ABSTRACT
BACKGROUND: Although it is traditionally believed that ATP binding cassette subfamily C member 2 (ABCC2) is a multidrug resistance-associated protein correlated with a worse prognosis, our previous and several other studies demonstrated the contrary to be true in gastric cancer (GC). We aim to explore the underlying mechanism of this discovery. METHODS: Our study utilized whole-exome sequencing (WES), RNA sequencing, and droplet digital PCR (ddPCR) analysis of 80 gastric cancer samples, along with comprehensive immunohistochemical (IHC) analysis of 1044 human GC tissue samples.By utilizing CRISPRCas9 to genetically modify cell lines with the ABCC2-24C > T (rs717620) point mutation and conducting dual-luciferase reporter assays, we identified that transcription factors SOX9 and ETS1 serve as negative regulators of ABCC2 expression. Seahorse assay and mass spectrometry were used to discover altered metabolic patterns. Gain and loss-of-function experiments in GC cell lines and preclinical models were carried out to validate ABCC2 biological function. RESULTS: ABCC2 high expression correlated with better prognosis, and rs717620 can influence ABCC2 expression by disrupting the binding of ETS1 and SOX9. Gain and loss-of-function experiments in GC cell lines demonstrated amino acid deprivation reduces proliferation, migration, and drug resistance in ABCC2-high GC cells. ABCC2 leads to reduced intracellular amino acid pools and disruption of cellular energy metabolism. This phenomenon depended on ABCC2-mediated GSH extrusion, resulting in alterations in redox status, thereby increasing the cell's susceptibility to ferroptosis. Furthermore, patient-derived organoids and patient-derived tumor-like cell clusters were used to observe impact of ABCC2 on therapeutic effect. In the xenograft model with high ABCC2 expression, we observed that constricting amino acid intake in conjunction with GPX4 inactivation resulted in notable tumor regression. CONCLUSIONS: Our findings demonstrate a significant role of ABCC2 in amino acid metabolism and ferroptosis by mediating GSH efflux in GC. This discovery underlines the potential of combining multiple ferroptosis targets as a promising therapeutic strategy for GC with high ABCC2 expression. HIGHLIGHTS: ABCC2 plays a crucial role in inducing metabolic vulnerability and ferroptosis in gastric cancer through enhanced glutathione efflux. The ABCC2 24C > T polymorphism is a key factor influencing its expression. These results highlight the potential of ABCC2 as a predictive biomarker and therapeutic target in gastric cancer.
Subject(s)
Ferroptosis , Glutathione , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins , Stomach Neoplasms , Humans , Stomach Neoplasms/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Ferroptosis/genetics , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Glutathione/metabolism , Animals , Mice , Cell Line, Tumor , Male , FemaleABSTRACT
P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance transporter 2 (MRP2) are efflux transporters involved in the absorption, excretion, and distribution of drugs. Bidirectional cell assays are recognized models for evaluating the potential of new drugs as substrates or inhibitors of efflux transporters. However, the assays are complicated by a lack of selective substrates and/or inhibitors, as well simultaneous expression of several efflux transporters in cell lines used in efflux models. This project aims to evaluate an in vitro efflux cell assay employing model substrates and inhibitors of P-gp, BCRP and MRP2 with knockout (KO) cell lines. The efflux ratios (ER) of P-gp (digoxin, paclitaxel), BCRP (prazosin, rosuvastatin), MRP2 (etoposide, olmesartan) and mixed (methotrexate, mitoxantrone) substrates were determined in wild-type C2BBe1 and KO cells. For digoxin and paclitaxel, the ER decreased to less than 2 in the cell lines lacking P-gp expression. The ER decreased to less than 3 for prazosin and less than 2 for rosuvastatin in the cell lines lacking BCRP expression. For etoposide and olmesartan, the ER decreased to less than 2 in the cell lines lacking MRP2 expression. The ER of methotrexate and mitoxantrone decreased in single- and double-KO cells without BCRP and MRP2 expression. These results show that KO cell lines have the potential to better interpret complex drug-transporter interactions without depending upon multi-targeted inhibitors or overlapping substrates. For drugs that are substrates of multiple transporters, the single- and double-KO cells may be used to assess their affinities for the different transporters.