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Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.

Purpose: Multidrug resistance-associated protein 1 (MRP1) is a transport protein with a widespread tissue distribution, which has been implicated in the pathophysiology of Alzheimer's and chronic respiratory disease. PET with 6-bromo-7-[11C]methylpurine ([11C]BMP) has been used to measure MRP1 function in rodents. In this study, [11C]BMP was for the first time characterised in humans to assess the function of MRP1 and other MRP subtypes in different tissues. Methods: Thirteen healthy volunteers (7 men, 6 women) underwent dynamic whole-body PET scans on a long axial field-of-view (LAFOV) PET/CT system after intravenous injection of [11C]BMP. Three subjects of each sex were scanned a second time to assess reproducibility. Volumes of interest were outlined for MRP-expressing tissues (cerebral cortex, cerebellum, choroid plexus, retina, lungs, myocardium, kidneys, and liver). From the time-activity curves, the elimination rate constant (kE, h− 1) was derived as a parameter for tissue MRP function and its test-retest variability (TRTV, %) was calculated. Radiation dosimetry was calculated using the Medical Internal Radiation Dose (MIRD) methodology. Results: Mean kE and corresponding TRTV values were: cerebral cortex: 0.055 ± 0.010 h− 1 (− 4 ± 24%), cerebellum: 0.033 ± 0.009 h− 1 (1 ± 39%), choroid plexus: 0.292 ± 0.059 h− 1 (0.1 ± 16%), retina: 0.234 ± 0.045 h− 1 (30 ± 38%), lungs: 0.875 ± 0.095 h− 1 (− 3 ± 11%), myocardium: 0.641 ± 0.105 h− 1 (11 ± 25%), kidneys: 1.378 ± 0.266 h− 1 (14 ± 16%), and liver: 0.685 ± 0.072 h− 1 (7 ± 9%). Significant sex differences were found for kE in the cerebellum, lungs and kidneys. Effective dose was 4.67 ± 0.18 µSv/MBq for men and 4.55 ± 0.18 µSv/MBq for women. Conclusion: LAFOV PET/CT with [11C]BMP potentially allows for simultaneous assessment of MRP function in multiple human tissues. Mean TRTV of kE in different tissues was in an acceptable range, except for the retina. The radiation dosimetry of [11C]BMP was in the typical range of 11C-tracers. LAFOV PET/CT holds great potential to assess at a whole-body, multi-tissue level molecular targets relevant for drug disposition in humans. Trial registration: EudraCT 2021-006348-29. Registered 15 December 2021. [ABSTRACT FROM AUTHOR]

The overexpression of ATP-binding cassette (ABC) transporters contributes to the failure of chemotherapies and symbolizes a great challenge in oncology, associated with the adaptation of tumor cells to anticancer drugs such that these transporters become less effective, a mechanism known as multidrug resistance (MDR). The aim of this review is to present the most widely used methodologies for induction and comprehension of in vitro models for detection of multidrug-resistant (MDR) modulators or inhibitors, including biochemical and morphological techniques for chemosensitivity studies. The overexpression of MDR proteins, predominantly, the subfamily glycoprotein-1 (P-gp or ABCB1) multidrug resistance, multidrug resistance-associated protein 1 (MRP1 or ABCCC1), multidrug resistance-associated protein 2 (MRP2 or ABCC2) and cancer resistance protein (ABCG2), in chemotherapy-exposed cancer lines have been established/investigated by several techniques. Amongst these techniques, the most used are (i) colorimetric/fluorescent indirect bioassays, (ii) rhodamine and efflux analysis, (iii) release of 3,30-diethyloxacarbocyanine iodide by fluorescence microscopy and flow cytometry to measure P-gp function and other ABC transporters, (iv) exclusion of calcein-acetoxymethylester, (v) ATPase assays to distinguish types of interaction with ABC transporters, (vi) morphology to detail phenotypic characteristics in transformed cells, (vii) molecular testing of resistance-related proteins (RT-qPCR) and (viii) 2D and 3D models, (ix) organoids, and (x) microfluidic technology. Then, in vitro models for detecting chemotherapy MDR cells to assess innovative therapies to modulate or inhibit tumor cell growth and overcome clinical resistance. It is noteworthy that different therapies including anti-miRNAs, antibody-drug conjugates (to natural products), and epigenetic modifications were also considered as promising alternatives, since currently no anti-MDR therapies are able to improve patient quality of life. Therefore, there is also urgency for new clinical markers of resistance to more reliably reflect in vivo effectiveness of novel antitumor drugs. [ABSTRACT FROM AUTHOR]

Objective: The metabolism- and transporter-based drug-drug interactions (DDIs) between mycophenolate mofetil (MMF) and co-administered medications may be key factors for the high individual variability in MMF exposure. This study systematically assessed the influence of co-medications on the mycophenolic acid (MPA) pharmacokinetic (PK) process in vitro, particularly to provide mechanistic evidence of the metabolic interaction among steroids, cyclosporine (CsA), and MMF. Methods: Based on a previous study, we hypothesized that there are three main DDI pathways affecting MMF PK in vivo. A human hepatocyte induction study, transporter substrate/inhibition study using human embryonic kidney 293 cells, and multidrug resistance-associated protein 2 (MRP2) substrate/inhibition study using vesicle membrane were conducted to assess the mechanistic evidence of the metabolic interaction in triple therapies. The potential DDI risks associated with seven medications commonly co-administered with MMF in clinical practice were further evaluated. Results: The in vitro results suggested that prednisolone, the active metabolite of prednisone, induces the enzymatic activity of uridine 5'-diphosphoglucuronosyltransferase (UGT), particularly the UGT1A9 and UGT2B7 isoforms, resulting in increased metabolism of MPA to MPA glucuronide (MPAG). This induction potential was not observed in CsA-treated human hepatocytes. CsA inhibits organic anion-transporting polypeptide (OATP) 1B1- and OATP1B3- mediated MPAG. Prednisolone and CsA showed no inhibitory effect on MRP2- mediated MPAG efflux. Salvia miltiorrhiza significantly inhibited organic aniontransporting polypeptide and OAT 3 activities, suggesting that it affects the hepatic uptake and renal excretion of MPAG, causing increased MPAG exposure in vivo. Conclusion: These identified factors may contribute to the high inter-individual variability in MMF exposure and facilitate further development of mechanistic MMF PK models and individualized therapies. [ABSTRACT FROM AUTHOR]

