Protein Mrp Research Articles

Non-steroidal anti-inflammatory drugs increase MRP4 expression in an endometriotic epithelial cell line in a PPARa dependent manner.

Endometriosis is a debilitating disease characterized by chronic inflammation. The transporter multidrug resistance-associated protein 4 (MRP4/ABCC4) is expressed in human endometrial tissue; it is overexpressed in ectopic endometrial tissue, and is modulated by the anti-inflammatory lipid Lipoxin A4 (LXA4). Recently, it was demonstrated that aspirin induces platelet MRP4 over-expression, through genomic modulation in megakaryocytes. Since patients with endometriosis frequently use aspirin or other non-aspirin Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), the aim of this study was to verify whether aspirin and other NSAIDs enhance MRP4 expression in 12Z human endometriotic epithelial cells and whether this was peroxisome proliferator-activated receptor alpha (PPARa) dependent. MRP4 and PPARa expression was analyzed by Q-RT-PCR using TaqMan® Master Mix and TaqMan® Assay Reagents (Life Technologies, Monza, Italy) and Western blot. In 12Z cells, aspirin and other NSAIDs enhanced MRP4 mRNA and protein expression; these treatments also induced PPARa expression. Aspirin and diclofenac-induced increases in MRP4 expression were not observed in cells where PPARa was knocked down using siRNA. NSAIDs-induced MRP4 expression was correlated with augmented PGE2 secretion, indicating functional relevance. MRP4 expression was increased in cells treated with NSAIDs and the nuclear receptor PPARa is involved. Elevated PGE2 levels in cell supernatants correlate with its increased transport by MRP4 after NSAID treatment. More importantly, we provide evidence that in endometriotic epithelial cells aspirin and non-aspirin NSAIDs treatments alter gene expression.

Inhibition of Multidrug Resistance Proteins by MK 571 Enhances Bladder, Prostate, and Urethra Relaxation through cAMP or cGMP Accumulation.

The biologic effect of cAMP and cGMP is terminated by phosphodiesterases and multidrug resistance proteins MRP4 and MRP5, which pump cyclic nucleotides out of the cell. Therefore, this study aimed to characterize the role of MRP inhibitor, MK 571 (3-[[[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propanoic acid), in the bladder, prostate, and urethra of male mice by means of functional assays, protein expression, and cyclic nucleotide quantification. The cumulative addition of MK 571 (1-30 µM) produced only small relaxation responses (approximately 25%) in all studied tissues. In the bladder, isoprenaline/fenoterol and forskolin concentration-dependently relaxed and MK 571 (20 µM) increased the maximal response values by 37% and 24%, respectively. When MK 571 was coincubated with fenoterol or forskolin, intracellular levels of cAMP and protein expression of phospho-vasodilator-stimulated phosphoprotein (p-VASP) Ser157 were significantly greater compared with bladders stimulated with fenoterol or forskolin alone. In the prostate and urethra, sodium nitroprusside concentration-dependently relaxed and MK 571 (20 µM) significantly increased relaxation responses by 70% and 56%, respectively, accompanied by greater intracellular levels of cGMP and protein expression of p-VASP Ser239 in the prostate. Tadalafil and BAY 41-2272 (5-cyclopropyl-2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-pyrimidinamine) also relaxed the prostate and urethra, respectively, and MK 571 markedly enhanced this response. The stable analog of cGMP (8-Br-cGMP) induced concentration-dependent relaxation responses in the prostate and urethra, and MK 571 significantly increased the relaxation response. In conclusion, to our knowledge, this is the first study to show that efflux transporters are physiologically active in the bladder, prostate, and urethra to control intracellular levels of cAMP or cGMP.

Influence of Orally Administered Borneol on the Expression of Hepatic Transporters in Rats.

