Multidrug Resistance-associated Protein Research Articles

Lipid-derived electrophiles inhibit the function of membrane channels during ferroptosis

The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO2). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis.

A metabolic mechanism analysis of Gentiana radix and wine-processed Gentiana radix effects on damp-heat jaundice syndrome in Rats

Ethnopharmacological relevanceGentiana radix (GR) and wine-processed Gentiana radix (WGR) are commonly used in folk medicine for the treatment of bile or liver disorders, including jaundice, hepatitis, swelling or inflammation for thousands of years. However, the therapeutic effects of gentian root (GR) and wine-made gentian root (WGR) treatment on damp-heat jaundice syndrome (DHJS) have not been studied in animal experiments.Aim of the study: This study aimed to investigate the protective effects and mechanisms of GR and WGR on DHJS in rats. Materials and methodsIn a high-fat and high-sugar diet in a humidified hot environment, hepatic injury induced by giving of alpha-naphthalene isothiocyanate (ANIT) in rats were used as a DHJS model. Histological analysis, enzyme-linked immunosorbent assay (ELISA), PCR analysis, and metabolomics were used to elucidate the mechanism of GR and WGR for DHJS. ResultsThe results indicated that GR and WGR affected DHJS by inhibiting the release of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), direct bilirubin (D-BIL), total bilirubin (TBIL), total bile acid (TBA), malondialdehyde (MDA), glutathione S-transferase (GST) (P < 0.05). In addition, they significantly reduced the gene expression levels of Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BESP), multidrug resistance-associated protein 2 (MRP2) and multidrug resistance-associated protein 3 (MRP3) (P < 0.05). The WGR group improved the above function indicators better than the GR group. GR and WGR could restore 11 potential biomarkers in rats with DHJS tended to return to normal levels, these biomarkers were involved in arachidonic acid metabolism, steroid hormone biosynthesis, biosynthesis of unsaturated fatty acids, porphyrin and chlorophyll metabolism, retinol metabolism, arginine biosynthesis. The results of the metabolic pathway showed that WGR was significantly better than GR in the improvement of porphyrin and chlorophyll metabolism. ConclusionsThese findings suggest that treatment with GR and WGR has a beneficial effect on DHJS in rats, the major mechanisms may be involved in improving functional indicators of the body and endogenous metabolism, and WGR is more effective than GR. It provides important evidence for the clinical application of GR and WGR in the treatment of DHJS.

Advancing 6-bromo-7-[11C]methylpurine to clinical use: improved regioselective radiosynthesis, non-clinical toxicity data and human dosimetry estimates

Background6-Bromo-7-[11C]methylpurine ([11C]BMP) is a radiotracer for positron emission tomography (PET) to measure multidrug resistance-associated protein 1 (MRP1) transport activity in different tissues. Previously reported radiosyntheses of [11C]BMP afforded a mixture of 7- and 9-[11C]methyl regioisomers. To prepare for clinical use, we here report an improved regioselective radiosynthesis of [11C]BMP, the results of a non-clinical toxicity study as well as human dosimetry estimates based on mouse PET data.Results[11C]BMP was synthesised by regioselective N7-methylation of 6-bromo-7H-purine (prepared under good manufacturing practice) with [11C]methyl triflate in presence of 2,2,6,6-tetramethylpiperidine magnesium chloride in a TRACERlab™ FX2 C synthesis module. [11C]BMP was obtained within a total synthesis time of approximately 43 min in a decay-corrected radiochemical yield of 20.5 ± 5.2%, based on starting [11C]methyl iodide, with a radiochemical purity > 99% and a molar activity at end of synthesis of 197 ± 130 GBq/μmol (n = 28). An extended single-dose toxicity study conducted in male and female Wistar rats under good laboratory practice after single intravenous (i.v.) administration of unlabelled BMP (2 mg/kg body weight) revealed no test item related adverse effects. Human dosimetry estimates, based on dynamic whole-body PET data in female C57BL/6J mice, suggested that an i.v. injected activity amount of 400 MBq of [11C]BMP will deliver an effective dose in the typical range of 11C-labelled radiotracers.Conclusions[11C]BMP can be produced in sufficient amounts and acceptable quality for clinical use. Data from the non-clinical safety evaluation showed no adverse effects and suggested that the administration of [11C]BMP will be safe and well tolerated in humans.