Endosymbionts provide essential nutrients for hosts, promoting growth, development, and reproduction. However, the molecular regulation of nutrient transport from endosymbiont to host is not well understood. Here, we used bioinformatic analysis, RNA-Sequencing, luciferase assays, RNA immunoprecipitation, and in situ hybridization to show that a bacteriocyte-distributed MRP4 gene (multidrug resistance-associated protein 4) is negatively regulated by a host (aphid)-specific microRNA (miR-3024). Targeted metabolomics, microbiome analysis, vitamin B6 (VB6) supplements, 3D modeling/molecular docking, in vitro binding assays (voltage clamp recording and microscale thermophoresis), and functional complementation of Escherichia coli were jointly used to show that the miR-3024/MRP4 axis controls endosymbiont (Serratia)-produced VB6 transport to the host. The supplementation of miR-3024 increased the mortality of aphids, but partial rescue was achieved by providing an external source of VB6. The use of miR-3024 as part of a sustainable aphid pest-control strategy was evaluated by safety assessments in nontarget organisms (pollinators, predators, and entomopathogenic fungi) using virus-induced gene silencing assays and the expression of miR-3024 in transgenic tobacco. The supplementation of miR-3024 suppresses MRP4 expression, restricting the number of membrane channels, inhibiting VB6 transport, and ultimately killing the host. Under aphids facing stress conditions, the endosymbiont titer is decreased, and the VB6 production is also down-regulated, while the aphid's autonomous inhibition of miR-3024 enhances the expression of MRP4 and then increases the VB6 transport which finally ensures the VB6 homeostasis. The results confirm that miR-3024 regulates nutrient transport in the endosymbiont-host system and is a suitable target for sustainable pest control. [ABSTRACT FROM AUTHOR]

Clotrimazole (CLZ), traditionally an antifungal agent, unveils promising avenues in cancer therapy, particularly in addressing bladder and prostate cancers. In vitro assessments underscore its remarkable efficacy as a standalone treatment, significantly diminishing cancer cell viability. Mechanistically, CLZ operates through multifaceted pathways, including the inhibition of Ca2+-dependent K+ channels, suppression of glycolysis-related enzymes, and modulation of the ERK-p65 signaling cascade, thus underscoring its potential as a versatile therapeutic agent. Our investigation sheds light on intriguing observations regarding the resilience of UM-UC-5 bladder cancer cells against high doses of paclitaxel (PTX), potentially attributed to heightened levels of the apoptosis-regulating protein Mcl-1. However, synergistic studies demonstrate that the combination of Doxorubicin (DOXO) and CLZ emerges as particularly potent, especially in prostate cancer contexts. This effectiveness could be associated with the inhibition of drug efflux mediated by multidrug resistance-associated protein 1 (MRP1), underscoring the importance of exploring combination therapies in cancer treatment paradigms. In essence, our findings shed light on the anticancer potential of CLZ, emphasizing the significance of tailored approaches considering specific cancer types and molecular pathways in drug repurposing endeavors. While further validation and clinical exploration are warranted, the insights gleaned from this study offer promising prospects for enhancing cancer therapy efficacy. [ABSTRACT FROM AUTHOR]

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multidrug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; Vmax) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a six-parameter version of this model to control time-course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only three unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT, found the mean estimated value for each parameter, and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of Vmax for MRP2 at the 30-min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP. NEW & NOTEWORTHY: We developed a computational model of sodium fluorescein (SF) biliary excretion in ex vivo machine perfusion and used this model to assess changes in model parameters associated with the activity of MRP2, a hepatocyte membrane transporter, in response to increasing warm ischemia time. We found a significant decrease in the parameter value describing MRP2 activity, consistent with a role of decreased MRP2 function in ischemia-reperfusion injury leading to decreased secretion of SF into bile. [ABSTRACT FROM AUTHOR]

Cyclosporine-A (CsA) is currently used to treat immune rejection after organ transplantation as a commonly used immunosuppressant. Liver injury is one of the most common adverse effects of CsA, whose precise mechanism has not been fully elucidated. Pregnane X receptor (PXR) plays a critical role in mediating drug-induced liver injury as a key regulator of drug and xenobiotic clearance. As a nuclear receptor, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters, including cytochrome P4503A (CPY3A) and multidrug resistance-associated protein 2 (MRP2). Our study established CsA-induced cytotoxic hepatocytes in an in vitro model, demonstrating that CsA dose-dependently increased the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) level secreted in the HepG2 cell supernatant, as well as viability and oxidative stress of HepG2 cells. CsA also dose-dependently decreased the PXR, CYP3A4, CPY3A5, and MRP2 levels of HepG2 cells. Mechanistically, altering the expression of PXR, CYP3A4, CYP3A5, and MRP2 affected the impact of CsA on AST and LDH levels. Moreover, altering the expression of PXR also changed the level of CYP3A4, CPY3A5, and MRP2 of HepG2 cells treated by CsA. Our presented findings provide experimental evidence that CsA-induced liver injury is PXR tightly related. We suggest that PXR represents an attractive target for therapy of liver injury due to its central role in the regulation of the metabolizing enzymes CYP3A and MRP2-mediated bile acid transport and detoxification. [ABSTRACT FROM AUTHOR]