Borneol, a traditional Chinese medicine (TCM), is often orally co-administered with other TCM and chemical drugs, but the drug-drug interactions between borneol and the other compounds remains unclear. This work investigates the effect of orally administered borneol on the transcription and expression of hepatic uptake transporters (Ntcp, Oatp2b1, Oatp1a1, Oatp1a4, Oct1, Oct2, Octn2 and Oat2) and efflux transporters (Mdrla, Mrp2, Mrp4 and Mrp5) in rats, aiming to obtain essential information to guide its clinical applications. Rats were administered borneol (33, 100 and 300mg/kg/day, respectively) and vehicle (control)orally via intragastric gavage for 7 consecutive days. The mRNA levels of rat hepatic uptake transporters (Ntcp, Oatp2b1, Oatp1a1, Oatp1a4, Oct1, Oct2, Octn2 and Oat2) and efflux transporters (Mdrla, Mrp2, Mrp4 and Mrp5) were determined using real-time quantitative PCR, while the hepatic Ntcp, Mdrla, Mrp2, Mrp4 and Mrp5 proteins were quantified using western blotting. The oral administration of borneol led to dose-dependent inhibition of mRNA and protein expression of Mrp4 and Mdr1a, dose-independent inhibition of mRNA and protein expression of Mrp2, and inverse dose-dependent inhibition of mRNA and protein expression of Ntcp. No significant effects were observed for mRNA expression of the other transporters tested following borneol administration. Oral administration of borneol may affect the metabolism of substances that are involved in bile acid enterohepatic circulation and substrates of Ntcp, Mdrla, Mrp2 and Mrp4 transporters.

Eukaryotic translation initiation factor 3 subunit G (EIF3G) resensitized HCT116/5-Fu to 5-fluorouracil (5-Fu) via inhibition of MRP and MDR1.

PurposeColorectal cancer (CRC) has become a predominant cancer and accounts for approximately 10% of cancer-related mortality. Drug resistance still remains a priority mortality factor for patients due to no available therapeutic alternatives. The purpose of the present study was to investigate the underlying molecular mechanisms how eukaryotic translation initiation factor 3 subunit G (EIF3G) resensitized 5-Fu-resistant human CRC cells (HCT116/5-Fu) to 5-fluorouracil (5-Fu).MethodsMultiple cellular and molecular biology experiments were performed in the present study, such as CCK-8, western blotting and flow cytometry.ResultsWe found that EIF3G is highly expressed at RNA and protein levels in HCT116/5-Fu cells compared with HCT116 cells using quantitative real-time polymerase chain reaction and Western blot analysis. In addition, silencing EIF3G enhanced 5-Fu-induced apoptosis in HCT116/5-Fu cells. Moreover, EIF3G silencing decreased the activity of the drug-related proteins MDR1 and MRP levels in HCT116/5-Fu cells. Finally, the xenograft tumor model further confirmed that EIF3G resensitized HCT116/5-Fu tumors to 5-Fu. We observed that EIF3G silencing followed by 5-Fu administration had a synergistic interaction effect on HCT116/5-Fu in vitro and in vivo.ConclusionThese findings demonstrate that EIF3G is a targetable regulator of chemoresistance in CRC, and inhibiting EIF3G in combination with 5-Fu might be a potential therapeutic strategy for colon cancer.

Abstract 1978: Antibody-drug conjugates with DNA inhibitor linker-payloads can overcome resistance in cultured tumor cells made resistant to conjugates with cleavable-linked auristatins