Inhibition of human drug transporter activities by succinate dehydrogenase inhibitors

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0–3.9 μM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 μM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 μM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

Physiologically based pharmacokinetics modeling and transporter proteomics to predict systemic and local liver and muscle disposition of statins.

Statins are used to reduce liver cholesterol levels but also carry a dose-related risk of skeletal muscle toxicity. Concentrations of statins in plasma are often used to assess efficacy and safety, but because statins are substrates of membrane transporters that are present in diverse tissues, local differences in intracellular tissue concentrations cannot be ruled out. Thus, plasma concentration may not be an adequate indicator of efficacy and toxicity. To bridge this gap, we used physiologically based pharmacokinetic (PBPK) modeling to predict intracellular concentrations of statins. Quantitative data on transporter clearance were scaled from invitro to invivo conditions by integrating targeted proteomics and transporter kinetics data. The developed PBPK models, informed by proteomics, suggested that organic anion-transporting polypeptide 2B1 (OATP2B1) and multidrug resistance-associated protein 1 (MRP1) play a pivotal role in the distribution of statins in muscle. Using these PBPK models, we were able to predict the impact of alterations in transporter function due to genotype or drug-drug interactions on statin systemic concentrations and exposure in liver and muscle. These results underscore the potential of proteomics-guided PBPK modeling to scale transporter clearance from invitro data to real-world implications. It is important to evaluate the role of drug transporters when predicting tissue exposure associated with on- and off-target effects.

Multi-functional D-alpha-tocopheryl polyethylene glycol succinate surface modified nanocrystals improve the stability and oral bioavailability of pueraria flavonoids

Pueraria flavonoids (PF), the main component of “Getong Tongluo capsule”, were applied in the clinical treatment of cardiovascular diseases. However, the complex nature of multiple-components, along with poor stability and low oral bioavailability of PF, hinders its widespread clinical use. This study aimed to develop D-alpha-tocopheryl polyethylene glycol succinate (TPGS) surface-modified PF-loaded nanocrystals (PF–NCs)，and investigate their physico-chemical properties and oral absorption mechanism. The prepared PF-NCs showed as rod shape crystal with an average particle size and polydispersity index of 259.0 ± 7.80 nm and 0.195 ± 0.046. Compared with PF, the stability of PF-NCs was significantly improved. Furthermore, the transport of PF-NCs in Madin-Darby canine kidney (MDCK) monolayers was time-, concentration- and energy-dependent. The endocytosis of PF-NCs was mediated mainly through macropinocytosis, caveolin, clathrin and lipid rafts/caveolae. The involvement of multidrug resistance-associated protein 2 (MRP2) transporter and P-gp in the transmembrane transport of PF-NCs was also significant. Pharmacokinetic studies demonstrated that PF-NCs exhibited a substantial enhancement of PF's oral bioavailability. Overall, the prepared PF-NCs demonstrated enhanced stability and bioavailability, offering valuable insights for the development of multi-component nanocrystals and investigation into their mechanisms of oral absorption.

Ataxia telangiectasia and Rad3-related (ATR) inhibition by VE-822 potently reversed 5-flourouracil resistance in colorectal cancer cells through targeting DNA damage response.