Simple Summary: Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptors, progesterone receptors, and human epidermal receptors. This lack of receptors renders TNBC unsuitable for targeted-based treatment, making it the most fatal and aggressive subtype of breast cancer. TNBC has a greater relapse rate, worse prognosis, and increased metastasis rate compared to non-TNBC because of its tendency to resist apoptosis, a programmed cell death triggered by most chemotherapeutic drugs, producing anticancer efficacy. This work describes two new drugs, TPH104c, and TPH104m, that induce a non-apoptotic form of cell death in TNBC. The incubation of TNBC cells with TPH104c or TPH104m causes cellular expansion and rupture without producing apoptotic characteristics, such as nuclear fragmentation, apoptotic blebbing, or caspase activation. TPH104c and TPH104m decreased the mitochondrial protein, division regulator, and dynamin-related protein 1 (DRP1). The level of DRP1 in TNBC cells affects the magnitude of cytotoxicity produced by TPH104c and TPH104m. Apoptosis induction with taxanes or anthracyclines is the primary therapy for TNBC. Cancer cells can develop resistance to anticancer drugs, causing them to recur and metastasize. Therefore, non-apoptotic cell death inducers could be a potential treatment to circumvent apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induced non-apoptotic cell death in TNBC cells. These lead compounds were 15- to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to that of normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death, characterized by the absence of cellular shrinkage and the absence of nuclear fragmentation and apoptotic blebs. Although TPH104c and TPH104m induced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase the levels of cytochrome c and intracellular reactive oxygen species (ROS) and caspase activation, and cell death was not rescued by incubating cells with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). Furthermore, TPH104c and TPH104m significantly downregulated the expression of the mitochondrial fission protein, DRP1, and their levels determined their cytotoxic efficacy. Overall, TPH104c and TPH104m induced non-apoptotic cell death, and further determination of their cell death mechanisms will aid in the development of new potent and efficacious anticancer drugs to treat TNBC. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporter is a gene superfamily in plants. ATP-binding cassette subfamily C (ABCC) protein is a multidrug resistance-associated (MRP) transporter. They play various roles in plant growth, development, and secondary metabolite transport. However, there are few studies on ABCC transporters in tea plants. In this study, genome-wide association study (GWAS) analysis of epigallocatechin gallate (EGCG) content in 108 strains of Kingbird revealed that CsABCCs may be involved in EGCG transport. We identified 25 CsABCC genes at the genomic level of the tea plant, their phylogenetic tree, gene structure, targeted miRNA and other bioinformatics were analyzed. The expression patterns of CsABCCs in eight different tissues and abiotic stress indicate that they have potential roles in regulating the growth, development, and defense of tea plants. The correlation analysis revealed that the expression of the CsABCC11 gene was closely related to the EGCG content in tea buds of 108 strains of the Kingbird, and the subcellular localization experiments in tobacco showed that CsABCC11 protein was localized on the plasma membrane. The virus-induced gene silencing (VIGS) strategy in tea plants further verified that CsABCC11 was involved in EGCG accumulation. Our study laid a foundation for studying the biological function of CsABCC and provided a new candidate molecular marker gene for further EGCG-related variety breeding, which will be of great interest to breeders. Supplementary Material Supplementary Material File [ABSTRACT FROM AUTHOR]

[Objectives] This study aimed to investigate whether bile acids could promote the excretion of lipopolysaccharides (LPS) in broiler chickens and alleviate LPS-induced inflammatory responses. [Methods] Sixty 1-day-old Ross 308 male broiler chickens were divided into four groups, each consisting of 15 chickens:the control group (CON), and the model group (LPS) fed with basal diet, the low-dose bile acid alleviation group (LBA) fed with a basal diet containing 160 mg·kg-1 of porcine bile acid (BA), and the high-dose bile acid alleviation group (HBA) fed with a basal diet containing 250 mg·kg-1 of porcine bile acid. On the 28th day of the experiment, after a 9-hour fasting period, the CON group received intraperitoneal injection of an equivalent volume of physiological saline, while the LPS group, LBA group and HBA group received intraperitoneal injection of LPS (1 mg·kg-1 BW). Three hours after LPS injection, blood was drawn from the wing vein, and all chickens were slaughtered for sampling to detect inflammatory related indicator level, LPS levels, and the expression levels of genes associated with the optake and excretion of LPS in liver. [Results] The liver in the CON group did not exhibit inflammatory cell infiltration. In the LPS group, the liver showed extensive inflammatory cell infiltration, and serum IL-1β levels significantly increased (P<0.05). Additionally, serum and liver LPS levels and liver mannose receptor C1 gene (MRC1), stabilin 1/2 gene (STAB 1/2) expression levels all significantly increased (P<0.05). Moreover, the concentrations of total bile acids (TBA) in serum, liver, bile, and cecal contents significantly decreased (P<0.05). The gene expression levels of liver bile salt export pump (BSEP), multidrug resistance protein 2 (MRP2), multidrug resistance-associated protein 1 (MDR1), liver X receptor α (LXRα), and farnesoid X receptor (FXR) all significantly decreased (P<0.05). Compared with the LPS group, the LBA group reduced inflammatory cell infiltration in the liver (P<0.05) and significantly decreased in serum IL-1β concentration (P<0.05). Furthermore, serum and liver LPS levels, as well as the gene expression levels of liver MRC1 and STAB1/2, significantly decreased (P<0.05). In the HBA group, inflammatory cell infiltration disappeared in the liver, and both serum IL-1β concentration and the gene expression levels of liver MRC1 and STAB1/2 significantly decreased (P<0.05). Serum LPS level significantly decreased (P<0.05), while bile LPS levels significantly increased (P<0.05). Additionally, the concentrations of total TBA in serum, bile, and cecal contents, as well as the gene expression levels of liver MDR1, ATP-binding cassette transporter G2 (ABCG2), and FXR protein expression levels, all significantly increased (P<0.05). [Conclusions] In summary, bile acids enhance liver uptake, synthesis, and excretion functions, improving the efficiency of LPS excretion in broiler chickens and consequently alleviating inflammatory response. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 in vitro to in vivo 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 in vitro 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria. Dynamic processes of bacterial genome amplification involving a non-replicable but inheritable circular DNA molecule encoding multiple drug resistance proteins. [ABSTRACT FROM AUTHOR]

This document contains abstracts from the journal Clinical Science. The first abstract discusses the potential of targeting molecular ET-1 receptors to identify new therapeutic vulnerabilities in cancer. The second abstract focuses on the role of endothelial cell health in preventing cardiovascular diseases, specifically atherosclerosis and coronary artery disease. The third abstract explores the association between alterations in the cardiac and renal endothelin system and cardiac dysfunction in a preclinical model of lupus. The text also briefly mentions the 18th International Conference on Endothelin, which covered various aspects of endothelin signaling and clinical trials related to endothelin-based therapies. [Extracted from the article]

Background: 6-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. [ABSTRACT FROM AUTHOR]

The disease spectrum of ABCB4 gene mutation involves various diseases such as progressive familial intrahepatic cholestasis type 3（PFIC3）, gallstone disease, intrahepatic cholestasis of pregnancy, portal hypertension, liver cirrhosis, and even primary hepatic and biliary malignancies. A young male patient was admitted to Department of Hepatobiliary Medicine, Eastern Hepatobiliary Surgery Hospital, and was initially diagnosed with liver cirrhosis and gallstones, and he was planned to receive laparoscopic cholecystectomy. Preoperative examination showed abnormal liver function, liver cirrhosis, splenomegaly, and mild esophageal varices, and next generation sequencing was performed to make a confirmed diagnosis of ABCB4 gene mutation-associated liver cirrhosis with gallstones. The liver function of the patient gradually returned to normal after cholagogic treatment with ursodeoxycholic acid capsules. [ABSTRACT FROM AUTHOR]

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. The ABC transporter MRP2/ABCC2 is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. Dysfunction leads to Dubin-Johnson syndrome. Here the authors provide structural and biochemical evidence on the modulation of MRP2 by intracellular kinases and inhibition by therapeutic drugs. [ABSTRACT FROM AUTHOR]