Abstract Antibody drug conjugates (ADCs) are targeted biotherapeutics delivering cytotoxic payloads to tumor-expressed antigens. The most common class of ADC payloads are microtubule inhibitors (MTI) and DNA inhibitors, exemplified by clinically approved ado-trastuzumab emtansine (TDM1; MTI DM1), brentuximab vedotin (MTI auristatin), gemtuzumab ozogamicin and inotuzumab ozogamicin (DNA double strand breaker calicheamicin). Auristatin payloads are in active development, primarily employing proteolytically cleavable linker mcValCitPABC. There are many benefits of cleavable-linked permeable payloads, including bystander activity in a heterogeneous tumor environment. However, due to the complexity of human cancer, tumors become refractory to most drug therapies. We hypothesized that cultured tumor cells chronically treated with mcValCitPABC-auristatin ADCs would acquire varied resistance mechanisms. As model ADCs, we used trastuzumab-mcValCitPABC-mono methyl auristatin E (TvcMMAE) and trastuzumab-mcValCitPABC-Aur0101 (Tvc0101). Her2 expressing gastric carcinoma line, N87, was chronically exposed to the respective ADCs at cytotoxic doses in a cyclical fashion (3 days on; ~1 - 3 weeks off). Resistance was acquired very slowly (over ~1 year), and cells were characterized for their resistance profile. N87-TvcMMAE cells had >1000X relative resistance (RR) to T-auristatin and TDM1 ADCs compared with parental N87, and showed moderate to high resistance (~50 – 300X) to unconjugated MTIs including auristatins and DM1. The cells were cross-resistant (>100 - 1000X) to T-ADCs delivering several payload classes, including NAc-calicheamicin and cyclopropylpyrroloindolone (CPI). Her2 and MDR1 levels were unchanged in N87-TvcMMAE cells, however, a slight increase of MRP1 was observed on immunoblots. Cells made resistant to Tvc0101 ADC showed a different profile. N87-Tvc0101 cells had >1000X RR to T-auristatin ADCs and >400X to TDM1, but were sensitive (<4X) to unconjugated auristatins and DM1. Interestingly, these cells retained sensitivity to T-ADCs delivering DNA inhibitor payloads, including NAc-calicheamicin (<5X) and CPI (<14X). These data are striking since N87-Tvc0101 cells had significantly reduced Her2 levels, yet responded to other Her2 ADCs. Immunoblots indicated no observed changes in MDR1 or MRP1 protein. The cross-resistance profile of each cell model is distinct, despite using the same parental cell background and ADCs differing in the auristatin. These data suggest that different resistance mechanisms develop upon treatment with structurally similar mcValCitPABC-auristatin ADCs, and that resistant tumors may respond to ADCs delivering a different linker-payload yet targeting the same antigen, even in cases where antigen levels have decreased. Citation Format: Xingzhi Tan, Matthew Sung, Frank Loganzo. Antibody-drug conjugates with DNA inhibitor linker-payloads can overcome resistance in cultured tumor cells made resistant to conjugates with cleavable-linked auristatins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1978.

Huatanjiangqi Capsule Upregulating NRF2 and MRP1 Expression in Chronic Obstructive Pulmonary Disease in Rats

The therapy of Huatanjiangqi capsule (HTJQ) has been used in the treatment of chronic obstructive pulmonary disease (COPD) clinically, with remarkable benefits. A lower functional activity of MRP1 is related to COPD development. However, the mechanism that contributes to MRP1 up-regulation by HTJQ was unclear. This study aimed to investigate the effects of HTJQ on the expression of Nrf2 and MRP1 in COPD rats. Rats were exposed to cigarette smoke plus lipopolysaccharide tracheal instillation to induce a COPD model. Then, Nrf2, HO-1, and MRP1 mRNA expression and their protein production in lung tissues were examined. The model group showed significant changes in the above assessments as compared to those of the control group (p < 0.05). The lung function of model group treated with low/high dose of HTJQ was significantly strengthened (both p < 0.05), and the inflammatory cells and goblet cells were decreased in lung tissues. Treatment with low/high dose of HTJQ resulted in obvious increase in Nrf2, HO-1, and MRP1 mRNA and protein expression levels (p < 0.05). HTJQ could up-regulate Nrf2 and MRP1 expression in lung tissues of COPD model rats. The mechanism by which HTJQ up-regulates MRP1 expression may be associated with Nrf2.