VE-822 is a novel inhibitor of ATR, a key kinase involved in the DNA damage response pathway. The role of ATR inhibition in reversing drug resistance in various cancer types has been investigated. Therefore, this study investigated the effects of ATR inhibition by VE-822 on reversing 5-fluorouracil (5-FU) resistance in colorectal cancer cell line (Caco-2). Caco-2 and 5-FU resistance Caco-2 (Caco-2/5-FU) cells were treated with 5-FU and VE-822, alone and in combination. Cell proliferation and viability were assessed by MTT assay and Trypan Blue staining. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) activities were measured by Rhodamine123 accumulation and uptake assay. The mRNA levels of P-gp, MRP-1, ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) were measured by qRT-PCR. Western blot was used to measure the protein levels of P-gp, MRP-1, γ-H2AX, ATR and CHK1 in cells. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) levels were determined via ELISA. Apoptosis was evaluated by ELISA death assay, DAPI staining and lactate dehydrogenase (LDH) assay. The Caco-2/5-FU cells showed lower levels of 5-FU mediated proliferation inhibition in comparison to Caco-2 cells. VE-822 decreased the IC50 value of 5-FU on resistant cells. In addition, the expression levels and activity of P-gp and MRP-1 were significantly decreased in resistant cells treated with VE-822 (P < 0.05). The combination of 5-FU and VE-822 increased apoptosis in Caco-2/5-FU cells by downregulating CHK1 and ATR and upregulating γ-H2AX and 8-oxo-dG. The simultaneous treatment of resistant colorectal cancer cells with 5-FU and ATR inhibitor, VE-822, was demonstrated to be effective in reversing drug resistance and potentiating 5-FU mediated anticancer effects via targeting DNA damage.

Unveiling synergistic QTLs associated with slow wilting in soybean (Glycine max [L.] Merr.)

Key messageA stable QTL qSW_Gm10 works with a novel locus, qSW_Gm01, in a synergistic manner for controlling slow-wilting traits at the early vegetative stage under drought stress in soybean.Drought is one of the major environmental factors which limits soybean yield. Slow wilting is a promising trait that can enhance drought resilience in soybean without additional production costs. Recently, a Korean soybean cultivar SS2-2 was reported to exhibit slow wilting at the early vegetative stages. To find genetic loci responsible for slow wilting, in this study, quantitative trait loci (QTL) analysis was conducted using a recombinant inbred line (RIL) population derived from crossing between Taekwangkong (fast-wilting) and SS2-2 (slow-wilting). Wilting score and leaf moisture content were evaluated at the early vegetative stages for three years. Using the ICIM-MET module, a novel QTL on Chr01, qSW_Gm01 was identified, together with a previously known QTL, qSW_Gm10. These two QTLs were found to work synergistically for slow wilting of the RILs under the water-restricted condition. Furthermore, the SNP markers from the SoySNP50K dataset, located within these QTLs, were associated with the wilting phenotype in 30 diverse soybean accessions. Two genes encoding protein kinase 1b and multidrug resistance-associated protein 4 were proposed as candidate genes for qSW_Gm01 and qSW_Gm10, respectively, based on a comprehensive examination of sequence variation and gene expression differences in the parental lines under drought conditions. These genes may play a role in slow wilting by optimally regulating stomatal aperture. Our findings provide promising genetic resources for improving drought resilience in soybean and give valuable insights into the genetic mechanisms governing slow wilting.

An acute herb-drug interaction of Magnoliae Officinalis Cortex with methotrexate via inhibiting multidrug resistance-associated protein 2.

Magnoliae Officinalis Cortex (MOC), an herbal drug, contains polyphenolic lignans mainly magnolol (MN) and honokiol (HK). Methotrexate (MTX), a critical drug for cancers and autoimmune deseases, is a substrate of multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). This study investigated the effect of coadministration of MOC on the pharmacokinetics of MTX and relevant mechanisms. Sprague-Dawley rats were orally administered MTX alone and with single dose (2.0 and 4.0 g/kg) and repeated seven doses of MOC (2.0 g/kg thrice daily for 2 days, the 7th dose given at 0.5 h before MTX). The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. The results showed that a single dose of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 352% and 308%, and a single dose at 4.0 g/kg significantly enhanced the AUC0-t and MRT by 362% and 291%, respectively. Likewise, repeated seven doses of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 461% and 334%, respectively. Mechanism studies indicated that the function of MRP2 was significantly inhibited by MN, HK and the serum metabolites of MOC (MOCM), whereas BCRP was not inhibited by MOCM. In conclusion, coadministration of MOC markedly enhanced the systemic exposure and mean residence time of MTX through inhibiting the MRP2-mediated excretion of MTX.