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

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

Background: Multidrug resistance-associated protein 1 (MRP1), an energy-dependent efflux pump, is expressed widely in various tissues and contributes to many physiological and pathophysiological processes. 6-Bromo-7-[11C]methylpurine ([11C]7m6BP) is expected to be useful for the assessment of MRP1 activity in the human brain and lungs. However, the radiochemical yield (RCY) in the synthesis of [11C]7m6BP was low, limiting its clinical application, because the methylation of the precursor with [11C]CH3I provided primarily the undesired isomer, 6-bromo-9-[11C]methylpurine ([11C]9m6BP). To increase the RCY of [11C]7m6BP, we investigated conditions for improving the [11C]7m6BP/[11C]9m6BP selectivity of the methylation reaction. Results: [11C]7m6BP was manually synthesized via the methylation of 6-bromopurine with [11C]CH3I in various solvents and at different temperatures in the presence of potassium carbonate for 5 min. Several less polar solvents, including tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-MeTHF), and ethyl acetate (AcOEt) improved the [11C]7m6BP/[11C]9m6BP selectivity from 1:1 to 2:1, compared with the conventionally used solvents for the alkylation of 6-halopurines, acetone, acetonitrile, and N,N-dimethylformamide. However, a higher temperature (140 °C or 180 °C) was needed to progress the 11C-methylation in the less polar solvents, and the manual conditions could not be directly translated to an automated synthesis. [11C]Methyl triflate ([11C]CH3OTf) was thus used as a methylating agent to increase the conversion at a lower temperature. The 11C-methylation using [11C]CH3OTf at 100 °C proceeded efficiently in THF, 2-MeTHF, and AcOEt with maintenance of the improved selectivity. Starting from 28 to 34 GBq [11C]CO2, [11C]7m6BP was produced with 2.3–2.6 GBq for THF, 2.7–3.3 GBq for AcOEt, and 2.8–3.9 GBq for 2-MeTHF at approximately 30 min after the end of bombardment (n = 3 per solvent). The isolated RCYs (decay corrected) for THF, 2-MeTHF, and AcOEt were 24–28%, 29–35%, and 22–31% (n = 3), respectively. Conclusions: The use of THF, 2-MeTHF, and AcOEt improved the [11C]7m6BP/[11C]9m6BP selectivity in the methylation reaction, and the improved method provided [11C]7m6BP with sufficient radioactivity for clinical use. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Appropriate interactions between antiretroviral therapies (ART) and drug transporters and metabolizing enzymes at the blood brain barrier (BBB) are critical to ensure adequate dosing of the brain to achieve HIV suppression. These proteins are modulated by demographic and lifestyle factors, including substance use. While understudied, illicit substances share drug transport and metabolism pathways with ART, increasing the potential for adverse drug:drug interactions. This is particularly important when considering the brain as it is relatively undertreated compared to peripheral organs and is vulnerable to substance use-mediated damage. Methods: We used an in vitro model of the human BBB to determine the extravasation of three first-line ART drugs, emtricitabine (FTC), tenofovir (TFV), and dolutegravir (DTG), in the presence and absence of cocaine, which served as our illicit substance model. The impact of cocaine on BBB integrity and permeability, drug transporters, metabolizing enzymes, and their master transcriptional regulators were evaluated to determine the mechanisms by which substance use impacted ART central nervous system (CNS) availability. Results: We determined that cocaine had a selective impact on ART extravasation, where it increased FTC's ability to cross the BBB while decreasing TFV. DTG concentrations that passed the BBB were below quantifiable limits. Interestingly, the potent neuroinflammatory modulator, lipopolysaccharide, had no effect on ART transport, suggesting a specificity for cocaine. Unexpectedly, cocaine did not breach the BBB, as permeability to albumin and 4 kDa FITC-dextran, as well as tight junction proteins and adhesion molecules remained unchanged. Rather, cocaine selectively decreased the pregnane-x receptor (PXR), but not constitutive androstane receptor (CAR). Consequently, drug transporter expression and activity decreased in endothelial cells of the BBB, including p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4). Further, cytochrome P450 3A4 (CYP3A4) enzymatic activity increased following cocaine treatment that coincided with decreased expression. Finally, cocaine modulated adenylate kinases that are required to facilitate biotransformation of ART prodrugs to their phosphorylated, pharmacologically active counterparts. Conclusion: Our findings indicate that additional considerations are needed in CNS HIV treatment strategies for people who use cocaine, as it may limit ART efficacy through regulation of drug transport and metabolizing pathways at the BBB. [ABSTRACT FROM AUTHOR]

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. SpragueeDawley 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. [ABSTRACT FROM AUTHOR]

Background and Purpose: Coproporphyrin (CP) I and III are byproducts of haem synthesis currently investigated as biomarkers for drug–drug interactions involving hepatic organic anion transporting polypeptide (OATP) 1B transporters. Another hepatically expressed OATP‐member is OATP2B1. The aim of this study was to test the impact of OATP2B1, which specifically transports CPIII, on CP serum levels, applying novel rat models. Experimental Approach: CPIII transport kinetics and the interplay between OATP2B1 and multidrug resistance‐associated proteins (MRPs) were determined in vitro using the vTF7 expression system. Novel rSlco2b1−/− and SLCO2B1+/+ rat models were characterized for physiological parameters and for CP serum levels. Hepatic and renal expression of transporters involved in CP disposition were determined by real‐time qPCR, Western blot analysis, and immunohistochemistry. Key Results: In vitro experiments revealed differences in transport kinetics comparing human and rat OATP2B1 and showed a consistent, species‐specific interplay with hMRP3/rMRP3. Deletion of rOATP2B1 was associated with a trend towards lower CPI serum levels compared with wildtype rats, while CPIII remained unchanged. Comparing SLCO2B1+/+ with knockout rats revealed an effect of sex: only in females the genetic modification influenced CP serum levels. Analysis of hepatic and renal transporters revealed marginal, but in part, statistically significant differences in rMRP2 abundance, which may contribute to the observed changes in CP serum levels. Conclusion and Implications: Our findings support that factors other than OATP1B transporters are of relevance for basal CP levels. Only in female rats, humanization of SLCO2B1 affects basal CPI and CPIII serum levels, despite isomer selectivity of OATP2B1. [ABSTRACT FROM AUTHOR]

