RESUMO
P-glycoprotein (P-gp) mediated multidrug resistance (MDR) is the leading cause of chemotherapy failure since it causes the efflux of chemotherapeutic drugs from the cancer cells. Solasodine, a steroidal alkaloid and oxaspiro compound, present in the Solanaceae family showed significant cytotoxic effects on various cancer cells. However, the effect of solasodine on reversing P-gp mediated drug resistance is still unknown. Primarily in this study, the integrative network pharmacology analysis found 71 common targets between solasodine and cancer MDR, among them NF-κB was found as a potential target. The results of immunofluorescence analysis showed that solasodine significantly inhibits NF-κB-p65 nuclear translocation which caused downregulated P-gp expression in KBChR-8-5 cells. Further, solasodine binds to the active sites of the TMD region of P-gp and inhibits P-gp transport activity. Moreover, solasodine significantly promotes doxorubicin intracellular accumulation in the drug resistant cells. Solasodine reduced the fold resistance and synergistically sensitized doxorubicin's therapeutic effects in KBChR-8-5 cells. Additionally, the solasodine and doxorubicin combination treatment increased the apoptotic cell populations and G2/M phase cell cycle arrest in KBChR-8-5 cells. The MDR tumor bearing xenograft mice showed tumor-suppressing characteristics and P-gp downregulation during the combination treatment of solasodine and doxorubicin. These results indicate that solasodine targets NF-κB signaling to downregulate P-gp overexpression, inhibit P-gp transport activity, and enhance chemosensitization in MDR cancer cells. Considering its multifaceted impact, solasodine represents a potent natural fourth-generation P-gp modulator for reversing MDR in cancer.
Assuntos
Apoptose , Doxorrubicina , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Camundongos Nus , NF-kappa B , Transdução de Sinais , Alcaloides de Solanáceas , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , Alcaloides de Solanáceas/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , NF-kappa B/metabolismo , Camundongos , Doxorrubicina/farmacologia , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proliferação de Células/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genéticaRESUMO
Zebrafish have proved to be invaluable for modeling complex physiological processes shared by all vertebrate animals. Resistance of cancers and other diseases to drug treatment can occur owing to expression of the ATP-dependent multidrug transporters ABCB1, ABCG2, and ABCC1, either because of expression of these transporters by the target cells to reduce intracellular concentrations of cytotoxic drugs at barrier sites such as the blood-brain barrier (BBB) to limit penetration of drugs into privileged compartments, or by affecting the absorption, distribution, and excretion of drugs administered orally, through the skin, or directly into the bloodstream. We describe the drug specificity, cellular localization, and function of zebrafish orthologs of multidrug resistance ABC transporters with the goal of developing zebrafish models to explore the physiological and pathophysiological functions of these transporters. Finally, we provide context demonstrating the utility of zebrafish in studying cancer drug resistance. Our ultimate goal is to improve treatment of cancer and other diseases which are affected by ABC multidrug resistance transporters.