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2 , Neoplasm Proteins , Humans , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Neoplasm Proteins/metabolism , Neoplasm Proteins/genetics , Multidrug Resistance-Associated Protein 2 , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Gene Knockout Techniques , Biological Transport , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Cell Line , Digoxin/pharmacology , Digoxin/pharmacokinetics , Digoxin/metabolism , Prazosin/pharmacology , Paclitaxel/pharmacology , AnimalsABSTRACT
BACKGROUND: Plasmodium vivax has become the predominant species in the border regions of Thailand. The emergence and spread of antimalarial drug resistance in P. vivax is one of the significant challenges for malaria control. Continuous surveillance of drug resistance is therefore necessary for monitoring the development of drug resistance in the region. This study aims to investigate the prevalence of the mutation in the P. vivax multidrug resistant 1 (Pvmdr1), dihydrofolate reductase (Pvdhfr), and dihydropteroate synthetase (Pvdhps) genes conferred resistance to chloroquine (CQ), pyrimethamine (P) and sulfadoxine (S), respectively. METHOD: 100 P. vivax isolates were obtained between January to May 2023 from a Kanchanaburi province, western Thailand. Nucleotide sequences of Pvmdr1, Pvdhfr, and Pvdhps genes were amplified and sequenced. The frequency of single nucleotide polymorphisms (SNPs)-haplotypes of drug-resistant alleles was assessed. The linkage disequilibrium (LD) tests were also analyzed. RESULTS: In Pvmdr1, T958M, Y976F, and F1076L, mutations were detected in 100%, 21%, and 23% of the isolates, respectively. In Pvdhfr, the quadruple mutant allele (I57R58M61T117) prevailed in 84% of the samples, followed by (L57R58M61T117) in 11%. For Pvdhps, the double mutant allele (G383G553) was detected (48%), followed by the triple mutant allele (G383M512G553) (47%) of the isolates. The most prevalent combination of Pvdhfr (I57R58M61T117) and Pvdhps (G383G553) alleles was sextuple mutated haplotypes (48%). For LD analysis, the association in the SNPs pairs was found between the intragenic and intergenic regions of the Pvdhfr and Pvdhps genes. CONCLUSION: The study has recently updated the high prevalence of three gene mutations associated with CQ and SP resistance. Genetic monitoring is therefore important to intensify in the regions to further assess the spread of drug resistant. Our data also provide evidence on the distribution of drug resistance for the early warning system, thereby threatening P. vivax malaria treatment policy decisions at the national level.
Subject(s)
Antimalarials , Drug Resistance , Malaria, Vivax , Plasmodium vivax , Polymorphism, Single Nucleotide , Plasmodium vivax/genetics , Plasmodium vivax/drug effects , Plasmodium vivax/isolation & purification , Thailand/epidemiology , Drug Resistance/genetics , Humans , Antimalarials/pharmacology , Malaria, Vivax/parasitology , Malaria, Vivax/epidemiology , Malaria, Vivax/drug therapy , Tetrahydrofolate Dehydrogenase/genetics , Linkage Disequilibrium , Mutation , Protozoan Proteins/genetics , Chloroquine/pharmacology , Dihydropteroate Synthase/genetics , Sulfadoxine/pharmacology , Pyrimethamine/pharmacology , Multidrug Resistance-Associated Proteins/genetics , Haplotypes , Male , Female , AdultABSTRACT