Membrane transporter multidrug resistance-associated protein 2 (MRP2/Abcc2) exhibits high pharmaco-toxicological relevance because it exports multiple cytotoxic compounds from cells. However, no detailed information about the gene expression and regulation of MRP2 in chickens is yet available. Here, we sought to investigate the expression distribution of Abcc2 in different tissues of chicken and then determine whether Abcc2 expression is induced by chicken xenobiotic receptor (CXR). The bioinformatics analyses showed that MRP2 transporters have three transmembrane structural domains (MSDs) and two highly conserved nucleotide structural domains (NBDs), and a close evolutionary relationship with turkeys. Tissue distribution analysis indicated that Abcc2 was highly expressed in the liver, kidney, duodenum, and jejunum. When exposed to metyrapone (an agonist of CXR) and ketoconazole (an antagonist of CXR), Abcc2 expression was upregulated and downregulated correspondingly. We further confirmed that Abcc2 gene regulation is dependent on CXR, by overexpressing and interfering with CXR, respectively. We also demonstrated the induction of Abcc2 expression and the activity of ivermectin, with CXR being a likely mediator. Animal experiments demonstrated that metyrapone and ivermectin induced Abcc2 in the liver, kidney, and duodenum of chickens. Together, our study identified the gene expression of Abcc2 and its regulation by CXR in chickens, which may provide novel targets for the reasonable usage of veterinary drugs. [ABSTRACT FROM AUTHOR]

• We found that renal distribution of nephrotoxic drugs including cisplatin and imatinib was increased in cholestatic mice under bile duct ligation. • Increased accumulation of nephrotoxic drugs may be attributed to down-regulation of drug efflux transporter MRP6 on renal basolateral membranes. • This renal adaption may provide important insights into the onset of drug-induced kidney injury during liver dysfunction. In hepatic dysfunction, renal pharmacokinetic adaptation can be observed, although information on the changes in drug exposure and the interorgan regulation of membrane transporters in kidney in liver diseases is limited. This study aimed to clarify the effects of renal exposure to nephrotoxic drugs during cholestasis induced by bile duct ligation (BDL). Among the 11 nephrotoxic drugs examined, the tissue accumulation of imatinib and cisplatin in kidney slices obtained from mice 2 weeks after BDL operation was higher than that in sham-operated mice. The uptake of imatinib in the kidney slices of BDL mice was slightly higher, whereas its efflux from the slices was largely decreased compared to that in sham-operated mice. Proteomic analysis revealed a reduction in renal expression of the efflux transporter multidrug resistance-associated protein 6 (Mrp6/ Abcc6) in BDL mice, and both imatinib and cisplatin were identified as Mrp6 substrates. Survival probability after cisplatin administration was reduced in BDL mice. In conclusion, the present study demonstrated that BDL-induced cholestasis leads to the downregulation of the renal basolateral efflux transporter Mrp6, resulting in drug accumulation in renal cells and promoting drug-induced renal injury. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) is one of the most common malignancies worldwide, causing thousands to die each year. Its complex molecular nature leads to significant heterogeneity and variable responses to therapy. ABC proteins, which for many years were regarded as the pillar of the resistance to chemotherapy because they export anticancer drugs from cancer cells, have recently been identified as interesting molecular markers associated with many other physiological functions. We previously reported that during the phenotypic transition, CRC differentially regulates the expression of two transporters, ABCC4 and ABCG2. In cells with a mesenchymal and invasive phenotype, ABCC4 is upregulated, and ABCG2 is downregulated. We have therefore decided to explore this phenomenon by analysing samples from CRC patients with high expression of either ABCC4 or ABCG2 to determine their potential use as markers of therapeutic outcome. Background: Our previous findings proved that ABCC4 and ABCG2 proteins present much more complex roles in colorectal cancer (CRC) than typically cancer-associated functions as drug exporters. Our objective was to evaluate their predictive/diagnostic potential. Methods: CRC patients' transcriptomic data from the Gene Expression Omnibus database (GSE18105, GSE21510 and GSE41568) were discriminated into two subpopulations presenting either high expression levels of ABCC4 (ABCC4 High) or ABCG2 (ABCG2 High). Subpopulations were analysed using various bioinformatical tools and platforms (KEEG, Gene Ontology, FunRich v3.1.3, TIMER2.0 and STRING 12.0). Results: The analysed subpopulations present different gene expression patterns. The protein–protein interaction network of subpopulation-specific genes revealed the top hub proteins in ABCC4 High: RPS27A, SRSF1, DDX3X, BPTF, RBBP7, POLR1B, HNRNPA2B1, PSMD14, NOP58 and EIF2S3 and in ABCG2 High: MAPK3, HIST2H2BE, LMNA, HIST1H2BD, HIST1H2BK, HIST1H2AC, FYN, TLR4, FLNA and HIST1H2AJ. Additionally, our multi-omics analysis proved that the ABCC4 expression correlates with substantially increased tumour-associated macrophage infiltration and sensitivity to FOLFOX treatment. Conclusions: ABCC4 and ABCG2 may be used to distinguish CRC subpopulations that present different molecular and physiological functions. The ABCC4 High subpopulation demonstrates significant EMT reprogramming, RNA metabolism and high response to DNA damage stimuli. The ABCG2 High subpopulation may resist the anti-EGFR therapy, presenting higher proteolytical activity. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that cancer cells can mimic characteristics of embryonic development, promoting their development and progression. Cancer cells share features with embryonic development, characterized by robust proliferation and differentiation regulated by signaling pathways such as Wnt, Notch, hedgehog, and Hippo signaling. In certain phase, these cells also mimic embryonic diapause and fertilized egg implantation to evade treatments or immune elimination and promote metastasis. Additionally, the upregulation of ATP‐binding cassette (ABC) transporters, including multidrug resistance protein 1 (MDR1), multidrug resistance‐associated protein 1 (MRP1), and breast cancer‐resistant protein (BCRP), in drug‐resistant cancer cells, analogous to their role in placental development, may facilitate chemotherapy efflux, further resulting in treatment resistance. In this review, we concentrate on the underlying mechanisms that contribute to tumor development and progression from the perspective of embryonic development, encompassing the dysregulation of developmental signaling pathways, the emergence of dormant cancer cells, immune microenvironment remodeling, and the hyperactivation of ABC transporters. Furthermore, we synthesize and emphasize the connections between cancer hallmarks and embryonic development, offering novel insights for the development of innovative cancer treatment strategies. [ABSTRACT FROM AUTHOR]

BackgroundAnti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1.MethodsImpacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted.ResultsCo-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact.ConclusionsThese results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum.