Design, synthesis and antineoplastic activity of novel hybrids of podophyllotoxin and indirubin against human leukaemia cancer cells as multifunctional anti-MDR agents

To overcome cancer drug resistance, in present study, a series of podophyllotoxin-indirubin hybrids were designed, synthesized, and evaluated for anticancer efficacy against two human chronic myeloid leukemia cell cultures. Among them, compound Da-1 was the most potent in resistent K562/VCR cells with an IC50 value of 0.076 ± 0.008 μM. Preliminary mechanism studies showed that Da-1 significantly induced apoptosis and cell cycle arrest at the G2 phase. Decrease in mitochondrial membrane potential, accompanied by activated PARP cleavage, was observed in K562/VCR cells after incubation with Da-1. Meanwhile, Da-1 caused the accumulation of intracellular ROS, regulated JNK and AKT signaling, and down-regulated the expression levels of P-gp and MRP1 proteins. Importantly, Western blotting revealed that Da-1 could induce K562/VCR cells autophagy, by increasing the levels of Beclin1 and LC3-II. Finally, Da-1 could disrupt microtubule organization, and binding mode to tubulin was investigated by using molecular modeling. Together, Da-1 was a novel hybrid with potent antiproliferative activity and might be a promising agent for the treatment of drug-resistant leukemia cancer.

Leflunomide Increases Hepatic Exposure to Methotrexate and Its Metabolite by Differentially Regulating Multidrug Resistance-Associated Protein Mrp2/3/4 Transporters via Peroxisome Proliferator-Activated Receptor α Activation.

Methotrexate (MTX) is the gold standard drug for the treatment of rheumatoid arthritis (RA), and it is frequently combined with leflunomide (LEF) to enhance its clinical efficacy. However, this combination can exacerbate liver toxicity, and the underlying mechanism has not yet been clarified. We investigated whether LEF affects the pharmacokinetics of MTX and its primary toxic metabolite, 7-hydroxyl methotrexate (7OH MTX), in mice. LEF significantly increased the plasma concentration (area under the plasma concentration-time curve) of MTX and 7OH MTX (2.4 and 4.5 times, respectively), decreased their bile excretion, and increased their accumulation in the liver and kidneys. When we investigated the effect of LEF on the MTX absorption, distribution, metabolism, and excretion process, we found that LEF had little effect on liver aldehyde oxidase and 7OH MTX formation. However, LEF significantly decreased the expression of the apical efflux transporter multidrug resistance-associated protein 2 (Mrp2) and increased that of the basolateral efflux transporters Mrp3/4, except there was no significant change in Mrp4 protein expression. Mrp2/3/4 alteration changed the distribution of MTX and 7OH MTX in plasma and tissues. Further studies suggested that LEF indirectly activated peroxisome proliferator-activated receptor α (PPARα), which was likely responsible for the Mrp2/3/4 alteration in the liver. The MTX plasma concentration change induced by LEF was reversed by the PPARα-specific antagonist GW6471. These results may partially explain the exacerbated liver toxicity caused by combination treatment with MTX and LEF and may raise concerns regarding the risk of potential drug-drug interactions between PPARα agonists and Mrp substrates in the clinic.

Cytotoxicity of η6-areneruthenium-based molecules to glioblastoma cells and their recognition by multidrug ABC transporters

A new series of amphiphilic η6-areneruthenium(II) compounds containing phenylazo ligands (group I: compounds 1a, 1b, 2a and 2b) and phenyloxadiazole ligands (group II: compounds 3a, 3b, 4a and 4b) were synthesized and characterized for their anti-glioblastoma activity. The effects of the amphiphilic η6-areneruthenium(II) complexes on the viability of three human glioblastoma cell lines, U251, U87MG and T98G, were evaluated. The azo-derivative ruthenium complexes (group I) showed high cytotoxicity to all cell lines, whilst most oxadiazole-derivative complexes (group II) were less cytotoxic, except for compound 4a. The cationic complexes 2a, 2b and 4b were more cytotoxic than the neutral complexes. Compounds 2a and 2b caused a significant reduction in the percentage of cells in the G0/G1 phase, with concomitant increases in the G2/M phase and fragmented DNA in the T98G cell line. The η6-areneruthenium(II) compounds were also tested in cell lines that overexpress the multidrug ABC transporters P-gp, MRP1 and ABCG2. Compounds 2b and 4a were substrates for the P-gp protein, with resistance indexes of 8.6 and 1.9, respectively. Compound 2b was also a substrate for ABCG2 and MRP1 proteins, with lower resistance indexes (1.8 and 1.6, respectively). The contribution of multidrug ABC transporters to the cytotoxicity of compound 2b in T98G cells was evidenced, since verapamil (a characteristic inhibitor of MRP1) increased the cytotoxicity of compound 2b at concentrations up to 20 μmol L−1, whilst GF120918 and Ko143 (specific inhibitors of P-gp and ABCG2, respectively) had no significant effect. In addition, we showed that compound 2b interacts with glutathione (GSH), which could explain its cellular efflux by MRP1. Our results showed that the amphiphilic η6-areneruthenium(II) complexes are promising anti-glioblastoma compounds, especially compound 2b, which was cytotoxic for all three cell lines, although it is transported by the three main multidrug ABC transporters.