Assuntos
Antineoplásicos , Neoplasias , Animais , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Membrana Transportadoras , Resistência a Múltiplos Medicamentos/genética , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Acridinas , Tetra-Hidroisoquinolinas , Humanos , Acridinas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Microscopia CrioeletrônicaRESUMO
Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2-knockdown (Adar2-KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2-KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Adenosina Desaminase , Doxorrubicina , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Edição de RNA , RNA Circular , Animais , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Feminino , RNA Circular/genética , RNA Circular/metabolismo , Doxorrubicina/farmacologia , Linhagem Celular Tumoral , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Antibióticos Antineoplásicos/farmacologiaRESUMO
The highly selective Spleen Tyrosine Kinase (SYK) inhibitors entospletinib and lanraplenib disrupt kinase activity and inhibit immune cell functions. They are developed for treatment of B-cell malignancies and autoimmunity diseases. The impact of P-gp/ABCB1 and BCRP/ABCG2 efflux transporters, OATP1a/1b uptake transporters and CYP3A drug-metabolizing enzymes on the oral pharmacokinetics of these drugs was assessed using mouse models. Entospletinib and lanraplenib were orally administered simultaneously at moderate dosages (10 mg/kg each) to female mice to assess the possibility of examining two structurally and mechanistically similar drugs at the same time, while reducing the number of experimental animals and sample-processing workload. The plasma pharmacokinetics of both drugs were not substantially restricted by Abcb1 or Abcg2. The brain-to-plasma ratios of entospletinib in Abcb1a/b-/-, Abcg2-/- and Abcb1a/b;Abcg2-/- mice were 1.7-, 1.8- and 2.9-fold higher, respectively, compared to those in wild-type mice. For lanraplenib these brain-to-plasma ratios were 3.0-, 1.3- and 10.4-fold higher, respectively. This transporter-mediated restriction of brain penetration for both drugs could be almost fully inhibited by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, without signs of acute toxicity. Oatp1a/b and human CYP3A4 did not seem to affect the pharmacokinetics of entospletinib and lanraplenib, but mouse Cyp3a may limit lanraplenib plasma exposure. Unexpectedly, entospletinib and lanraplenib increased each other's plasma exposure by 2.6- to 2.9-fold, indicating a significant drug-drug interaction. This interaction was, however, unlikely to be mediated through any of the studied transporters or CYP3A. The obtained insights may perhaps help to further improve the safety and efficacy of entospletinib and lanraplenib.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Encéfalo , Indazóis , Morfolinas , Inibidores de Proteínas Quinases , Pirazinas , Animais , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Feminino , Camundongos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Camundongos Knockout , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Administração OralRESUMO
Morphine is a widely used opioid for the treatment of pain. Differences in drug transporter expression and activity may contribute to variability in morphine pharmacokinetics and response. Using appropriate mouse models, we investigated the impact of the efflux transporters ABCB1 and ABCG2 and the OATP uptake transporters on the pharmacokinetics of morphine, morphine-3-glucuronide (M3G), and M6G. Upon subcutaneous administration of morphine, its plasma exposure in Abcb1a/1b-/-;Abcg2-/--, Abcb1a/1b-/-;Abcg2-/-;Oatp1a/1b-/-;Oatp2b1-/- (Bab12), and Oatp1a/1b-/-;Oatp2b1-/- mice was similar to that found in wild-type mice. Forty minutes after dosing, morphine brain accumulation increased by 2-fold when mouse (m)Abcb1 and mAbcg2 were ablated. Relative recovery of morphine in small intestinal content was significantly reduced in all the knockout strains. In the absence of mOatp1a/1b and mOatp2b1, plasma levels of M3G were markedly increased, suggesting a lower elimination rate. Moreover, Oatp-deficient mice displayed reduced hepatic and intestinal M3G accumulation. Mouse Oatps similarly affected plasma and tissue disposition of subcutaneously administered M6G. Human OATP1B1/1B3 transporters modestly contribute to the liver accumulation of M6G. In summary, mAbcb1, in combination with mAbcg2, limits morphine brain penetration and its net intestinal absorption. Variation in ABCB1 activity due to genetic polymorphisms/mutations and/or environmental factors might, therefore, partially affect morphine tissue exposure in patients. The ablation of mOatp1a/1b increases plasma exposure and decreases the liver and small intestinal disposition of M3G and M6G. Since the contribution of human OATP1B1/1B3 to M6G liver uptake was quite modest, the risks of undesirable drug interactions or interindividual variation related to OATP activity are likely negligible.