We present the case of a probable pharmacokinetic drug-drug interaction (DDI) between fluconazole (FLU) and vancomycin (VCM), in a patient who underwent hepatobiliary surgery, and biliary drainage, and was under treatment with vancomycin, meropenem, and fluconazole, due to infectious pathology. Changes in the kinetic process of VCM and FLU has not been fully elucidated. Therefore, understanding the changes in renal transporters involved in the renal excretion pathway of VCM and FLU (e.g. multidrug resistance-associated proteins, organic cation transporters, P-glycoprotein) is beneficial in predicting better drug disposition and optimize optimizing dosing. Inhibition of the aforementioned transporters is a common mechanism underlying DDIs. After pharmacokinetic monitoring of VCM treatment, the trough concentration values and the half-life of VCM showed an abnormally unexpected increase when VCM was used in combination with FLU, compared to data obtained when vancomycin was used without fluconazole. Said changes, of the pharmacokinetic parameters studied, would support a DDI between FLU and VCM. Because of this, special caution is required whenever VCM is co-administered with fluconazole, and close monitoring of VCM trough concentrations is recommended to minimize the risk of toxicity. [ABSTRACT FROM AUTHOR]

Nonalcoholic steatohepatitis (NASH) and hepatic fibrosis are leading causes of cirrhosis with rising morbidity and mortality worldwide. Currently, there is no appropriate treatment for NASH and hepatic fibrosis. Many studies have shown that oxidative stress is a main factor inducing NASH. Nomilin (NML) and obacunone (OBA) are limonoid compounds naturally occurring in citrus fruits with various biological properties. However, whether OBA and NML have beneficial effects on NASH remains unclear. Here, we demonstrated that OBA and NML inhibited hepatic tissue necrosis, inflammatory infiltration and liver fibrosis progression in methionine and choline‐deficient (MCD) diet, carbon tetrachloride (CCl4)‐treated and bile duct ligation (BDL) NASH and hepatic fibrosis mouse models. Mechanistic studies showed that NML and OBA enhanced anti‐oxidative effects, including reduction of malondialdehyde (MDA) level, increase of catalase (CAT) activity and the gene expression of glutathione S‐transferases (GSTs) and Nrf2‐keap1 signaling. Additional, NML and OBA inhibited the expression of inflammatory gene interleukin 6 (Il‐6), and regulated the bile acid metabolism genes Cyp3a11, Cyp7a1, multidrug resistance‐associated protein 3 (Mrp3). Overall, these findings indicate that NML and OBA may alleviate NASH and liver fibrosis in mice via enhancing antioxidant and anti‐inflammation capacity. Our study proposed that NML and OBA may be potential strategies for NASH treatment. [ABSTRACT FROM AUTHOR]

The large-conductance Ca2+-activated K+ channel, KCa1.1, plays a pivotal role in cancer progression, metastasis, and the acquisition of chemoresistance. Previous studies indicated that the pharmacological inhibition of KCa1.1 overcame resistance to doxorubicin (DOX) by down-regulating multidrug resistance-associated proteins in the three-dimensional spheroid models of human prostate cancer LNCaP, osteosarcoma MG-63, and chondrosarcoma SW-1353 cells. Investigations have recently focused on the critical roles of intratumoral, drug-metabolizing cytochrome P450 enzymes (CYPs) in chemoresistance. In the present study, we examined the involvement of CYPs in the acquisition of DOX resistance and its overcoming by inhibiting KCa1.1 in cancer spheroid models. Among the CYP isoforms involved in DOX metabolism, CYP3A4 was up-regulated by spheroid formation and significantly suppressed by the inhibition of KCa1.1 through the transcriptional repression of CCAAT/enhancer-binding protein, CEBPB, which is a downstream transcription factor of the Nrf2 signaling pathway. DOX resistance was overcome by the siRNA-mediated inhibition of CYP3A4 and treatment with the potent CYP3A4 inhibitor, ketoconazole, in cancer spheroid models. The phosphorylation levels of Akt were significantly reduced by inhibiting KCa1.1 in cancer spheroid models, and KCa1.1-induced down-regulation of CYP3A4 was reversed by the treatment with Akt and Nrf2 activators. Collectively, the present results indicate that the up-regulation of CYP3A4 is responsible for the acquisition of DOX resistance in cancer spheroid models, and the inhibition of KCa1.1 overcame DOX resistance by repressing CYP3A4 transcription mainly through the Akt-Nrf2-CEBPB axis. [ABSTRACT FROM AUTHOR]

ATP‐binding cassette C‐family drug membrane transporters play an important role in local pharmacokinetics, that is, drug concentration in cellular compartments. From the structural point of view, only the bovine ortholog of the multidrug resistance‐associated protein 1 (bMRP1) has been resolved. We here used μs‐scaled molecular dynamics simulations to investigate the structure and dynamics of the bovine multidrug resistance‐associated protein 1 in pre‐ and post‐hydrolysis functional states. The present work aims to examine the slight but likely relevant structural differences between pre‐ and post‐hydrolysis states of outward‐facing conformations as well as the interactions between the multidrug resistance‐associated protein 1 and the surrounding lipid bilayer. Global conformational dynamics show unfavourable extracellular opening associated with nucleotide‐binding domain dimerization indicating that the post‐hydrolysis state adopts a close‐cleft conformation rather than an outward‐open conformation. Our present simulations also highlight persistent interactions with annular cholesterol molecules and the expected active role of lipid bilayer in the allosteric communication between distant domains of the transporter. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette transporter (ABCC1) is associated with poor survival and chemotherapy drug resistance in high grade serous ovarian cancer (HGSOC). The mechanisms driving ABCC1 expression are poorly understood. Alternative polyadenylation (APA) can give rise to ABCC1 mRNAs which differ only in the length of their 3′untranslated regions (3′UTRs) in a process known as 3′UTR-APA. Like other ABC transporters, shortening of the 3′UTR of ABCC1 through 3′UTR-APA would eliminate microRNA binding sites found within the longer 3′UTRs, hence eliminating miRNA regulation and altering gene expression. We found that the HGSOC cell lines Caov-3 and Ovcar-3 express higher levels of ABCC1 protein than normal cells. APA of ABCC1 occurs in all three cell lines resulting in mRNAs with both short and long 3′UTRs. In Ovcar-3, mRNAs with shorter 3′UTRs dominate resulting in a six-fold increase in protein expression. We were able to show that miR-185-5p and miR-326 both target the ABCC1 3′UTR. Hence, 3′UTR-APA should be considered as an important regulator of ABCC1 expression in HGSOC. Both HGSOC cell lines are cisplatin resistant, and we used erastin to induce ferroptosis, an alternative form of cell death. We showed that we could induce ferroptosis and sensitize the cisplatin resistant cells to cisplatin by using erastin. Knocking down ABCC1 resulted in decreased cell viability, but did not contribute to erastin induced ferroptosis. [ABSTRACT FROM AUTHOR]