IFN‑γ induces apoptosis in gemcitabine‑resistant pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and aggressive form of pancreatic cancer. Gemcitabine (GEM), the first‑line treatment for PDAC, which alleviates symptoms and enhances the quality of life of patients. However, it is prone to lead to the development of drug resistance during treatment. Interferon (IFN)‑γ exhibits antitumor and immunomodulatory properties. The present study aimed to explore the impact of IFN‑γ on the viability, migration and apoptosis of GEM‑resistant pancreatic cancer cells. Firstly, a GEM‑resistant pancreatic cancer cell line, named PANC‑1/GEM, was constructed. Hematoxylin and eosin staining analyzed the cell morphology, whereas reverse transcription‑quantitative PCR (RT‑qPCR) assessed the expression levels of the drug‑resistance genes multidrug resistance‑associated protein (MRP) and breast cancer resistance protein (BCRP). The MTT assay and cell counting techniques were used to determine the appropriate concentration of IFN‑y and its effects on cell viability. The IFN‑γ‑induced apoptosis of PANC‑1/GEM cells was assessed using an Apoptosis Detection Kit, whereas the impact of IFN‑γ on the migration of these cells was evaluated using a wound‑healing assay. The MTT assay revealed a resistance index of 22.4 in the PANC‑1/GEM cell line. RT‑qPCR indicated that, compared with in wild‑type cells, the PANC‑1/GEM resistant strain exhibited lower MRP and higher BCRP mRNA expression levels. The optimal concentration of IFN‑γ for affecting PANC‑1/GEM cells was determined to be 0.3 µg/ml. At this concentration, IFN‑γ induced PANC‑1/GEM cell apoptosis, along with a notable reduction in migration. Following treatment of PANC‑1/GEM cells with IFN‑γ, MRP expression increased whereas BCRP mRNA expression decreased, indicating a reversal in their drug‑resistance gene expression. In conclusion, IFN‑γ exhibited antitumor immune properties by upregulating MRP and downregulating BCRP expression, reversing drug‑resistance gene expression, and reducing cell viability and migration, while promoting apoptosis in PANC‑1/GEM cells. IFN‑γ could potentially serve as a treatment option for patients with GEM‑resistant pancreatic cancer.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes.

Doxorubicin is a commonly used anti-cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance-associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell-derived cardiomyocytes (hiPSC-CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC-CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.

PIM Kinase Inhibition Sensitizes Neuroblastoma to Doxorubicin

BackgroundChemoresistance contributes to relapse in high-risk neuroblastoma. Cancer cells acquire resistance through multiple mechanisms, including drug efflux pumps. In neuroblastoma, multidrug resistance-associated protein-1 (MRP1/ABCC1) efflux pump expression correlates with worse outcomes. These pumps are regulated by PIM kinases, a family of serine–threonine kinases, overexpressed in neuroblastoma. We hypothesized PIM kinase inhibition would sensitize neuroblastoma cells by modulating MRP1. MethodsKocak database query evaluated ABCC1, PIM1, PIM2, and PIM3 expression in neuroblastoma patients. SK-N-AS and SK-N-BE(2) cells were treated with doxorubicin or the pan-PIM kinase inhibitor, AZD1208. Flow cytometry assessed intracellular doxorubicin accumulation. AlamarBlue assay measured viability. The lethal dose 50% (LD50) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy. ResultsKocak database query demonstrated positive correlation between PIM genes and ABCC1. PIM kinase inhibition increased intracellular doxorubicin accumulation in both cell lines, suggesting PIM kinase regulation of MRP1. Isobolograms showed synergy between AZD1208 and doxorubicin. ConclusionsThe correlation between PIM and ABCC1 gene expression suggests PIM kinases may contribute to neuroblastoma chemotherapeutic resistance. PIM kinase inhibition increased intracellular doxorubicin accumulation. Combination treatment with AZD1208 and doxorubicin decreased neuroblastoma cell viability in a synergistic fashion. These findings support further investigations of PIM kinase inhibition in neuroblastoma. Type of StudyBasic Science Research. Level of EvidenceNA.

