grown crystals were of type II, with poor diffraction behaviour and weak crystal contacts. LCP lipid screening resulted in type I crystals that diffracted to 3.4 Å resolution and belonged to the hexagonal space group P6122.

Lapatinib potentiates cytotoxicity of \xa0YM155 in neuroblastoma via inhibition of the ABCB1 efflux transporter

Adverse side effects of cancer agents are of great concern in the context of childhood tumors where they can reduce the quality of life in young patients and cause life-long adverse effects. Synergistic drug combinations can lessen potential toxic side effects through lower dosing and simultaneously help to overcome drug resistance. Neuroblastoma is the most common cancer in infancy and extremely heterogeneous in clinical presentation and features. Applying a systematic pairwise drug combination screen we observed a highly potent synergy in neuroblastoma cells between the EGFR kinase inhibitor lapatinib and the anticancer compound YM155 that is preserved across several neuroblastoma variants. Mechanistically, the synergy was based on a lapatinib induced inhibition of the multidrug-resistance efflux transporter ABCB1, which is frequently expressed in resistant neuroblastoma cells, which allowed prolonged and elevated cytotoxicity of YM155. In addition, the drug combination (i.e. lapatinib plus YM155) decreased neuroblastoma tumor size in an in vivo model.

Structural Biology Meets Drug Resistance: An Overview on Multidrug Resistance Transporters

Structural biology provides snapshots of biological function of molecular- and atomic-level structures of macromolecules, and holds great promise in addressing the emerging problems of biomedical science. Since the discovery of penicillin in early twentieth century, mankind has become aware and confronted with the emergence of antibiotic-resistant pathogens. In parallel to the failure of antibiotic therapy against infectious pathogens, there had been continuous reports of cancerous cells not responding to chemotherapy with increase in the duration of therapy. Research on the underlying causes of multidrug resistance in cancerous cells and later on in infectious bacteria revealed the involvement of integral membrane transporters, capable of recognizing a broad range of structurally different molecules as substrates and exporting them from the cell using cellular energy. Structural biologists succeeded in determining the structure of AcrB from Escherichia coli in 2002, the first structure of a multidrug resistance (MDR) transporter, and since then rapid progress has been made in the structural elucidation of these transporters. To date, structures of these transporters in apo- and substrate/inhibitor-bound state have been determined and deposited in the protein databank. This repository is a valuable source for structure-based drug design against MDR pumps. In this review, major findings related to structural biology of MDR transporters belonging to three major superfamilies, viz., ATP-binding cassette superfamily, major facilitator superfamily and resistance nodulation division superfamily are presented. Further, the future role of structural biology in improving our understanding of drug–transporter interactions and in designing novel inhibitors against MDR pump are discussed.

Synthesis and SAR Study of Anticancer Protoflavone Derivatives: Investigation of Cytotoxicity and Interaction with ABCB1 and ABCG2 Multidrug Efflux Transporters.

There is a constant need for new therapies against multidrug-resistant (MDR) cancer. Natural compounds are a promising source of novel anticancer agents. We recently showed that protoflavones display activity in MDR cancer cell lines that overexpress the P-glycoprotein (P-gp) drug efflux pump. In this study, 52 protoflavones, including 22 new derivatives, were synthesized and tested against a panel of drug-sensitive parental cells and their MDR derivatives obtained by transfection with the human ABCB1 or ABCG2 genes, or by adaptation to chemotherapeutics. With the exception of protoapigenone, identified as a weak ABCG2 substrate, all protoflavones bypass resistance conferred by these two transporters. The majority of the compounds were found to exhibit mild to strong (up to 13-fold) selectivity against the MCF-7Dox and KB-V1 cell lines, but not to transfected MDR cells engineered to overexpress the MDR transporters. Our results suggest that protoflavones can overcome MDR cancer by evading P-gp-mediated efflux.