We conducted a retrospective analysis of GRP94 immunohistochemical (IHC) staining, an ER stress protein, on large B-cell lymphoma (LBCL) cells, intracellular p53, and 15 factors involved in the metabolism of the CHOP regimen: AKR1C3 (HO metabolism), CYP3A4 (CHOP metabolism), and HO efflux pumps (MDR1 and MRP1). The study subjects were 42 patients with LBCL at our hospital. The IHC staining used antibodies against the 17 factors. The odds ratios by logistic regression analysis used a dichotomous variable of CR and non-CR/relapse were statistically significant for MDR1, MRP1, and AKR1C3. The overall survival (OS) after R-CHOP was compared by the log-rank test. The four groups showed that Very good (5-year OS, 100%) consisted of four patients who showed negative IHC staining for both GRP94 and CYP3A4. Very poor (1-year OS, 0%) consisted of three patients who showed positive results in IHC for both GRP94 and CYP3A4. The remaining 35 patients comprised two subgroups: Good (5-year OS 60–80%): 15 patients who showed negative staining for both MDR1 and AKR1C3 and Poor (5-year OS, 10–20%): 20 patients who showed positive staining for either MDR, AKR1C3, MRP1, or p53. The Histological Prognostic Index (HPI) (the four groups: Very poor, Poor, Good, and Very good) is a breakthrough method for stratifying patients based on the factors involved in the development of treatment resistance. [ABSTRACT FROM AUTHOR]

Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident. [Display omitted] • Aagenaes syndrome/lymphedema cholestasis syndrome 1 is caused by a variant in the 5'-untranslated region of UNC45A. • Patients with Aagenaes syndrome have reduced expression of mRNA and lower UNC45A protein levels than controls. • A CRISPR/Cas9-created variant in the 5'untranslated region reproduced the reduction in mRNA and protein levels of UNC45A. • Liver biopsies demonstrated paucity of bile ducts, cholestasis, giant cell transformation and variable inflammation. • Aberrant localization of BSEP and MRP2 was observed in liver biopsies. [ABSTRACT FROM AUTHOR]

The eradication of cancer stem cells (CSCs) is vital to successful cancer treatment and overall disease-free survival. CSCs are a sub-population of cells within a tumor that are defined by their capacity for continuous self-renewal and recapitulation of new tumors, demonstrated in vitro through spheroid formation. Flavonoids are a group of phytochemicals with potent anti-oxidant and anti-cancer properties. This paper explores the impact of the flavonoid precursor phloridzin (PZ) linked to the ω-3 fatty acid docosahexaenoate (DHA) on the growth of MCF-7 and paclitaxel-resistant MDA-MB-231-TXL breast cancer cell lines. Spheroid formation assays, acid phosphatase assays, and Western blotting were performed using MCF-7 cells, and the cell viability assays, Annexin-V-488/propidium iodide (PI) staining, and 7-aminoactinomycin D (7-AAD) assays were performed using MDA-MB-231-TXL cells. PZ-DHA significantly reduced spheroid formation, as well as the metabolic activity of MCF-7 breast cancer cells in vitro. Treatment with PZ-DHA also suppressed the metabolic activity of MDA-MB-231-TXL cells and led to apoptosis. PZ-DHA did not have an observable effect on the expression of the drug efflux transporters ATP-binding cassette super-family G member 2 (ABCG2) and multidrug resistance-associated protein 1 (MRP1). PZ-DHA is a potential treatment avenue for chemo-resistant breast cancer and a possible novel CSC therapy. Future pre-clinical studies should explore PZ-DHA as a chemo-preventative agent. [ABSTRACT FROM AUTHOR]

Poly ADP‐ribose polymerase (PARP) inhibitors have been approved for the treatment of various cancers. They share a similar mechanism of action but have differences in pharmacokinetic characteristics and potential for drug–drug interactions (DDI). This study evaluated the potential ATP‐binding cassette transporter‐mediated interactions between PARP inhibitors (niraparib, olaparib and rucaparib) and statins (atorvastatin and rosuvastatin). We studied the inhibitory activity of PARP inhibitors on breast cancer resistance protein (BCRP), multidrug resistance‐associated protein 3 (MRP3) and P‐glycoprotein (P‐gp) using vesicular transport assays and determined the concentrations required for 50% inhibition (IC50). Then, we predicted the increase of statin exposure followed by the administration of PARP inhibitors using a mechanistic static model. Rucaparib was the strongest inhibitor of BCRP‐mediated rosuvastatin transport (IC50 13.7 μM), followed by niraparib (42.6 μM) and olaparib (216 μM). PARP inhibitors did not affect MRP3. While niraparib appeared to inhibit P‐gp, the inhibition showed large variability. The inhibition of intestinal BCRP by rucaparib, niraparib and olaparib was predicted to elevate rosuvastatin exposure by 52%, 37% and 24%, respectively. The interactions between PARP inhibitors and rosuvastatin are probably of minor clinical significance alone, but combined with other predisposing factors, they may increase the risk of rosuvastatin‐associated adverse effects. [ABSTRACT FROM AUTHOR]

Renal tubular secretion mediated by organic anion transporters (OATs) and the multidrug resistance-associated protein 4 (MRP4) is an important means of drug and toxin excretion. Unfortunately, there are no biomarkers to evaluate their function. The aim of this study was to identify and characterize an endogenous biomarker of the renal tubular OATs-MRP4 channel. Twenty-six uremic toxins were selected as candidate compounds, of which kynurenic acid was identified as a potential biomarker by assessing the protein-binding ratio and the uptake in OAT1-, OAT3-, and MRP4-overexpressing cell lines. OAT1/3 and MRP4 mediated the transcellular vectorial transport of kynurenic acid in vitro. Serum kynurenic acid concentration was dramatically increased in rats treated with a rat OAT1/3 (rOAT1/3) inhibitor and in rOAT1/3 double knockout (rOAT1/3−/−) rats, and the renal concentrations were markedly elevated by the rat MRP4 (rMRP4) inhibitor. Kynurenic acid was not filtered at the glomerulus (99% of albumin binding), and was specifically secreted in renal tubules through the OAT1/3-MRP4 channel with an appropriate affinity (K m) (496.7 μM and 382.2 μM for OAT1 and OAT3, respectively) and renal clearance half-life (t 1/2) in vivo (3.7 ± 0.7 h). There is a strong correlation in area under the plasma drug concentration-time curve (AUC 0– t) between cefmetazole and kynurenic acid, but not with creatinine, after inhibition of rOATs. In addition, the phase of increased kynurenic acid level is earlier than that of creatinine in acute kidney injury process. These results suggest that albumin-bound kynurenic acid is an appropriate endogenous biomarker for adjusting the dosage of drugs secreted by this channel or predicting kidney injury. [Display omitted] • A strategy of screening biomarker of renal tubular transport channels was proposed. • Kynurenic acid as a biomarker of OATs-MRP4 has a good specificity and sensitivity. • Kidney injury and drug excretion can be predicted via serum kynurenic acid level. [ABSTRACT FROM AUTHOR]