Functional investigation of a highly conserved aspartate residue in the anti -cancer drug efflux transport protein MRP1

Multidrug Resistant Protein 1 (MRP1 or ABCC1) belongs to a subclass of ATP-binding cassette (ABC) transporters that export a wide range of metabolites and xenobiotics across the plasma membrane. Increased expression of MRP1 in cancer cells enhances efflux of many anti-cancer agents, giving rise to multidrug resistant tumours. The purpose of this study was to investigate the function of an aspartate (Asp) amino acid that is highly conserved in all MRP-related proteins by mutating it and determining the consequences of doing so. Asp430 lies at the interface of the cytoplasm and a transmembrane helix in the first membrane-spanning domain of MRP1. Previous studies have shown that when Asp430 is mutated, the protein becomes unstable and is degraded.Because this Asp430 is highly conserved in many MRP-related ABC transporters and because structural homology models of human MRP1 predict that Asp430 is in close proximity to Arg433, we hypothesized that a salt bridge between these two a mino acids could be essential for proper folding and stability of the protein during its biosynthesis. Using site -directed mutagenesis, these two amino acids were interchanged to probe the existence of such an interaction. Thus a double mutant where Asp430 was mutated to Arg, and Arg433 was mutated to Asp was created, and the resultant mutant protein (D430R/R433D) was tested for its ability to be detected in mammalian&#x0D; cells by gel electrophoresis and immunoblotting. Our results show differences between the migration patterns of double and single mutants that are compatible with differences in the&#x0D; glycosylation levels of MRP1. However the fact that D430R and the R433D mutants don’t share the same migration pattern, together with the variation in migration bet&#x0D; ween D430 wild type and Supported by CIHR MOP-10519the double mutant D430R/R433D indicate that the possibility of a salt bridge can be discarded.Supported by CIHR MOP-10519

ATP Binding Enables Substrate Release from Multidrug Resistance Protein 1

The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellularsubstrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.

Effect of heme oxygenase-1 on the protection of ischemia reperfusion injury of bile duct in rats after liver transplantation

To investigate the effect of heme oxygenase-1 (HO-1) on the ischemic reperfusion injury (IRI) of bile duct in rat models after liver transplantation. 320 SD rats were equally and randomly divided into 5 groups, which were group A receiving injection of 3×108/pfu/ml adenovirus (adv), group B with donor receiving Adv-HO-1 and recipient receiving Adv-HO-1-siRNA, group C with donor and recipient both receiving Adv-HO-1, group D with donor receiving Adv-HO-1-siRNA and recipient receiving Adv-HO-1, and group E with donor and recipient both receiving Adv-HO-1-siRNA at 24h before liver transplantation. Donor liver was stored in UW liquid at 4°C followed by measuring HO-1 level by western blot before transplantation. On d1, d3, d7 and d14, serum and liver was isolated for analysis of liver function, inflammatory cell infiltration by H&E staining, ultrastructure of liver by transmission electron microscopy as well as the expression of HO-1, Bsep, Mrp2 and Ntcp by western blot. Compared with group D and E, group B and C displayed improved liver function as demonstrated by lower level of ALT, AST, LDH, TBIL, ALP and GGT, increased secretion of TBA and PL as well as expression of transporter proteins (Bsep, Mrp2 and Ntcp), reduced inflammatory cells infiltration and liver injury. Our study demonstrated that overexpression of HO-1 in donor liver can ameliorate the damage to bile duct and liver, and improved liver function, suggesting HO-1 might be a new therapeutic target in the treatment of IRI after liver transplantation.