The drug efflux pump is a crucial mechanism implicated in resistance to multiple antimicrobials. Thymoquinone (TQ) has evidently demonstrated multiple activities, antibacterial being the most effective. Knowledge about TQ activity against multidrug-resistant Staphylococcus aureus is very scarce. Therefore, the present study was conducted to investigate TQ resistance modulation in ciprofloxacin (CIP) and doxycycline (DO) multidrug-resistant S. aureus. Forty-seven samples were collected from different sources, and S. aureus was isolated and identified. Then, S. aureus resistance profiles to antimicrobials, N. sativa essential oil, and TQ; the correlation between TQ-MIC readings and disc diffusion; cartwheel and ethidium bromide (EtBr) accumulation assays; and norA gene expression were all described within silico molecular docking for TQ interactions with norA efflux pump protein. TQ-MICs ranged from 5-320 µg/ml. TQ down-regulated norA gene expression, resulting in a drop in efflux pump activity of 77.5-90.6% in the examined strains, comparable to that observed with verapamil. Exposure of S. aureus strains to CIP and DO raises the initial basal efflux pumping expression to 34.2 and 22.9 times, respectively. This induced efflux pumping overexpression was substantially reduced by 97.7% when TQ was combined with CIP or DO. There was a significant reduction of MICs of CIP and DO MICs by 2-15 and 2-4 folds, respectively, after treatment with 0.5XMIC-TQ in resistance modulation assays. These results refer to TQ ligand inhibitory interactions with NorA protein in molecular docking. Interpretations of inhibition zone diameters (IZDs) of disc diffusion and TQ-MICs exhibit independence of MICs from IZDs, as indicated by invalid linear regression analysis. TQ significantly reduced efflux pumping S. aureus induced by CIP and DO, but further investigations are needed to improve TQ-pharmacokinetics to restore CIP and DO activity and suppress fluoroquinolone and doxycycline-resistant S. aureus selection in clinical and animal settings.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Benzoquinones , Ciprofloxacin , Drug Resistance, Multiple, Bacterial , Microbial Sensitivity Tests , Molecular Docking Simulation , Multidrug Resistance-Associated Proteins , Staphylococcus aureus , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Benzoquinones/pharmacology , Benzoquinones/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/pharmacology , Ciprofloxacin/pharmacology , Doxycycline/pharmacology , Gene Expression Regulation, Bacterial/drug effectsABSTRACT
BACKGROUND: and Purpose: Chemotherapy-induced peripheral neuropathy (CIPN) constitutes a significant health problem due to the increasing prevalence and lack of therapies for treatment and prevention. While pivotal for routine cancer treatment, paclitaxel and vincristine frequently cause CIPN and impact the quality of life among cancer patients and survivors. Here, we investigate molecular mechanisms and drug transport in CIPN. EXPERIMENTAL APPROACH: Human sensory neurons were derived from induced pluripotent stem cells (iPSC-SNs), which were characterized using flow cytometry and immunolabeling. These iPSC-SNs were exposed to different concentrations of the two microtubule-targeting agents, paclitaxel and vincristine, with and without pre-exposure to inhibitors and inducers of efflux transporters. Neuronal networks were quantified via fluorescent staining against sensory neuron markers. Transcriptional effects of the chemotherapeutics were examined using quantitative polymerase chain reactions (qPCR). KEY RESULTS: Paclitaxel exposure resulted in axonal retraction and thickening, while vincristine caused fragmentation and abolishment of axons. Both agents increased the mRNA expression of the pain receptor, transient receptor potential vanilloid (TRPV1), and highly induced neuronal damage, as measured by activating transcription factor 3 (ATF3) mRNA. iPSC-SNs express the efflux transporters, P-glycoprotein (P-gp, encoded by ABCB1) and multidrug resistance-associated protein 1 (MPR1, encoded by ABCC1). Modulation of efflux transporters indicate that P-gp and MRP1 play a role in modulating neuronal accumulation and neurotoxicity in preliminary experiments. CONCLUSION: and Implications: iPSC-SNs are a valuable and robust model to study the role of efflux transporters and other mechanistic targets in CIPN. Efflux transporters may play a role in CIPN pathogenesis as they regulate the disposition of chemotherapy to the peripheral nervous system, and they may present potential therapeutic targets for CIPN.