Lovastatin impairs cellular proliferation and enhances hyaluronic acid production in fibroblast-like synoviocytes

IntroductionStatins have demonstrated chondroprotective effects by reducing inflammation and mitigating extracellular matrix degradation. However, statins are also reported to be cytotoxic to several types of cells. Early-onset osteoarthritis (OA) is characterized by synovial inflammation, which adversely affects hyaluronan (HA) production in fibroblast-like synoviocytes (FLSs). Nevertheless, the precise effects of statins on the synovium remain unclear. MethodsThis study investigated the impact of lovastatin on human FLSs, and HA secretion-related genes, signaling pathways, and production were evaluated. ResultsThe findings revealed that high doses of lovastatin (20 or 40 μM) decreased FLS viability and increased cell death. FLS proliferation ceased when cultured in a medium containing 5 or 10 μM lovastatin. mRNA expression analysis demonstrated that lovastatin (5 and 10 μM) upregulated the gene level of hyaluronan synthase 1 (HAS1), HAS2, and proteoglycan 4 (PRG4), but not HAS3. While the expression of multidrug resistance-associated protein 5 transporter gene remained unaffected, both inward-rectifying potassium channel and acid-sensing ion channel 3 were upregulated. Western blot further confirmed that lovastatin increased the production of HAS1 and PRG4, and activated the PKC-α, ERK1/2, and p38-MAPK signaling pathways. Additionally, lovastatin elevated intracellular cAMP levels and HA production in FLSs. ConclusionLovastatin impairs cellular proliferation but enhances HA production in human FLSs.

Extracellular cAMP and MRP4 activity influence in vitro capacitation and fertilizing ability of pig spermatozoa

cAMP has been reported to be an essential driver of sperm capacitation. In bovine sperm cAMP efflux through multidrug resistance-associated protein 4 (MRP4) has been suggested to maintain intracellular cAMP homeostasis and generate extracellular signaling able to regulate capacitation. The aim of this work was to determine whether extracellular cAMP may influence in vitro pig sperm capacitation and acquisition of fertilizing ability and to evaluate the role of MRP4. In vitro sperm capacitation and gamete coincubation were performed in Brackett and Oliphant's medium (BO) in presence of caffeine (Ctr+) or in BO without caffeine (Ctr-) supplemented with 0, 8, 9, 10 mM cAMP. Despite the percentage of capacitated sperm, assayed by immunolocalization of tyrosine-phosphorylated proteins, was significantly lower in Ctr- compared to Ctr+, it increased supplementing 10 mM cAMP to Ctr- reaching values similar to Ctr+. The absence of caffeine during gamete coincubation reduced the fertilization rate compared to Ctr+, while 10 mM cAMP supplementation to Ctr- increased the fertilization rate reaching values similar to Ctr + .The presence of MRP4 in pig spermatozoa was detected for the first time by western blot and immunohistochemistry assays. To evaluate MRP4 role on pig sperm capacitation, in vitro capacitation and gamete coincubation were performed in Ctr + in presence of MK571, a MRP4 selective inhibitor. MK571 reduced the percentage of capacitated cells and the fertilization rate, while cAMP addition fully reversed MRP4 blockade consequences.Present findings suggest that, under our in vitro conditions, extracellular cAMP and MRP4 activity influence pig sperm capacitating events.

Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport.

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Chishao (Paeoniae Radix Rubra) alleviates intra-hepatic cholestasis by modulating NTCP in rats.