Assuntos
Camundongos Knockout , Derivados da Morfina , Morfina , Animais , Morfina/farmacocinética , Morfina/metabolismo , Derivados da Morfina/metabolismo , Derivados da Morfina/sangue , Camundongos , Distribuição Tecidual , Masculino , Encéfalo/metabolismo , Analgésicos Opioides/farmacocinética , Analgésicos Opioides/metabolismo , Analgésicos Opioides/sangue , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/genética , Fígado/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genéticaRESUMO
Investigations on placental P-glycoprotein (P-gp) regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. The role of long noncoding RNA (lncRNA) on placental P-gp regulation is lacking. The present study was carried out to investigate the regulation and underlying mechanisms of lncRNA urothelial carcinoma associated 1 (UCA1) on P-gp in Bewo cells. lncRNA UCA1 inhibition or overexpression could decrease or increase ABCB1 mRNA expression, P-gp expression and its cellular efflux function, respectively. RNA-FISH revealed that lncRNA UCA1 was mainly located in the cytoplasm of Bewo cells. MicroRNA array was applied and 10 significant miRNAs was identified after lncRNA UCA1 inhibition. Databases of LncTarD, LncRNA2Target, and miRcode were further used to search potential target miRNAs of lncRNA UCA1 and miR-16-5p was screened out. Thereafter, we confirmed that miR-16-5p expression was significantly upregulated or reduced after lncRNA UCA1 knockdown or overexpression, respectively. Furthermore, we also proved that ABCB1 mRNA expression, P-gp expression and its cellular efflux function was enhanced or reduced after miR-16-5p inhibition or overexpression, respectively. The rescue experiment further indicated that miR-16-5p was involved in the positive regulation of lncRNA UCA1 on the expression and function of P-gp. Lastly, dual-luciferase reporter system, RNA-binding protein immunoprecipitation and RNA pull-down assays were performed to explore the relationships among lncRNA UCA1, miR-16-5p, and ABCB1. It was found that lncRNA UCA1(1103-1125) could directly interact with miR-16-5p and miR-16-5p could directly target ABCB1 coding DNA sequence region (882-907). In conclusion, LncRNA UCA1 could promote the expression and function of P-gp by sponging miR-16-5p in BeWo cells.
Assuntos
Carcinoma de Células de Transição , MicroRNAs , RNA Longo não Codificante , Neoplasias da Bexiga Urinária , Gravidez , Humanos , Feminino , RNA Longo não Codificante/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Placenta , Subfamília B de Transportador de Cassetes de Ligação de ATP , MicroRNAs/genética , RNA MensageiroRESUMO
P-glycoprotein (P-gp, encoded in humans by the ABCB1 gene and in rodents by the Abcb1a/b genes) is a membrane transporter that can restrict the intestinal absorption and tissue distribution of many drugs and may also contribute to renal and hepatobiliary drug excretion. The aim of this study was to compare the performance and sensitivity of currently available radiolabeled P-gp substrates for positron emission tomography (PET) with the single-photon emission computed tomography (SPECT) radiotracer [99mTc]Tc-sestamibi for measuring the P-gp function in the kidneys and liver. Wild-type, heterozygous (Abcb1a/b(+/-)), and homozygous (Abcb1a/b(-/-)) Abcb1a/b knockout mice were used as models of different P-gp abundance in excretory organs. Animals underwent either dynamic PET scans after intravenous injection of [11C]N-desmethyl-loperamide, (R)-[11C]verapamil, or [11C]metoclopramide or consecutive static SPECT scans after intravenous injection of [99mTc]Tc-sestamibi. P-gp in the kidneys and liver of the mouse models was analyzed with immunofluorescence labeling and Western blotting. In the kidneys, Abcb1a/b() mice had intermediate P-gp abundance compared with wild-type and Abcb1a/b(-/-) mice. Among the four tested radiotracers, renal clearance of radioactivity (CLurine,kidney) was significantly reduced (-83%) in Abcb1a/b(-/-) mice only for [99mTc]Tc-sestamibi. Biliary clearance of radioactivity (CLbile,liver) was significantly reduced in Abcb1a/b(-/-) mice for [11C]N-desmethyl-loperamide (-47%), [11C]metoclopramide (-25%), and [99mTc]Tc-sestamibi (-79%). However, in Abcb1a/b(+/-) mice, CLbile,liver was significantly reduced (-47%) only for [99mTc]Tc-sestamibi. Among the tested radiotracers, [99mTc]Tc-sestamibi performed best in measuring the P-gp function in the kidneys and liver. Owing to its widespread clinical availability, [99mTc]Tc-sestamibi represents a promising probe substrate to assess systemic P-gp-mediated drug-drug interactions and to measure renal and hepatic P-gp function under different (patho-)physiological conditions.