Effect of Levetiracetam add-on therapy on expression of multidrug resistant-associated protein 1 and 2 in peripheral blood mononuclear cells of children with intractable epilepsy

Objective To explore the changes of multidrug resistant-associated protein (MRP)1 expression and MRP2 expression in the peripheral blood mononuclear cells in intractable epileptic children after accepting the Levetiracetam (LEV) add-on therapy. Methods From June 2012 to September 2015, 68 intractable epileptic children who had been diagnosed at the Outpatient Department of the Xuzhou Children′s Hospital were randomly selected and given the LEV add-on therapy.The expressions of MRP1 and MRP2 in their peripheral blood mononuclear cells were detected by real-time fluorescence quantitative polymerase chain reaction and Western blot before the LEV add-on therapy and 6 months after the LEV add-on therapy. Results (1) Compared to the expressions of MRP1 mRNA and MRP2 mRNA in the peripheral blood mononuclear of intractable epileptic children before therapy, the expressions of MRP1 mRNA (0.23±0.02) and MRP2 mRNA (0.17±0.03) after therapy were obviously down-regulated with significant differences(tMRP1=32.77, P<0.01; tMRP2=26.73, P<0.01). (2)The relative expressions of protein of MRP1 and MRP2 in the peripheral blood mononuclear of intractable epileptic children before therapy were 0.59±0.03, 0.53±0.02 respectively, and they were 0.39±0.03, 0.38±0.02 after therapy.Compared with those before therapy, the protein expressions of MRP1 and MRP2 were down-regulated, and the differences were significant (tMRP1=11.55, P<0.01; tMRP2=9.87, P<0.01). Conclusions The expressions of MRP1 and MRP2 mRNA and protein in the peripheral blood mononuclear cells in some intractable epileptic children can be down-regulated if patients receive the LEV add-on therapy, and LEV may control the seizures of intractable epileptic children by down-regulating the expressions of MRP1 and MRP2, then reduce the occurrence and development of drug-resistant epilepsy. Key words: Intractable epilepsy; Levetiracetam; Multidrug resistant-associated protein; Child

The phytoestrogens daidzein and equol inhibit the drug transporter BCRP/ABCG2 in breast cancer cells: potential chemosensitizing effect.

The soy isoflavone genistein has been described to up-regulate breast cancer resistance protein (BCRP) and, thus, enhance chemoresistance in breast cancer cells. The aim of this work was to assess the effect of long- and short-term incubation with daidzein, the second most abundant soy isoflavone and its metabolite equol on the expression and activity of P-glycoprotein, multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and BCRP in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with phytoestrogen concentrations within the range achieved in individuals with a high isoflavone intake. Transporter expression was evaluated at protein and mRNA level through western blot and qRT-PCR, respectively. Transporter activity was determined using doxorubicin, mitoxantrone and carboxy-dichlorofluorescein as substrates. Daidzein (5µM) up-regulated MRP2- and down-regulated MRP1 protein expressions in MCF-7 and MDA-MB-231 cells, respectively. Both effects were ER-dependent, as determined using the antagonist ICI 182,780. The decrease in MRP1 mRNA in MDA-MB-231 cells indicates a transcriptional mechanism. On the contrary, MRP2 induction in MCF-7 cells takes place post-transcriptionally. Whereas changes in the transporter expression had a minor effect on the transporter activity, acute incubation with daidzein, R-equol and S-equol led to a strong inhibition of BCRP activity and an increase in the IC50 of BCRP substrates. In contrast to previous reports for genistein, daidzein and equol do not provoke a major up-regulation of the transporter expression but instead an inhibition of BCRP activity and sensitization to BCRP substrates.