Subject(s)
Induced Pluripotent Stem Cells , Multidrug Resistance-Associated Proteins , Paclitaxel , Peripheral Nervous System Diseases , Sensory Receptor Cells , Vincristine , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Humans , Paclitaxel/toxicity , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/metabolism , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Antineoplastic Agents/adverse effects , Antineoplastic Agents/toxicity , TRPV Cation Channels/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , Cells, CulturedABSTRACT
Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum multi-drug resistance protein 1 (Pfmrp1) gene have previously been reported to confer resistance to Artemisinin-based Combination Therapies (ACTs) in Southeast Asia. A total of 300 samples collected from six sites between 2008 and 2019 under an ongoing malaria drug sensitivity patterns in Kenya study were evaluated for the presence of SNPs at Pfmrp1 gene codons: H191Y, S437A, I876V, and F1390I using the Agena MassARRAY® platform. Each isolate was further tested against artemisinin (ART), lumefantrine (LU), amodiaquine (AQ), mefloquine (MQ), quinine (QN), and chloroquine (CQ) using malaria the SYBR Green I-based method to determine their in vitro drug sensitivity. Of the samples genotyped, polymorphism at Pfmrp1 codon I876V was the most frequent, with 59.3% (163/275) mutants, followed by F1390I, 7.2% (20/278), H191Y, 4.0% (6/151), and S437A, 3.3% (9/274). A significant decrease in median 50% inhibition concentrations (IC50s) and interquartile range (IQR) was noted; AQ from 2.996 ng/ml [IQR = 2.604-4.747, n = 51] in 2008 to 1.495 ng/ml [IQR = 0.7134-3.318, n = 40] (P<0.001) in 2019, QN from 59.64 ng/ml [IQR = 29.88-80.89, n = 51] in 2008 to 18.10 ng/ml [IQR = 11.81-26.92, n = 42] (P<0.001) in 2019, CQ from 35.19 ng/ml [IQR = 16.99-71.20, n = 30] in 2008 to 6.699 ng/ml [IQR = 4.976-9.875, n = 37] (P<0.001) in 2019, and ART from 2.680 ng/ml [IQR = 1.608-4.857, n = 57] in 2008 to 2.105 ng/ml [IQR = 1.266-3.267, n = 47] (P = 0.0012) in 2019, implying increasing parasite sensitivity to the drugs over time. However, no significant variations were observed in LU (P = 0.2692) and MQ (P = 0.0939) respectively, suggesting stable parasite responses over time. There was no statistical significance between the mutation at 876 and parasite sensitivity to selected antimalarials tested, suggesting stable sensitivity for the parasites with 876V mutations. These findings show that Kenyan parasite strains are still sensitive to AQ, QN, CQ, ART, LU, and MQ. Despite the presence of Pfmrp1 mutations in parasites among the population.
Subject(s)
Antimalarials , Artemether, Lumefantrine Drug Combination , Malaria, Falciparum , Plasmodium falciparum , Polymorphism, Single Nucleotide , Antimalarials/pharmacology , Antimalarials/therapeutic use , Humans , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Artemether, Lumefantrine Drug Combination/therapeutic use , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Multidrug Resistance-Associated Proteins/genetics , Kenya , Mefloquine/pharmacology , Mefloquine/therapeutic use , Amodiaquine/pharmacology , Amodiaquine/therapeutic use , Drug Resistance/genetics , Artemisinins/pharmacology , Artemisinins/therapeutic use , Chloroquine/pharmacology , Chloroquine/therapeutic use , Quinine/pharmacology , Quinine/therapeutic use , Male , FemaleABSTRACT
Human multidrug resistance protein 5 (hMRP5) effluxes anticancer and antivirus drugs, driving multidrug resistance. To uncover the mechanism of hMRP5, we determine six distinct cryo-EM structures, revealing an autoinhibitory N-terminal peptide that must dissociate to permit subsequent substrate recruitment. Guided by these molecular insights, we design an inhibitory peptide that could block substrate entry into the transport pathway. We also identify a regulatory motif, comprising a positively charged cluster and hydrophobic patches, within the first nucleotide-binding domain that modulates hMRP5 localization by engaging with membranes. By integrating our structural, biochemical, computational, and cell biological findings, we propose a model for hMRP5 conformational cycling and localization. Overall, this work provides mechanistic understanding of hMRP5 function, while informing future selective hMRP5 inhibitor development. More broadly, this study advances our understanding of the structural dynamics and inhibition of ABC transporters.