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Metoclopramida , Humanos , Camundongos , Animais , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Tomografia Computadorizada por Raios X , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Fígado/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único , Rim/diagnóstico por imagem , Nitrilas , Compostos de Organotecnécio , Camundongos KnockoutRESUMO
Cancer cells frequently develop resistance to chemotherapeutic therapies and targeted drugs, which has been a significant challenge in cancer management. With the growing advances in technologies in isolation and identification of natural products, the potential of natural products in combating cancer multidrug resistance has received substantial attention. Importantly, natural products can impact multiple targets, which can be valuable in overcoming drug resistance from different perspectives. In the current review, we will describe the well-established mechanisms underlying multidrug resistance, and introduce natural products that could target these multidrug resistant mechanisms. Specifically, we will discuss natural compounds such as curcumin, resveratrol, baicalein, chrysin and more, and their potential roles in combating multidrug resistance. This review article aims to provide a systematic summary of recent advances of natural products in combating cancer drug resistance, and will provide rationales for novel drug discovery.
Assuntos
Antineoplásicos , Produtos Biológicos , Neoplasias , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Neoplasias/tratamento farmacológico , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos AntineoplásicosRESUMO
AIM: We aimed to assess if dicloxacillin/flucloxacillin reduces the therapeutic efficacy of direct oral anticoagulants (DOACs) and the underlying molecular mechanism. METHODS: In a randomized, crossover study, we assessed whether dicloxacillin reduces oral absorption of drugs through P-glycoprotein (P-gp) during 10 and 28 days of treatment. To study the impact of dicloxacillin/flucloxacillin on intestinal and hepatic expression of P-gp in vitro, we usd LS174T cells and 3D spheroids of primary human hepatocytes. Finally, we used nationwide Danish health registries and the UK's Clinical Practice Research Datalink to estimate hazard ratios (HRs) for the risk of stroke and systemic embolism following dicloxacillin/flucloxacillin exposure among DOAC users, using phenoxymethylpenicillin and amoxicillin as active comparators. RESULTS: Dicloxacillin reduced the area under the curve of dabigatran to a geometric mean ratio 10 days of 0.67 (95% confidence interval [CI]: 0.42-1.1) and geometric mean ratio 28 days of 0.72 (95% CI: 0.39-1.4), suggesting reduced oral absorption via increased P-gp expression. In vitro, dicloxacillin raised P-gp expression in both intestinal and liver cells, while flucloxacillin only affected liver cells. In the pharmacoepidemiologic study, dicloxacillin and flucloxacillin were not associated with increased risk of stroke/systemic embolism (dicloxacillin vs. phenoxymethylpenicillin HR: 0.93, 95% CI: 0.72-1.2; flucloxacillin vs. amoxicillin HR: 0.89, 95% CI: 0.51-1.5). CONCLUSIONS: Dicloxacillin increases expression of intestinal P-gp, leading to reduced oral absorption of dabigatran. However, concomitant use of dicloxacillin/flucloxacillin was not associated with stroke and systemic embolism among DOAC users, suggesting no clinical impact from the drug-drug interaction between dicloxacillin/flucloxacillin and DOACs.