Objective: MicroRNAs (miRNAs) from mesenchymal stem cells (MSC)-derived extracellular vesicles (MSCs-EVs), including exosomes, are known to participate in different diseases. However, the function of miR-301b-3p from MSCs-EVs on the chemoresistance of gastric cancer (GC) cells remains poorly characterized. Thus, we aim to explore the role of MSCs-EVs-derived miR-301b-3p in multidrug resistance of GC cells. Methods: Cisplatin (DDP)/vincristine (VCR)-resistant and sensitive GC clinical samples were harvested to detect expression of miR-301b-3p and thioredoxin interacting protein (TXNIP). MSCs were respectively transfected with miR-301b-3p oligonucleotides and/or TXNIP plasmids to extract the EVs, which were then co-cultured with multidrug-resistant GC cells. Then, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), IC50, proliferation, migration, and apoptosis of resistant GC cells were determined. The tumor growth was observed in nude mice. Targeting relationship between miR-301b-3p and TXNIP was confirmed. Results: miR-301b-3p was upregulated, and TXNIP was downregulated in DDP/VCR-resistant GC tissues and cells. MSC-EVs induced drug resistance, proliferation, and migration and inhibited apoptosis of DDP/VCR-resistant GC cells in vitro, as well as facilitated tumor growth in vivo. Inhibition of miR-301b-3p or upregulation of TXNIP reversed the promoting effect of MSC-EVs on DDP/VCR resistant GC cells to DDP/VCR resistance and malignant behaviors. The effects of MSC-EVs carrying miR-301b-3p inhibition on DDP/VCR-resistant GC cells were reversed by TXNIP downregulation. TXNIP was confirmed as a target gene of miR-301b-3p. Conclusion: miR-301b-3p from MSCs-EVs inhibits TXNIP to promote multidrug resistance of GC cells, providing a novel insight for chemotherapy in GC. [ABSTRACT FROM AUTHOR]

Introduction. With the increase of hospital infections due to the indiscriminate use of antibiotics, multidrug resistance has increased, decreasing the effectiveness of antibiotics against these infections. For this reason, the identification of alternative agents such as probiotics has been considered. The aim of this study was to isolate and identify effective probiotics against carbapenem-resistant Pseudomonas aeruginosa strains. Material and Methods. During a period of eight months, isolates of P. aeruginosa were collected from patients in three hospitals in Isfahan. The presence of metallo-beta-lactamase enzymes was determined by the combination disc test (CDT). The inhibitory and antimicrobial activities of 20 probiotic bacteria isolated from local dairy products against these strains were investigated by agar dilution. Two probiotic strains that showed broader inhibition results were selected, and the values of the lowest inhibitory concentration (MIC) and the lowest lethal concentration (MBC) and their antibiofilm effect were determined using the microtiter plate method. The concentration of organic acids was done by HPLC. Findings. Of the 100 samples isolated and identified, 61 samples (61%) exhibited multiple drug resistance (MDR) and were selected for further investigation. Phenotypic diagnosis of the presence of metallo-beta-lactamase enzymes revealed that 74.5% of the strains were positive. The results showed that these two probiotics killed P. aeruginosa strains after only one hour, and the inhibition mechanism was due to the presence of lactic acid and acetic acid. The antibiofilm effect of these two probiotics was at concentrations of 1/2 and 1/4. Conclusion. The two Lactobacillus isolates had potential antimicrobial and antibiofilm properties against all carbapenem-resistant P. aeruginosa strains, even at thinner dilutions. Considering the broad activity of this strain, it can potentially be used for biocontrol of these strains. [ABSTRACT FROM AUTHOR]

Pharmacological interventions for hepatocellular carcinoma (HCC) are hindered by complex factors, and rational combination therapy may be developed to improve therapeutic outcomes. Very recently, we have identified a bioengineered microRNA let-7c-5p (or let-7c) agent as an effective inhibitor against HCC in vitro and in vivo. In this study, we sought to identify small-molecule drugs that may synergistically act with let-7c against HCC. Interestingly, we found that let-7c exhibited a strong synergism with 5-fluorouracil (5-FU) in the inhibition of HCC cell viability as manifested by average combination indices of 0.3 and 0.5 in Hep3B and Huh7 cells, respectively. By contrast, coadministration of let-7c with doxorubicin or sorafenib inhibited HCC cell viability with, rather surprisingly, no or minimal synergy. Further studies showed that protein levels of multidrug resistance-associated protein (MRP) ATP-binding cassette subfamily C member 5 (MRP5/ABCC5), a 5-FU efflux transporter, were reduced around 50% by let-7c in HCC cells. This led to a greater degree of intracellular accumulation of 5-FU in Huh7 cells as well as the second messenger cyclic adenosine monophosphate, an endogenous substrate of MRP5. Since 5-FU is an irreversible inhibitor of thymidylate synthetase (TS), we investigated the interactions of let-7c with 5-FU at pharmacodynamic level. Interestingly, our data revealed that let-7c significantly reduced TS protein levels in Huh7 cells, which was associated with the suppression of upstream transcriptional factors as well as other regulatory factors. Collectively, these results indicate that let-7c interacts with 5-FU at both pharmacokinetic and pharmacodynamic levels, and these findings shall offer insight into molecular mechanisms of synergistic drug combinations. SIGNIFICANCE STATEMENT: Combination therapy is a common strategy that generally involves pharmacodynamic interactions. After identifying a strong synergism between let-7c-5p and 5-fluorouracil (5-FU) against hepatocellular carcinoma cell viability, we reveal the involvement of both pharmacokinetic and pharmacodynamic mechanisms. In particular, let-7c enhances 5-FU exposure (via suppressing ABCC5/MRP5 expression) and cotargets thymidylate synthase with 5-FU (let-7c reduces protein expression, whereas 5-FU irreversibly inactivates enzyme). These findings provide insight into developing rational combination therapies based on pharmacological mechanisms.