Background: Cholestasis is a common pathological manifestation dominated by accumulation of potentially toxic biliary compounds. Na+-taurocholate cotransporting polypeptide (NTCP) plays a critical role in protection from cholestasis and can be targeted therapeutically. Chishao (Paeoniae Radix Rubra) is a clinically efficacious agent for treating cholestasis, but the underlying mechanism has not been fully clarified. Objective: To evaluate the effects of Chishao on the expression of NTCP in rats with alpha-naphthylisothiocyanate (ANIT)-induced cholestasis. Methods: Chishao extracts were obtained by water decoction. Cholestasis model induced by ANIT in rats were established. Thirty rats were divided into five groups: control group (C), ANIT model group (M), 10g/kg Chishao group (LD), 20g/kg Chishao group (MD) and 40g/kg Chishao group (HD). The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), direct bilirubin (DB), alkaline phosphatase (ALP) and total bile acid (TBA) were detected. The mRNA and protein expression of NTCP, multidrug resistance associated protein 2 (MRP2) and bile salt export pump (BSEP) were detected by reverse transcription qPCR and Western blotting respectively. To assess the effects of Chishao on NTCP, MRP2 and BSEP localized at the membrane of hepatocytes, an in vitro experiment involving primary hepatocytes was conducted via the utilization of laser scanning confocal microscopy. Results: The extracts of Chishao significantly improved serum ALT, AST, ALP, TB, DB and TBA (p < 0.05), especially ALP in the HD group (p < 0.01). The histological pathological findings were also reversed in LD, MD and HD groups. The mRNA level of MRP2 was significantly downregulated after treatment with ANIT, whereas it was reversed in MD and HD groups (p < 0.05). The mRNA expression of NTCP was significantly downregulated after ANIT treatment, but dramatically upregulated in the HD group. The expressions of BSEP and MRP2 were similar, but that of NTCP decreased after ANIT treatment, which was reversed significantly by Chishao extracts in a dose-dependent manner. The expression of NTCP in hepatocytes from rats increased dose-dependently after Chishao treatment in vitro. Conclusion: Chishao extracts can improve the serum and histological performances of intra-hepatic cholestasis caused by ANIT, probably by working on transport proteins in liver cell membranes.

TMEM97 knockdown inhibits 5-fluorouracil resistance by regulating epithelial-mesenchymal transition and ABC transporter expression via inactivating the Akt/mTOR pathway in 5-fluorouracil-resistant colorectal cancer cells.

Resistance to 5-fluorouracil (5-FU) is still a primary setback to the success of colorectal cancer (CRC) chemotherapy. Transmembrane protein 97 (TMEM97) functions as an oncogene in CRC. However, the role and mechanism of TMEM97 in regulating 5-FU resistance in CRC cells remains unclear. TMEM97 expression in CRC samples was analyzed by GEPIA and human protein atlas (HPA) databases. TMEM97, E-cadherin, Vimentin, N-cadherin, P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1)/ABCC1, ABCC2, and the changes of protein kinase B/mammalian target of rapamycin (mTOR) pathway were explored by western blot analysis. IC50 value for 5-FU and cell viability was examined by MTT assay. Apoptosis was evaluated by flow cytometry. TMEM97 was highly expressed in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) based on GEPIA and HPA databases. TMEM97 knockdown attenuated 5-FU resistance in HCT116/R and SW480/R cells, as evidenced by the reduced IC50 value for 5-FU and the increased apoptosis. TMEM97 knockdown suppressed epithelial-mesenchymal transition (EMT), expression of ATP-binding cassette (ABC) transporters, and the Akt/mTOR pathway. Mechanistically, activation of Akt/mTOR pathway abolished the inhibitory effects of TMEM97 knockdown on 5-FU resistance, EMT, and ABC transporter expression. In conclusion, TMEM97 knockdown inhibited 5-FU resistance in CRC by regulating EMT and ABC transporter expression via inactivating the Akt/mTOR pathway.