RESUMO
PURPOSE: Serotonin (5-HT3) receptor antagonists are promising agents for treatment of neuropathic pain. However, insufficient drug exposure at the central nervous system (CNS) might result in lack of efficacy. The goal of this study was to evaluate the impact of administration of a Pgp inhibitor (tariquidar) on ondansetron exposure in the brain, spinal cord, and cerebrospinal fluid in a wild-type rat model. METHODS: Ondansetron (10 mg/kg) and tariquidar (7.5 mg/kg) were administered intravenously, plasma and tissue samples were collected and analyzed by HPLC. A mathematical model with brain, spinal cord, cerebrospinal fluid and two systemic disposition compartments was developed to describe the data. RESULTS: The results demonstrate that tariquidar at 7.5 mg/kg resulted in a complete inhibition of Pgp efflux of ondansetron in the brain and spinal cord. The compartmental model successfully captured pharmacokinetics of ondansetron in wild type and Pgp knockout (KO) animals receiving the drug alone or in wild type animals receiving the ondansetron and tariquidar combination. CONCLUSIONS: The study provided important quantitative information on enhancement of CNS exposure to ondansetron using co-administration of Pgp Inhibitor in a rat model, which will be further utilized in conducting a clinical study. Tariquidar co-administration resulted in ondansetron CNS exposure comparable to observed in Pgp KO rats. Results also highlighted the effect of tariquidar on plasma disposition of ondansetron, which may not be dependent on Pgp inhibition, and should be evaluated in future studies.
Assuntos
Ondansetron , Quinolinas , Medula Espinal , Animais , Ondansetron/farmacocinética , Ratos , Masculino , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Quinolinas/farmacocinética , Quinolinas/administração & dosagem , Ratos Sprague-Dawley , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Modelos Biológicos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Antagonistas do Receptor 5-HT3 de Serotonina/farmacocinética , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologiaRESUMO
BACKGROUND: Individuals with Alzheimer's disease (AD) often require many medications; however, these medications are dosed using regimens recommended for individuals without AD. This is despite reduced abundance and function of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in AD, which can impact brain exposure of drugs. The fundamental mechanisms leading to reduced P-gp abundance in sporadic AD remain unknown; however, it is known that the apolipoprotein E (apoE) gene has the strongest genetic link to sporadic AD development, and apoE isoforms can differentially alter BBB function. The aim of this study was to assess if apoE affects P-gp abundance and function in an isoform-dependent manner using a human cerebral microvascular endothelial cell (hCMEC/D3) model. METHODS: This study assessed the impact of apoE isoforms on P-gp abundance (by western blot) and function (by rhodamine 123 (R123) uptake) in hCMEC/D3 cells. Cells were exposed to recombinant apoE3 and apoE4 at 2 - 10 µg/mL over 24 - 72 hours. hCMEC/D3 cells were also exposed for 72 hours to astrocyte-conditioned media (ACM) from astrocytes expressing humanised apoE isoforms. RESULTS: P-gp abundance in hCMEC/D3 cells was not altered by recombinant apoE4 relative to recombinant apoE3, nor did ACM containing human apoE isoforms alter P-gp abundance. R123 accumulation in hCMEC/D3 cells was also unchanged with recombinant apoE isoform treatments, suggesting no change to P-gp function, despite both abundance and function being altered by positive controls SR12813 (5 µM) and PSC 833 (5 µM), respectively. CONCLUSIONS: Different apoE isoforms have no direct influence on P-gp abundance or function within this model, and further in vivo studies would be required to address whether P-gp abundance or function are reduced in sporadic AD in an apoE isoform-specific manner.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Barreira Hematoencefálica , Encéfalo , Células Endoteliais , Isoformas de Proteínas , Humanos , Doença de Alzheimer/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E4/metabolismo , Apolipoproteína E4/genética , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/irrigação sanguínea , Linhagem Celular , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Microvasos/metabolismo , Microvasos/citologia , Isoformas de Proteínas/metabolismo , Rodamina 123/metabolismoRESUMO