Subject(s)
Cryoelectron Microscopy , Humans , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/chemistry , Biological Transport , HEK293 Cells , Models, Molecular , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Multidrug Resistance-Associated Proteins/genetics , Peptides/metabolism , Peptides/chemistry , Protein ConformationABSTRACT
The ABCC1 gene belongs to the ATP-binding cassette membrane transporter superfamily, which plays a crucial role in the efflux of various endogenous and exogenous substances. Mutations in ABCC1 can result in autosomal dominant hearing loss. However, the specific roles of ABCC1 in auditory function are not fully understood. Through immunofluorescence, we found that ABCC1 was expressed in microvascular endothelial cells (ECs) of the stria vascularis (StV) in the murine cochlea. Then, an Abcc1 knockout mouse model was established by using CRISPR/Cas9 technology to elucidate the role of ABCC1 in the inner ear. The ABR threshold did not significantly differ between WT and Abcc1-/- mice at any age studied. After noise exposure, the ABR thresholds of the WT and Abcc1-/- mice were significantly elevated. Interestingly, after 14 days of noise exposure, ABR thresholds largely returned to pre-exposure levels in WT mice but not in Abcc1-/- mice. Our subsequent experiments showed that microvascular integrity in the StV was compromised and that the number of outer hair cells and the number of ribbons were significantly decreased in the cochleae of Abcc1-/- mice post-exposure. Besides, the production of ROS and the accumulation of 4-HNE significantly increased. Furthermore, StV microvascular ECs were cultured to elucidate the role of ABCC1 in these cells under glucose oxidase challenge. Notably, 30 U/L glucose oxidase (GO) induced severe oxidative stress damage in Abcc1-/- cells. Compared with WT cells, the ROS and 4-HNE levels and the apoptotic rate were significantly elevated in Abcc1-/- cells. In addition, the reduced GSH/GSSG ratio was significantly decreased in Abcc1-/- cells after GO treatment. Taken together, Abcc1-/- mice are more susceptible to noise-induced hearing loss, possibly because ABCC1 knockdown compromises the GSH antioxidant system of StV ECs. The exogenous antioxidant N-acetylcysteine (NAC) may protect against oxidative damage in Abcc1-/- murine cochleae and ECs.
Subject(s)
Antioxidants , Cochlea , Hearing Loss, Noise-Induced , Mice, Knockout , Multidrug Resistance-Associated Proteins , Oxidative Stress , Animals , Mice , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Cochlea/metabolism , Cochlea/pathology , Hearing Loss, Noise-Induced/metabolism , Hearing Loss, Noise-Induced/genetics , Antioxidants/metabolism , Disease Models, Animal , Reactive Oxygen Species/metabolism , Endothelial Cells/metabolismABSTRACT
Vascular calcification is prevalent in chronic kidney disease (CKD). Genetic causes of CKD account for 10-20% of adult-onset disease. Vascular calcification is thought to be one of the most important risk factors for increased cardiovascular morbidity and mortality in CKD patients and is detectable in 80% of patients with end stage kidney disease (ESKD). Despite the high prevalence of vascular calcification in CKD, no single gene cause has been described. We hypothesized that variants in vascular calcification genes may contribute to disease pathogenesis in CKD, particularly in families who exhibit a predominant vascular calcification phenotype. We developed a list of eight genes that are hypothesized to play a role in vascular calcification due to their involvement in the ectopic calcification pathway: ABCC6, ALPL, ANK1, ENPP1, NT5E, SLC29A1, SLC20A2, and S100A12. With this, we assessed exome data from 77 CKD patients, who remained unsolved following evaluation for all known monogenic causes of CKD. We also analyzed an independent cohort (Ontario Neurodegenerative Disease Research Initiative (ONDRI), n = 520) who were screened for variants in ABCC6 and compared this to a control cohort of healthy adults (n = 52). We identified two CKD families with heterozygous pathogenic variants (R1141X and A667fs) in ABCC6. We identified 10 participants from the ONDRI cohort with heterozygous pathogenic or likely pathogenic variant in ABCC6. Replication in a healthy control cohort did not reveal any variants. Our study provides preliminary data supporting the hypothesis that ABCC6 may play a role in vascular calcification in CKD. By screening CKD patients for genetic causes early in the diagnostic pathway, patients with genetic causes associated with vascular calcification can potentially be preventatively treated with new therapeutics with aims to decrease mortality.
Subject(s)
Multidrug Resistance-Associated Proteins , Renal Insufficiency, Chronic , Vascular Calcification , Humans , Multidrug Resistance-Associated Proteins/genetics , Vascular Calcification/genetics , Vascular Calcification/pathology , Renal Insufficiency, Chronic/genetics , Male , Female , Middle Aged , Adult , Aged , Genetic Predisposition to DiseaseABSTRACT
Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.
Subject(s)
Malaria, Falciparum , Multidrug Resistance-Associated Proteins , Plasmodium falciparum , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Plasmodium falciparum/genetics , Humans , Real-Time Polymerase Chain Reaction/methods , Multidrug Resistance-Associated Proteins/genetics , Malaria, Falciparum/parasitology , Malaria, Falciparum/diagnosis , Antimalarials/pharmacology , Antimalarials/therapeutic use , Mutation , Drug Resistance/geneticsABSTRACT
Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.