Analysis of multidrug resistant transporter expression in tumor blood vessels of urothelial carcinoma during chemotherapy.

e23004 Background: ABCB1, a multidrug transporter, is encoded by multidrug resistance (MDR) 1 gene and plays a major role in drug resistance. Recently, we have reported that not only tumor cells, tumor vascular endothelial cells (TECs) also confer cancer drug resistance. We isolated TECs and found that they have various abnormalities such as aneuploidy or stemness characteristics. TECs showed resistance to Paclitaxel (PTX) with ABCB1 up-regulation. Furthermore, their resistance to PTX was abrogated by ABCB1 inhibition of TECs in vivo. In clinical urology, gemcitabine / cisplatin (GC) is standard 1st line chemotherapy for metastatic urothelial carcinoma (mUC). PTX is often selected in 2nd line chemotherapy for GC resistant cases, however the therapeutic outcomes are limited. We hypothesized that ABCB1 inhibitor/PTX combination would be more effective strategy for mUC, if TEC ABCB1 expression is upregulated. In this study, we investigated ABCB1 expression in tumor blood vessels of UC during 1st line chemotherapy. Methods: Paraffin-embedded samples were corrected from 50 patients who were performed tumor resection before and after 1st line chemotherapy. ABCB1 expressions were analyzed by immunohistochemical staining and the ratio of ABCB1 positive (+) vessels in total vessels were quantified. In vitro assays were performed to address how endothelial cells (ECs) are affected by change of tumor microenvironment during chemotherapy. Results: The ABCB1 (+) vessels were 0.00 – 16.80% (Median 0.00%) and 0.00-58.94% (Median 4.49%), before and after chemotherapy, respectively. In 32 of 50 cases (64%), the ratio of ABCB1 (+) vessels increased after 1st line chemotherapy. Additionally, gemcitabine induced MDR1 mRNA expression in ECs via NF-kB activation. Furthermore, gemcitabine and cisplatin induced IL-8 secretion from tumor cells, and MDR1 mRNA expression level was elevated in ECs. Conclusions: It was suggested that conventional chemotherapy may cause inflammatory change in tumor tissues, which causes in ABC transporter induction in tumor blood vessels. It was suggested that inhibition of ABC transporter in TECs is one of important new strategy to overcome cancer drug resistance.

Combined treatment of apatinib with docetaxel in non-small-cell lung cancer mice and its material basis of pharmacokinetics.

e14069 Background: Apatinib, a small-molecule inhibitor of vascular endothelial growth factor receptor-2, has attracted many attentions due to its encouraging anticancer activity in clinic third-line treatment across a broad range of malignancies, including gastric cancer, non-small-cell lung cancer (NSCLC) and colorectal cancer. Its usage in second-line with chemotherapeutic drugs is still under exploration. This study is focus on the antitumor effect of apatinib combined with docetaxel (DTX) on NSCLC and its pharmacokinetic material basis. Methods: Apatinib (150 mg/kg, every day, p.o.) with or without DTX (8 mg/kg, q3d for four times, i.p.) was administered in A549 xenograft nude mice. The antitumor effect, histopathology of important organs and serum transaminases for liver function were detected. The metabolomic changes in serum were profiled by GC/MS and analyzed for the treatment outcome. The effect of apatinib on DTX concentrations in plasma, tumor and other tissues were determined by LC-MS/MS. The expressions of multidrug resistant transporters were detected. Effects of apatinib on the uptake, efflux and subcellular distribution of DTX in A549 cell and A549/DTX (DTX resistant) cell were investigated. Results: Apatinib enhanced the antitumor effect of DTX with alleviated DTX-induced inflammatory cells infiltration in liver and decreased serum transaminases (ALT and AST). Metabolomic results showed that serum endogenic molecules of apatinib and DTX co-treatment mice were closer to that of normal nude mice when compared to the DTX group. Apatinib significantly increased DTX concentration in tumor (up to 1.82 times) without enhancement in serum, heart, liver, lung and kidney. Additionally, apatinib decreased the DTX-induced upregulation of P-glycoprotein in tumor. In vitro, apatinib increased the cell/subcellular accumulation (especially in cytosol) and decreased the efflux of DTX in A549/DTX cells, while apatinib exerted no significant effect on cellular pharmacokinetics of DTX in A549 cells. Conclusions: Apatinib has a potential application prospect in second-line therapy combined with DTX for NSCLC patients, especially for multidrug resistant patients.