Despite the availability of various 11C-labeled positron emission tomography (PET) tracers for assessing P-glycoprotein (P-gp) function, there are still limitations related to complex metabolism, high lipophilicity, and low baseline uptake. This study aimed to address these issues by exploring a series of customized dihydropyridines (DHPs) with enhanced stability and reduced lipophilicity as alternative PET tracers for P-gp dysfunction. Compared with verapamil and the rest DHPs, dimethyl 4-(4-fluorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1) exhibited superior cellular uptake differences between the human gastric cancer cell line SGC7901 and its drug-resistant counterpart. [18F]1 is successfully synthesized using a novel "hot-Hantzsch" approach in 22.1 ± 0.1 % radiochemical yields. MicroPET/CT imaging demonstrated that the uptake of [18F]1 in the brains of P-gp blocked mice increased by > 3 times compared to the control group. Additionally, [18F]1 displayed favorable lipophilicity (log D = 2.3) and excellent clearance characteristics, making it a promising tracer candidate with low background noise and high contrast.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Di-Hidropiridinas , Radioisótopos de Flúor , Tomografia por Emissão de Pósitrons , Di-Hidropiridinas/química , Di-Hidropiridinas/síntese química , Di-Hidropiridinas/farmacologia , Humanos , Animais , Radioisótopos de Flúor/química , Camundongos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Linhagem Celular Tumoral , Estrutura Molecular , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacologia , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
The P-glycoprotein (P-gp) efflux pump plays a major role in xenobiotic detoxification. The inhibition of its activity by environmental contaminants remains however rather little characterised. The present study was designed to develop a combination of different approaches to identify P-gp inhibitors among a large number of pesticides using in silico and in vitro models. First, the prediction performance of four web tools was evaluated alone or in combination using a set of recently marketed drugs. The best combination of web tools-AdmetSAR2.0/PgpRules/pkCSM-was next used to predict P-gp activity inhibition by 762 pesticides. Among the 187 pesticides predicted to be P-gp inhibitors, 11 were tested in vitro for their ability to inhibit the efflux of reference substrates (rhodamine 123 and Hoechst 33342) in P-gp overexpressing MCF7R cells and to inhibit the efflux of the reference substrate rhodamine 123 in the Caco-2 cell monolayer. In MCF7R cell assays, ivermectin B1a, emamectin B1 benzoate, spinosad, dimethomorph and tralkoxydim inhibited P-gp activity; ivermectin B1a, emamectin B1 benzoate and spinosad were determined to be stronger inhibitors (half-maximal inhibitory concentration [IC50 ] of 3 ± 1, 5 ± 1 and 7 ± 1 µM, respectively) than dimethomorph and tralkoxydim (IC50 of 102 ± 7 and 88 ± 7 µM, respectively). Ivermectin B1a, emamectin B1 benzoate, spinosad and dimethomorph also inhibited P-gp activity in Caco-2 cell monolayer assays, with dimethomorph being a weaker P-gp inhibitor. These combined approaches could be used to identify P-gp inhibitors among food contaminants, but need to be optimised and adapted for high-throughput screening.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Cicloexanonas , Dissacarídeos , Iminas , Praguicidas , Humanos , Ivermectina/farmacologia , Rodamina 123 , Células CACO-2 , Praguicidas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , BenzoatosRESUMO
It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.