MRP1 protein expression in leukemic stem cells as a negative prognostic marker in acute myeloid leukemia patients.

It is well established that expression of multi-drug resistance (MDR) proteins (MDR1, BCRP, MDR3, MRP1, and LRP) in leukemic blasts correlates with acute myeloid leukemia (AML) patients' clinical response. Assuming that leukemic stem cells (LSC) are resistant to chemotherapy and responsible for relapse, it might be clinically relevant to evaluate the expression level of MDR proteins in LSC and relate it to the clinical outcome. Bone marrow samples from 26 patients with de novo AML were labeled with antibodies to distinguish CD34+CD38-CD123+ LSC population and with antibodies against MDR1, BCRP, MDR3, MRP1, or LRP proteins. Multicolor flow cytometry was applied to evaluate the expression of MDR proteins in blasts and LSC. Nine of 26 patients with AML attained CR (30%). High negative correlation was found between MDR1 and LRP expression in blasts and the patient's remission. MDR proteins were expressed more frequently in LSC than in leukemic blasts. High negative correlation was also observed between remission achievement and MRP1 expression in LSC. Our data present for the very first time the high negative correlation between MRP1 protein expression in LSC and AML patients' remission. It does strongly suggest that MRP1 expression in LSC is an adverse prognostic marker in patients with de novo AML.

Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier

Poor antiretroviral drug (ARV) penetration in the testes could be due, in part, to the presence of ATP-binding cassette (ABC) membrane-associated drug efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs) expressed at the blood-testis barrier (BTB). The functional expression of these transporters is known to be regulated by ligand-activated nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in various tissues. This study aimed to investigate in vitro and ex vivo the role of PXR and CAR in the regulation of ABC transporters at the BTB. Both PXR and CAR proteins were expressed in human testicular tissue and in mouse TM4 Sertoli cells (an in vitro cell line model of the BTB). In addition, we demonstrated an upregulation of P-gp, Bcrp, and Mrp4 mRNA and protein expression, after exposure to PXR or CAR ligands in TM4 cells. Small interfering RNA downregulation of PXR or CAR attenuated the expression of these transporters, suggesting the direct involvement of these nuclear receptors in regulating P-gp, Bcrp, and Mrp4 in this system. In an ex vivo study using freshly isolated mouse seminiferous tubules, we found that exposure to PXR or CAR ligands, including ARVs, significantly increased P-gp expression and function. Together, our data suggest that ABC transporters could be regulated at the BTB during chronic treatment with ARVs that can serve as ligands for PXR and CAR, which could in turn further limit testicular ARV concentrations.

Graphene Quantum Dots Downregulate Multiple Multidrug‐Resistant Genes via Interacting with Their C‐Rich Promoters

Multidrug resistance (MDR) is the major factor in the failure of many forms of chemotherapy, mostly due to the increased efflux of anticancer drugs that mediated by ATP-binding cassette (ABC) transporters. Therefore, inhibiting ABC transporters is one of effective methods of overcoming MDR. However, high enrichment of ABC transporters in cells and their broad substrate spectra made to circumvent MDR are almost insurmountable by a single specific ABC transporter inhibitor. Here, this study demonstrates that graphene quantum dots (GQDs) could downregulate the expressions of P-glycoprotein, multidrug resistance protein MRP1, and breast cancer resistance protein genes via interacting with C-rich regions of their promoters. This is the first example that a single reagent could suppress multiple MDR genes, suggesting that it will be possible to target multiple ABC transporters simultaneously with a single reagent. The inhibitory ability of the GQDs to these drug-resistant genes is validated further by reversing the doxorubicin resistance of MCF-7/ADR cells. Notably, GQDs have superb chemical and physical properties, unique structure, low toxicity, and high biocompatibility; hence, their capability of inhibiting multiple drug-resistant genes holds great potential in cancer therapy.