Synthesis of a Fluorescent Analogue of Paclitaxel That Selectively Binds Microtubules and Sensitively Detects Efflux by P-Glycoprotein.

The anticancer drug paclitaxel (Taxol) exhibits paradoxical and poorly understood effects against slow-growing tumors. To investigate its biological activity, fluorophores such as Oregon Green have been linked to this drug. However, this modification increases its polarity by approximately 1000-fold and reduces the toxicity of Taxol towards cancer cell lines by over 200-fold. To construct more drug-like fluorescent probes suitable for imaging by confocal microscopy and analysis by flow cytometry, we synthesized derivatives of Taxol linked to the drug-like fluorophore Pacific Blue (PB). We found that PB-Gly-Taxol bound the target protein β-tubulin with both high affinity in vitro and high specificity in living cells, exhibited substantial cytotoxicity towards HeLa cells, and was a highly sensitive substrate of the multidrug resistance transporter P-glycoprotein (P-gp).

Synthesis of a Fluorescent Analogue of Paclitaxel That Selectively Binds Microtubules and Sensitively Detects Efflux by P‐Glycoprotein

AbstractThe anticancer drug paclitaxel (Taxol) exhibits paradoxical and poorly understood effects against slow‐growing tumors. To investigate its biological activity, fluorophores such as Oregon Green have been linked to this drug. However, this modification increases its polarity by approximately 1000‐fold and reduces the toxicity of Taxol towards cancer cell lines by over 200‐fold. To construct more drug‐like fluorescent probes suitable for imaging by confocal microscopy and analysis by flow cytometry, we synthesized derivatives of Taxol linked to the drug‐like fluorophore Pacific Blue (PB). We found that PB‐Gly‐Taxol bound the target protein β‐tubulin with both high affinity in vitro and high specificity in living cells, exhibited substantial cytotoxicity towards HeLa cells, and was a highly sensitive substrate of the multidrug resistance transporter P‐glycoprotein (P‐gp).

GSK1904529A, a Potent IGF-IR Inhibitor, Reverses MRP1-Mediated Multidrug Resistance.

Overexpression of multidrug-resistant efflux transporters is one of the major causes of chemotherapy failure. MRP1, a 190 kDa efflux transporter, confers resistance to a wide of range of chemotherapeutic drugs. Here we study the cellular effects of GSK1904529A in reversing MRP1-mediated drug resistance. Cytotoxicity of GSK1904529A was determined by MTT assay. Reversal effects of GSK1904529A in combination with MRP1 substrates were determined. The intracellular accumulation and efflux of MRP1 substrate was measured by scintillation counter and protein expression was determined by Western blotting analysis. Cell cycle effects of GSK1904529A in combination with MRP1 substrates were determined by flow cytometric analysis. GSK1904529A, at non-toxic concentrations, enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 cells. Furthermore, GSK1904529A increased the intracellular accumulation of [3 H]-vinblastine by inhibiting the efflux function of MRP1. GSK1904529A did not alter the expression level of MRP1, induced a G0/G1 phase cell cycle arrest. Our results indicated that GSK1904529A significantly increased the sensitivity of MRP1 overexpressing cells to chemotherapeutic agents. Furthermore, GSK1904529A enhanced the efficacy of chemotherapeutic drugs that are substrates of MRP1. J. Cell. Biochem. 118: 3260-3267, 2017. © 2017 Wiley Periodicals, Inc.

Efflux Transporters Regulate Arsenite-Induced Genotoxicity in Double Negative and Double Positive T Cells.

