RESUMEN
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.
Asunto(s)
Ferroptosis , Glutatión , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Ferroptosis/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Glutatión/metabolismo , Animales , Ratones , Línea Celular Tumoral , Masculino , FemeninoRESUMEN
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.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Proteínas de Neoplasias , Humanos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Técnicas de Inactivación de Genes , Transporte Biológico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Línea Celular , Digoxina/farmacología , Digoxina/farmacocinética , Digoxina/metabolismo , Prazosina/farmacología , Paclitaxel/farmacología , AnimalesRESUMEN
Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.
Asunto(s)
Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Análisis Espectral/métodos , Proteínas Portadoras/metabolismo , Proteínas Portadoras/química , Transporte Biológico , LipoproteínasRESUMEN
AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domainsâTM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.
Asunto(s)
Proteínas de Escherichia coli , Simulación de Dinámica Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Regulación Alostérica , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Sitios de Unión , Ligandos , Sitio Alostérico , Conformación Proteica , Unión Proteica , Multimerización de ProteínaRESUMEN
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.
Asunto(s)
Antibacterianos , Proteínas Bacterianas , Benzoquinonas , Ciprofloxacina , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Staphylococcus aureus , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Benzoquinonas/farmacología , Benzoquinonas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Doxiciclina/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacosRESUMEN
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.
Asunto(s)
Alopurinol , Febuxostat , Proteínas Facilitadoras del Transporte de la Glucosa , Células Endoteliales de la Vena Umbilical Humana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Ácido Úrico , Humanos , Alopurinol/farmacología , Febuxostat/farmacología , Ácido Úrico/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Transporte Biológico/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación de la Expresión Génica/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antioxidantes , Cóclea , Pérdida Auditiva Provocada por Ruido , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Estrés Oxidativo , Animales , Ratones , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Cóclea/metabolismo , Cóclea/patología , Pérdida Auditiva Provocada por Ruido/metabolismo , Pérdida Auditiva Provocada por Ruido/genética , Antioxidantes/metabolismo , Modelos Animales de Enfermedad , Especies Reactivas de Oxígeno/metabolismo , Células Endoteliales/metabolismoRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Humanos , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transporte Biológico , Células HEK293 , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Péptidos/metabolismo , Péptidos/química , Conformación ProteicaRESUMEN
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.
Asunto(s)
Tejido Adiposo , Corticosterona , Dieta Alta en Grasa , Resistencia a la Insulina , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Músculo Esquelético , Obesidad , Animales , Masculino , Dieta Alta en Grasa/efectos adversos , Ratones , Obesidad/metabolismo , Obesidad/genética , Obesidad/etiología , Tejido Adiposo/metabolismo , Resistencia a la Insulina/fisiología , Corticosterona/sangre , Corticosterona/metabolismo , Músculo Esquelético/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Ratones Noqueados , Ratones Endogámicos C57BL , Glucosa/metabolismoRESUMEN
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.
Asunto(s)
GMP Cíclico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Vejiga Urinaria , Animales , Vejiga Urinaria/metabolismo , Vejiga Urinaria/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , GMP Cíclico/metabolismo , Porcinos , Quinolinas/farmacología , AMP Cíclico/metabolismo , Relajación Muscular/efectos de los fármacos , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Femenino , Transducción de Señal , Inhibidores de Fosfodiesterasa/farmacología , PropionatosRESUMEN
OBJECTIVE: To screen interleukin (IL)-1ß secretion-related membrane transporters by macrophage experiment in vitro and conventional knockout mice. METHODS: THP-1 cell line was differentiated to obtain human THP-1-derived macrophages, and the primary macrophages were obtained from human peripheral blood. FVB wild-type mice with the same sex and age were used as the controls of MRP1 knockout mice. The macrophages in abdominal cavity and bone marrow of mice were cultivated. The cells were treated with ABCC1/MRP1, ABCG2/BCRP, ABCB1/P-gp, OATP1B1, and MATE transporter inhibitors, then stimulated by lipopolysaccharide and adenosine triphosphate. The secretion level of IL-1ß was detected by ELISA, Western blot, and immunofluorescence. RESULTS: After inhibiting ABCC1/MRP1 transporter, the secretion of IL-1ß decreased significantly, while inhibition of the other 4 transporters had no effect. In animal experiment, the level of IL-1ß secreted by macrophages in bone marrow of MRP1 knockout mice was significantly lower than control group (P < 0.05). CONCLUSION: ABCC1/MRP1 transporter is a newly discovered IL-1ß secretion pathway, which is expected to become a new target for solving clinical problems such as cytokine release syndrome.