Subject(s)
Adipose Tissue , Corticosterone , Diet, High-Fat , Insulin Resistance , Multidrug Resistance-Associated Proteins , Muscle, Skeletal , Obesity , Animals , Male , Diet, High-Fat/adverse effects , Mice , Obesity/metabolism , Obesity/genetics , Obesity/etiology , Adipose Tissue/metabolism , Insulin Resistance/physiology , Corticosterone/blood , Corticosterone/metabolism , Muscle, Skeletal/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Mice, Knockout , Mice, Inbred C57BL , Glucose/metabolismABSTRACT
Uric acid induces radical oxygen species formation, endothelial inflammation, and endothelial dysfunction which contributes to the progression of atherosclerosis. Febuxostat inhibits BCRP- and allopurinol stimulates MRP4-mediated uric acid efflux in human embryonic kidney cells. We hypothesized that endothelial cells express uric acid transporters that regulate intracellular uric acid concentration and that modulation of these transporters by febuxostat and allopurinol contributes to their different impact on cardiovascular mortality. The aim of this study was to explore a potential difference between the effect of febuxostat and allopurinol on uric acid uptake by human umbilical vein endothelial cells. Febuxostat increased intracellular uric acid concentrations compared with control. In contrast, allopurinol did not affect intracellular uric acid concentration. In line with this observation, febuxostat increased mRNA expression of GLUT9 and reduced MRP4 expression, while allopurinol did not affect mRNA expression of these uric acid transporters. These findings provide a possible pathophysiological pathway which could explain the higher cardiovascular mortality for febuxostat compared to allopurinol but should be explored further.
Subject(s)
Allopurinol , Febuxostat , Glucose Transport Proteins, Facilitative , Human Umbilical Vein Endothelial Cells , Multidrug Resistance-Associated Proteins , Uric Acid , Humans , Allopurinol/pharmacology , Febuxostat/pharmacology , Uric Acid/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Glucose Transport Proteins, Facilitative/metabolism , Glucose Transport Proteins, Facilitative/genetics , Biological Transport/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Gene Expression Regulation/drug effectsABSTRACT
Newly arrived refugees offer insights into malaria epidemiology in their countries of origin. We evaluated asymptomatic refugee children within 7 days of arrival in Uganda from South Sudan and the Democratic Republic of Congo (DRC) in 2022 for parasitemia, parasite species, and Plasmodium falciparum drug resistance markers. Asymptomatic P. falciparum infections were common in both populations. Coinfection with P. malariae was more common in DRC refugees. Prevalences of markers of aminoquinoline resistance (PfCRT K76T, PfMDR1 N86Y) were much higher in South Sudan refugees, of antifolate resistance (PfDHFR C59R and I164L, PfDHPS A437G, K540E, and A581G) much higher in DRC refugees, and of artemisinin partial resistance (ART-R; PfK13 C469Y and A675V) moderate in both populations. Prevalences of most mutations differed from those seen in Ugandans attending health centers near the refugee centers. Refugee evaluations yielded insights into varied malaria epidemiology and identified markers of ART-R in 2 previously little-studied countries.
Subject(s)
Antimalarials , Drug Resistance , Malaria, Falciparum , Plasmodium falciparum , Protozoan Proteins , Refugees , Humans , Uganda/epidemiology , Antimalarials/therapeutic use , Antimalarials/pharmacology , Drug Resistance/genetics , Prevalence , Child, Preschool , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Malaria, Falciparum/drug therapy , Female , Male , Child , Protozoan Proteins/genetics , Infant , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Sudan/epidemiology , Biomarkers/blood , Artemisinins/therapeutic use , Artemisinins/pharmacology , Parasitemia/epidemiology , Parasitemia/drug therapy , Plasmodium malariae/genetics , Plasmodium malariae/drug effectsABSTRACT
OBJECTIVE: Intermittent Preventive Treatment of schoolchildren (IPTsc) is recommended by WHO as a strategy to protect against malaria; to explore whether IPTsc with dihydroartemisinin-piperaquine (DP) or artesunate-amodiaquine (ASAQ) cause a selection of molecular markers in Plasmodium falciparum genes associated with resistance in children in seven schools in Tanga region, Tanzania. METHODS: SNPs in P. falciparum genes Pfmdr1, Pfexo, Pfkelch13, and Pfcrt and copy number variations in Pfplasmepsin-2 and Pfmdr1 were assessed in samples collected at 12 months (visit 4, n=74) and 20 months (visit 6, n=364) after initiation of IPTsc and compared with the baseline prevalence (n=379). RESULTS: The prevalence of Pfmdr1 N86 and Pfexo 415G was >99% and 0%, respectively without any temporal differences observed. The prevalence of Pfmdr1 184F changed significantly from baseline (52.2%) to visit 6 (64.6%) (χ2=6.11, P=0.013), but no differences were observed between the treatment arms (χ2=0.05, P=0.98). Finally, only minor differences in the amplification of Pfmdr1 were observed; from 10.2% at baseline to 16.7% at visit 6 (χ2=0.98, P=0.32). CONCLUSIONS: The IPTsc strategy does not seem to pose a risk for the selection of markers associated with DP or ASAQ resistance. Continuously and timely surveillance of markers of antimalarial drug resistance is recommended.