Analyzing the hepatoprotective effect of the Swertia cincta Burkillextract against ANIT-induced cholestasis in rats by modulating the expression of transporters and metabolic enzymes

Ethnopharmacological relevanceSwertia cincta Burkill was traditionally used for treating jaundice and various types of chronic and acute hepatitis in Yunnan and Tibet in China for hundreds of years. This study aims to investigate the protective effect of S. cincta Burkill (ESC) extract on alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity and cholestasis in rats. Materials and methodsCrude extracts were prepared using 90% ethanol and by vacuum drying. We utilized an ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS) system to conduct a phytochemical analysis of the active components of ESC. Liver function was evaluated by measuring the serum levels of enzymes and components and by analyzing the liver histology. We also measured the expression of bile metabolism-related transporters and metabolic enzymes at both protein and mRNA levels to elucidate the underlying mechanisms. ResultsESC analysis using an UHPLC/Q-TOF-MS revealed eight compounds. Oral administration of ESC to ANIT-treated rats can significantly reduce the increases in serum levels of ALT, AST, ALP, TBIL, and TBA. It can also improve liver pathology and bile flow. Western blot and qRT-PCR analyses showed that ESC upregulated the protein and mRNA expression of Fxr, Ntcp, Bsep, Cyp7a1, Mrp2, and Mdr2. ConclusionESC could alleviate liver injury by reducing enzyme activities of serums, improving liver pathology and bile flow. The protective mechanism was associated with regulation of the expression of hepatic transporters and metabolic enzymes.

Aldose reductase inhibitor increases doxorubicin-sensitivity of colon cancer cells and decreases cardiotoxicity

Anthracycline drugs such as doxorubicin (DOX) and daunorubicin remain some of the most active wide-spectrum and cost-effective drugs in cancer therapy. However, colorectal cancer (CRC) cells are inherently resistant to anthracyclines which at higher doses cause cardiotoxicity. Our recent studies indicate that aldose reductase (AR) inhibitors such as fidarestat inhibit CRC growth in vitro and in vivo. Here, we show that treatment of CRC cells with fidarestat increases the efficacy of DOX-induced death in HT-29 and SW480 cells and in nude mice xenografts. AR inhibition also results in higher intracellular accumulation of DOX and decreases the expression of drug transporter proteins MDR1, MRP1, and ABCG2. Further, fidarestat also inhibits DOX–induced increase in troponin-I and various inflammatory markers in the serum and heart and restores cardiac function in mice. These results suggest that fidarestat could be used as adjuvant therapy to enhance DOX sensitivity of CRC cells and to reduce DOX-associated cardiotoxicity.

ABCC4 functional SNP in the 3' splice acceptor site of exon 8 (G912T) is associated with unfavorable clinical outcome in children with acute lymphoblastic leukemia.

ATP-binding cassette subfamily C member 4 (ABCC4) encoding MRP4 protein is involved in pediatric acute lymphoblastic leukemia (ALL) drug resistance. The nonsynonymous single nucleotide polymorphism (SNP) rs2274407 (G912T; K304N) is located in the 3' splice acceptor site of exon 8 of ABCC4 pre-mRNA. The aim of this study was to investigate the prognostic value of rs2274407 in childhood ALL and its possible functional effect on MRP4. ABCC4 G912T SNP was genotyped in 145 Iranian Philadelphia-negative (Ph-) children with ALL using modified tetra-primer ARMS PCR and evaluated for possible association with 3-year disease-free survival (3DFS). In addition, functional impact of rs2274407 on the MRP4 activity and possible post-transcriptional modifications were bioinformatically and experimentally studied. ABCC4 912T allele carriers (G/T and T/T genotypes) are associated with worse 3DFS in Pre-B cell ALL [P=0.00019, OR (95% CI)=13.17 (2.55-68.11)]. In addition, computational studies showed that K304N alteration has no impact on the MRP4 activity. However, it may disrupt the normal splicing process of ABCC4 pre-mRNA. To date, this is the first study that shows the potential functional impact of rs2274407 SNP on the aberrant splicing of ABCC4 mRNA. We also demonstrated a robust association between G912T and pediatric ALL negative outcome, which may be explained by the novel computational studies performed in this study.