Arsenite (As+3) exposure is known to cause immunotoxicity in human and animal models. Our previous studies demonstrated that As+3 at 50-500 nM concentrations induced both genotoxicity and nongenotoxicity in mouse thymus cells. Developing T cells at CD4-CD8- double negative (DN) stage, the first stage after early T cells are transported from bone marrow to thymus, were found to be more sensitive to As+3 toxicity than the T cells at CD4 + CD8 + double positive (DP) stage in vitro. Induction of Mdr1 (Abcb1) and Mrp1 (Abcc1), 2 multidrug resistance transporters and exporters of As+3, was associated with the reversal of As+3-induced double strand breaks and DNA damage. In order to confirm that the thymus cell populations have different sensitivity to As+3in vivo, male C57BL/6J mice were exposed to 0, 100, and 500 ppb As+3 in drinking water for 30 d. A significant decrease in DN cell percentage was observed with exposure to 500 ppb As+3. Low to moderate concentrations of As+3 were shown to induce higher genotoxicity in sorted DN than DP cells in vitro. Calcein AM uptake and Mdr1/Mrp1 mRNA quantification results revealed that DN cells not only had limited As+3 exporter activity, but also lacked the ability to activate these exporters with As+3 treatments, resulting in a higher accumulation of intracellular As+3. Knockdown study of As+3 exporters in the DN thymic cell line, D1 using siRNA, demonstrated that Mdr1 and Mrp1 regulate intracellular As+3 accumulation and genotoxicity. Taken together, the results indicate that transporter regulation is an important mechanism for differential genotoxicity induced by As+3 in thymocytes at different developmental stages.

Dimeric isoxazolyl-1,4-dihydropyridines have enhanced binding at the multi-drug resistance transporter

A series of dimeric isoxazolyl-1,4-dihydropyridines (IDHPs) were prepared by click chemistry and examined for their ability to bind the multi-drug resistance transporter (MDR-1), a member of the ATP-binding cassette superfamily (ABC). Eight compounds in the present study exhibited single digit micromolar binding to this efflux transporter. One monomeric IDHP m-Br-1c, possessed submicromolar binding of 510nM at MDR-1. Three of the dimeric IDHPs possessed <1.5µM activity, and 4b and 4c were observed to have superior binding selectivity compared to their corresponding monomers verses the voltage gated calcium channel (VGCC). The dimer with the best combination of activity and selectivity for MDR-1 was analog 4c containing an m-Br phenyl moiety in the 3-position of the isoxazole, and a tether with five ethyleneoxy units, referred to herein as Isoxaquidar. Two important controls, mono-triazole 5 and pyridine 6, also were examined, indicating that the triazole – incorporated as part of the click assembly as a spacer – contributes to MDR-1 binding. Compounds were also assayed at the allosteric site of the mGluR5 receptor, as a GPCR 7TM control, indicating that the p-Br IDHPs 4d, 4e and 4f with tethers of from n=2 to 5 ethylenedioxy units, had sub-micromolar affinities with 4d being the most efficacious at 193nM at mGluR5. The results are interpreted using a docking study using a human ABC as our current working hypothesis, and suggest that the distinct SARs emerging for these three divergent classes of biomolecular targets may be tunable, and amenable to the development of further selectivity.

Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry.

The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segregation of the transporter-rich cells into a distinct cell cohort tilted towards the shorter wavelength axis of the plot, the cohort is generically known as the side population (SP). This feature has facilitated the surface marker-independent isolation of live stem cells. A drawback, though, is the known toxicity of Hoechst dyes. In this study we show that JC1, a bathochromic mitochondrial membrane potential-sensitive dye applied at proper concentration, can yield flow cytometry fluorescent emission bivariate plots containing a low JC1 accumulation (JC1low) cohort. Using a combination of multiple cell lines, ABC-transporter inhibitors and viral vector-driven insertion of the ABCG2 gene or ABCG2 and ABCB1 shRNAs we demonstrate that JC1low can be generated by either of the two aforementioned multidrug resistance transporters. Complete wash out of mitochondrial bound JC1 required more than 24 h. In spite of this tight binding, the dye did not affect either the mitochondrial membrane potentials or the proliferation rate. In contrast, contemporaneous with its nuclear accumulation, Hoechst 33342 or DVC, caused changes in the fluorescent emission of mitochondrial membrane potential sensitive dyes resembling the effects caused by the mitochondrial uncoupler FCCP. In a number of cell lines exposure to Hoechst resulted in marked slow-down of proliferation and abolition of ABCG2 transport activity during the subsequent 2 days but in K562 cells the exposure induced cell extended death. Overall, its lack of toxicity vis. a vis. the toxicity and genotoxicity of the DNA intercalating dyes makes JC1 an ideal tool for isolating live cells expressing high multidrug resistance transport activity.