Subject(s)
Amodiaquine , Antimalarials , Artemisinins , Drug Combinations , Drug Resistance , Malaria, Falciparum , Plasmodium falciparum , Humans , Antimalarials/therapeutic use , Antimalarials/pharmacology , Tanzania/epidemiology , Child , Malaria, Falciparum/epidemiology , Malaria, Falciparum/prevention & control , Malaria, Falciparum/parasitology , Malaria, Falciparum/drug therapy , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Drug Resistance/genetics , Male , Female , Amodiaquine/therapeutic use , Artemisinins/therapeutic use , Polymorphism, Single Nucleotide , Quinolines/therapeutic use , Child, Preschool , Protozoan Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Genetic Markers , DNA Copy Number Variations , PiperazinesABSTRACT
Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.
Subject(s)
Cyclic GMP , Multidrug Resistance-Associated Proteins , Urinary Bladder , Animals , Urinary Bladder/metabolism , Urinary Bladder/drug effects , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Cyclic GMP/metabolism , Swine , Quinolines/pharmacology , Cyclic AMP/metabolism , Muscle Relaxation/drug effects , Male , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Female , Signal Transduction , Phosphodiesterase Inhibitors/pharmacology , PropionatesABSTRACT
BACKGROUND: Antiretrovirals have the potential to cause drug interactions leading to inefficacy or toxicity via induction of efflux transporters through nuclear receptors, altering drug concentrations at their target sites. RESEARCH DESIGN AND METHODS: This study used molecular dynamic simulations and qRT-PCR to investigate bictegravir's interactions with nuclear receptors PXR and CAR, and its effects on efflux transporters (P-gp, BCRP, MRP1) in rat PBMCs. PBMC/plasma drug concentrations were measured using LC-MS/MS to assess the functional impact of transporter expression. RESULTS: Bictegravir significantly increased the expression of ABC transporters, with Car identified as a key mediator. This suggests that bictegravir's influence on nuclear receptors could affect drug transport and efficacy at the cellular level. CONCLUSIONS: Bictegravir activates nuclear receptors enhancing efflux transporter expression. Understanding these interactions is crucial for preventing drug-drug interactions and reducing toxicity in clinical use. Combining CAR antagonists with bictegravir may prevent drug resistance and toxicity. However, these findings are based on preclinical data and necessitate further clinical trials to confirm their applicability in clinical settings.
Subject(s)
Drug Interactions , Heterocyclic Compounds, 4 or More Rings , Leukocytes, Mononuclear , Tandem Mass Spectrometry , Animals , Rats , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/drug effects , Male , Heterocyclic Compounds, 4 or More Rings/pharmacology , Heterocyclic Compounds, 4 or More Rings/pharmacokinetics , Heterocyclic Compounds, 3-Ring/pharmacology , Heterocyclic Compounds, 3-Ring/pharmacokinetics , Heterocyclic Compounds, 3-Ring/administration & dosage , Piperazines/pharmacology , Pregnane X Receptor/genetics , Pregnane X Receptor/metabolism , Molecular Dynamics Simulation , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Gene Expression Regulation/drug effects , Constitutive Androstane Receptor , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Chromatography, Liquid/methods , Rats, Sprague-Dawley , Dioxolanes/pharmacology , Dioxolanes/pharmacokinetics , Dioxolanes/administration & dosage , Amides , PyridonesABSTRACT
OBJECTIVE: To explore the clinical manifestations and genetic basis for a rare case of Generalized arterial calcification of infancy (GACI). METHODS: A 44-day-old female infant who was treated at Baoding Hospital of Beijing Children's Hospital Affiliated to Capital Medical University on August 26, 2022 was selected as the study subject. Clinical data of the child was collected, and Trio-whole exome sequencing (Trio-WES), whole genome copy number variation sequencing (CNV-seq) and minigene splicing assay were carried out to analyze the pathogenicity of the variants. RESULTS: The child had presented with fever and high inflammatory indicators, for which treatment with various antibiotics was ineffective. Ultrasound had revealed extensive arterial calcification and arterial wall thickening. The child was suspected for GACI with arteritis related to the primary disease. Her fever was relieved by treatment with glucocorticoid and biological agents. Trio-WES revealed that she has harbored compound heterozygous variants of the ABCC6 gene, namely c.4404-1G>A and c.4041+5G>T, for which the latter was unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the variants were classified as likely pathogenic (PVS1+PM2_Supporting) and variant of unknown significance (PM2_Supporting+PM3+PP3), respectively. The result of CNV-seq was negative. And the minigene splicing assay has further verified that both variants can result in alternative splicing. CONCLUSION: For pyrexia with unknown causes and refractory to conventional treatment, it is necessary to recommend early genetic testing to avoid missed diagnosis of GACI.