Asunto(s)
Regulación hacia Abajo , Interleucina-1beta , Macrófagos , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Animales , Humanos , Ratones , Interleucina-1beta/metabolismo , Lipopolisacáridos , Macrófagos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Células THP-1RESUMEN
BACKGROUND: Altered glycosylation plays a role in carcinogenesis. GALNT14 promotes cancer stem-like properties and drug resistance. GDF-15 is known to induces drug resistance and stemness markers for maintenance of breast cancer (BC) stem-like cell state. Currently there is lack of data on association of GDF-15 and GALNTs. In this study, the expression and interaction of GALNT14 and GDF-15 with stemness (OCT4 and SOX2) and drug resistance (ABCC5) markers were evaluated in BC. METHODS: We investigated tumour tissue from 30 BC patients and adjacent non-tumour tissues. Expression of serum GALNT14 from BC patients and matched healthy controls was evaluated. Expression of GALNT14, GDF-15, OCT4, SOX2, ABCC5, and ß-catenin in BC tissue was determined by RT-PCR. Knockdown of GALNT14 and GDF-15 in the MCF-7 cell line was done through siRNA, gene expression and protein expression of ß-catenin by western blot were determined. RESULTS: A significant increase in the expression of GALNT14, GDF-15, OCT4, SOX2, ABCC5, and ß-catenin was observed in BC tumour tissues compared to adjacent non-tumour tissues. The serum level of GALNT14 was significantly high in BC patients (80.7 ± 65.3 pg/ml) compared to healthy controls (12.2 ± 9.12 pg/ml) (p < 0.000). To further analyse the signalling pathway involved in BC stemness and drug resistance, GALNT14 and GDF-15 were knocked down in the MCF-7 cell line, and it was observed that after knockdown, the expression level of OCT4, SOX2, ABCC5, and ß-catenin was decreased, and co-knockdown with GALNT14 and GDF-15 further decreased the expression of genes. CONCLUSION: It can be concluded that GALNT14, in association with GDF-15, promotes stemness and intrinsic drug resistance in BC, possibly through the ß-catenin signalling pathway.
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Neoplasias de la Mama , Resistencia a Antineoplásicos , Factor 15 de Diferenciación de Crecimiento , N-Acetilgalactosaminiltransferasas , Células Madre Neoplásicas , beta Catenina , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/metabolismo , Resistencia a Antineoplásicos/genética , beta Catenina/metabolismo , beta Catenina/genética , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Células MCF-7 , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Adulto , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Transducción de Señal , Vía de Señalización Wnt/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Línea Celular Tumoral , AncianoRESUMEN
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.
Asunto(s)
Aldehídos , Ferroptosis , Peroxidación de Lípido , Ferroptosis/efectos de los fármacos , Humanos , Peroxidación de Lípido/efectos de los fármacos , Aldehídos/farmacología , Aldehídos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Glutatión/metabolismoRESUMEN
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.
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Transportadoras de Casetes de Unión a ATP , Supervivencia Celular , Ganglios Espinales , Compuestos de Metilmercurio , Células de Schwann , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Nervios Periféricos/metabolismo , Nervios Periféricos/efectos de los fármacos , Masculino , Ratas , Proteína 2 Asociada a Resistencia a Múltiples MedicamentosRESUMEN
Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.
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Proteínas Bacterianas , Microscopía por Crioelectrón , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Protones , Staphylococcus aureus , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Staphylococcus aureus/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Antibacterianos/farmacología , Antibacterianos/metabolismo , Antibacterianos/química , Modelos Moleculares , Transporte Biológico , Sitios de Unión , Enlace de Hidrógeno , Conformación ProteicaRESUMEN
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.
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Fagopyrum , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Rutina , Rutina/metabolismo , Fagopyrum/genética , Fagopyrum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Transporte Biológico , Flavonoides/metabolismo , Filogenia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
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.
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Interacciones Farmacológicas , Compuestos Heterocíclicos de 4 o más Anillos , Leucocitos Mononucleares , Espectrometría de Masas en Tándem , Animales , Ratas , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Compuestos Heterocíclicos con 3 Anillos/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacocinética , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Piperazinas/farmacología , Receptor X de Pregnano/genética , Receptor X de Pregnano/metabolismo , Simulación de Dinámica Molecular , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Receptor de Androstano Constitutivo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Cromatografía Liquida/métodos , Ratas Sprague-Dawley , Dioxolanos/farmacología , Dioxolanos/farmacocinética , Dioxolanos/administración & dosificación , Amidas , PiridonasRESUMEN
Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.
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Proteínas de Escherichia coli , Escherichia coli , Animales , Virulencia , Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Antibacterianos/farmacología , Antibacterianos/metabolismo , Farmacorresistencia Microbiana , Bacterias/metabolismo , División Celular , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad Microbiana , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
Purpose: To describe the ocular findings of murine pseudoxanthoma elasticum (PXE) models with ATP-binding cassette subfamily C member 6 (Abcc6) gene knockout. Methods: This experiment was conducted in four Abcc6-/- rats and compared with six wild-type Abcc6+/+ control rats. The animals underwent necropsy at 6 months of age. Histological examination of the eyes was performed. Results: Histological examination of eight eyes from four Abcc6-/- rats revealed multiple nodular foci of calcification in the uvea, sclera, and conjunctiva, focally in perivascular distribution, as well as linear and nodular calcification of Bruch's membrane. Calcific foci were not associated with inflammation in the knockout rats. There was no evidence of calcification in control eyes. Discussion: The Abcc6-/- rat model shows that PXE can affect multiple ocular tissues beyond the calcification in Bruch's membrane noted in human eyes. Nodular calcific foci probably correspond to comet lesions seen in patients with PXE. The presence of ectopic calcium without inflammation distinguishes it from inflammatory calcium deposition in atherosclerosis. Further studies are needed to determine why PXE does not cause inflammatory infiltration. Translational Relevance: The Abcc6-/- murine model may be suitable for studying ocular PXE pathophysiology and ectopic calcification and developing effective therapies.
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Modelos Animales de Enfermedad , Seudoxantoma Elástico , Animales , Masculino , Ratas , Lámina Basal de la Coroides/patología , Lámina Basal de la Coroides/metabolismo , Calcinosis/patología , Calcinosis/genética , Técnicas de Inactivación de Genes , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/patología , Seudoxantoma Elástico/metabolismoRESUMEN
The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.