L‐Tryptophan and Bacterial Modulation of Intestinal and Hepatic Gene Expression

Host and microbial metabolites of L‐Tryptophan (Trp) have been shown in vitro to activate aryl hydrocarbon receptor (AhR) and upregulate its prototypical target genes: Cytochrome P450 (CYP) 1A1 and 1A2. Additionally, the Trp microbial metabolite indole‐3‐propionic acid (IPA) produced by Clostridium sporogenes has been shown to be an activator of pregnane X receptor (PXR) in mouse intestine to maintain mucosal integrity. However, very little is known regarding the effect of Trp and its metabolites on host xenobiotic biotransformation in liver and intestine. The objective of this study was to determine the potential crosstalk between microbial Trp metabolism and host xenobiotic biotransformation. To investigate the effect of Trp on the host expression of drug‐processing genes (DPGs), 60‐day‐old conventional (CV) male C57BL/6J mice were orally gavaged 0, 10, 20, or 40 mg/kg L‐Trp once daily for 6 days. Bacterial 16S rRNA qPCR showed that Trp did not alter the abundance of 5 known Trp‐metabolizing bacteria in intestine of CV mice; however, Trp at all 3 doses down‐regulated Bifidobacterium bifidum, which has been shown to be involved in bile acid metabolism. Unexpectedly, Trp did not up‐regulate Cyp1a1 mRNA in any sections of the intestine; in fact, all 3 doses of Trp decreased Cyp1a1 mRNA in jejunum. Conversely, Trp dose‐dependently up‐regulated the PXR‐target gene Cyp3a11, and the constitutive androstane receptor (CAR)‐target gene Cyp2b10 in duodenum at all doses, as well as the PXR‐target gene multidrug resistance transporter 1a (Mdr1a) at the highest dose. In liver, Trp oral treatment did not affect the mRNAs of the prototypical targeted DPGs for AhR, CAR, PXR, or peroxisome proliferator‐activated receptor α (PPARα), although the CAR mRNA was downregulated at the medium and high doses. Furthermore, control germ‐free (GF) mice have lower expression of Cyp3a11. To test that replacing the Trp microbial metabolite IPA in GF mice normalizes the expression of PXR‐targeted DPGs in liver and intestine, 90‐day‐old male GF C57BL/6 mice were administered 0 or 40 mg/kg IPA p.o. or i.p. once daily for 4 days. Neither route of IPA exposure markedly affected Cyp3a11 mRNA expression in intestine or liver. However, Mdr1a was up‐regulated by IPA in jejunum (p.o.) and large intestine (i.p.). The PPARα‐target gene Cyp4a14 was down‐regulated by IPA (i.p.) in duodenum, whereas Cyp1a2 mRNA was up‐regulated by IPA (p.o.) in liver. To recapitulate the effect of Trp oral exposure on human liver, differentiated HepaRG cells were treated with Trp or IPA at a concentration range from 10 to 250 μM for 24h. IPA, but not Trp, dose‐dependently up‐regulated CYP1A1, 2B6, and 3A4 in HepaRG cells, suggesting that the microbial metabolism of Trp is necessary to modulate a hepatic transcriptional response in DPGs. In conclusion, oral exposure to Trp has minimal effect on DPG expression in mouse liver, and preferably up‐regulates the PXR‐ and CAR‐targeted but not the AhR‐targeted DPGs in mouse intestine. The Trp microbial metabolite IPA does not appear to be a major inducer for PXR‐targeted DPGs in liver or intestine of mice, but it may be important for activating AhR, CAR, and PXR pathways in human hepatocytes.Support or Funding InformationNational Institutes of Health grants ES025708, GM111381, ES019487 and the University of Washington Center for Exposures, Diseases, Genomics, and Environment (P30 ES007033)