Subject(s)
Multidrug Resistance-Associated Proteins , Vascular Calcification , Humans , Female , Vascular Calcification/genetics , Multidrug Resistance-Associated Proteins/genetics , Infant , Genetic Testing , Exome Sequencing , DNA Copy Number Variations , MutationABSTRACT
While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs (pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.
Subject(s)
Antimalarials , DNA Copy Number Variations , Drug Resistance , Malaria, Falciparum , Plasmodium falciparum , Protozoan Proteins , Antimalarials/pharmacology , Mozambique , Plasmodium falciparum/genetics , Plasmodium falciparum/drug effects , Humans , Drug Resistance/genetics , DNA Copy Number Variations/genetics , Malaria, Falciparum/parasitology , Malaria, Falciparum/drug therapy , Protozoan Proteins/genetics , Polymerase Chain Reaction/methods , Quinolines/pharmacology , Amodiaquine/pharmacology , Multidrug Resistance-Associated Proteins/genetics , Aspartic Acid Endopeptidases/genetics , Artemisinins/pharmacology , Lumefantrine/pharmacology , PiperazinesABSTRACT
Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.
Subject(s)
ATP-Binding Cassette Transporters , Cell Survival , Ganglia, Spinal , Methylmercury Compounds , Schwann Cells , Animals , Ganglia, Spinal/metabolism , Ganglia, Spinal/drug effects , Methylmercury Compounds/toxicity , Schwann Cells/drug effects , Schwann Cells/metabolism , Cell Survival/drug effects , Cells, Cultured , Large Neutral Amino Acid-Transporter 1/metabolism , Large Neutral Amino Acid-Transporter 1/genetics , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Peripheral Nerves/metabolism , Peripheral Nerves/drug effects , Male , Rats , Multidrug Resistance-Associated Protein 2ABSTRACT
Tartary buckwheat (Fagopyrum tataricum) is an annual coarse cereal from the Polygonaceae family, known for its high content of flavonoid compounds, particularly rutin. But so far, the mechanisms of the flavonoid transport and storage in Tartary buckwheat (TB) remain largely unexplored. This study focuses on ATP-binding cassette transporters subfamily C (ABCC) members, which are crucial for the biosynthesis and transport of flavonoids in plants. The evolutionary and expression pattern analyses of the ABCC genes in TB identified an ABCC protein gene, FtABCC2, that is highly correlated with rutin synthesis. Subcellular localization analysis revealed that FtABCC2 protein is specifically localized to the vacuole membrane. Heterologous expression of FtABCC2 in Saccharomyces cerevisiae confirmed that its transport ability of flavonoid glycosides such as rutin and isoquercetin, but not the aglycones such as quercetin and dihydroquercetin. Overexpression of FtABCC2 in TB hairy root lines resulted in a significant increase in total flavonoid and rutin content (P < 0.01). Analysis of the FtABCC2 promoter revealed potential cis-acting elements responsive to hormones, cold stress, mechanical injury and light stress. Overall, this study demonstrates that FtABCC2 can efficiently facilitate the transport of rutin into vacuoles, thereby enhancing flavonoids accumulation. These findings suggest that FtABCC2 is a promising candidate for molecular-assisted breeding aimed at developing high-flavonoid TB varieties.