Assuntos
Eliminação Hepatobiliar , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Humanos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Hepatócitos/metabolismo , RNA Mensageiro/metabolismoRESUMO
Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. P-glycoprotein (P-gp) one of the ATP-binding cassette (ABC) transporters plays an important role in MDR. In this study, we examined the sensitizing property of andrographolide (Andro) to reverse MDR in the drug-resistant KBChR 8-5 cells. Andro exhibited increased cytotoxicity in a concentration-dependent manner in the P-gp overexpressing KBChR 8-5 cells. Furthermore, Andro showed synergistic interactions with PTX and DOX in this drug-resistant cells. Andro co-administration enhanced PTX- and DOX-induced cytotoxicity and reduced cell proliferation in the MDR cancer cells. Moreover, reactive oxygen species (ROS) were elevated with a decrease in the mitochondrial membrane potential (MMP) during Andro and chemotherapeutic drugs combination treatment in the drug-resistant cells. Furthermore, Andro and PTX-induced cell cycle arrest was observed in the drug-resistant cell. We also noticed that the expression of ABCB1 and AKT were downregulated during Andro (4 µM) treatment. Furthermore, Andro treatment enhanced the expression of caspase 3 and caspase 9 in the combinational groups that support the enhanced apoptotic cell death in drug-resistant cancer cells. Therefore, the results reveal that Andro plays a role in the reversal of P-gp-mediated MDR in KBChR 8-5 cells which might be due to regulating ABCB1/AKT signaling pathway.
Assuntos
Diterpenos , Resistencia a Medicamentos Antineoplásicos , Proteínas Proto-Oncogênicas c-akt , Resistência a Múltiplos Medicamentos , Transdução de Sinais , Linhagem Celular TumoralRESUMO
Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.
Assuntos
Rifabutina , Rifampina , Rifampina/farmacologia , Rifabutina/farmacologia , Rodamina 123/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Simulação de Acoplamento MolecularRESUMO
P-glycoprotein (P-gp), a multidrug efflux pump encoded by the ABCB1 (formerly MDR1) gene, plays a crucial role in limiting drug absorption and eliminating toxic compounds in both humans and dogs. However, species-specific differences in P-gp substrates necessitate the development of canine-specific evaluation systems. Canine intestinal organoids derived monolayers offer a promising platform for studying drug transport, yet P-gp-mediated transport in these models remains unexplored.We generated canine colonoid-derived 2D monolayers to investigate ABCB1 gene expression and P-gp function. We employed widely recognised P-gp substrates, Rhodamine 123 and Doxorubicin, in conjunction with the P-gp inhibitor PSC833 at Days 5 and 10 of culture.A significant increase in gene expression of P-gp encoded by the ABCB1 was noted on Day 10 compared to Day 5 of culture. Despite this disparity in gene expression, the transport activity of P-gp, as assessed by the efflux of Rhodamine 123 and Doxorubicin with PSC833 inhibition, did not exhibit significant differences between these two time points. However, the inhibition of P-gp function by PSC833 confirms the presence of functional P-gp in our model.Canine intestinal organoid-derived monolayers provide a valuable tool for investigating P-gp-mediated drug transport. These findings highlight the potential for predicting drug bioavailability and adverse reactions in veterinary medicine, aligning with principles of ethical and sustainable research.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Doxorrubicina , Organoides , Rodamina 123 , Animais , Cães , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Rodamina 123/metabolismo , Organoides/metabolismo , Doxorrubicina/farmacologia , Mucosa Intestinal/metabolismo , Ciclosporinas/farmacologia , Transporte BiológicoRESUMO
This study aimed to determine changes in the hydrolysis of vicagrel, a substrate drug of arylacetamide deacetylase (Aadac) and carboxylesterase 2 (Ces2), in P-glycoprotein (P-gp)-deficient or P-gp-inhibited mice and to elucidate the mechanisms involved.Male wild-type (WT) and P-gp knock-out (KO) mice were used to investigate the systemic exposure of vicagrel thiol active metabolite H4 and platelet response to vicagrel, and the mRNA and protein expression levels of intestinal Aadac and Ces2. Moreover, WT mice were administered vicagrel alone or in combination with elacridar (a potent P-gp inhibitor) to determine drug-drug interactions.Compared with WT mice, P-gp KO mice exhibited significant increases in the systemic exposure of H4, the protein expression levels of intestinal Aadac and Ces2, and inhibition of ADP-induced platelet aggregation by vicagrel. Further, the H4 exposure was positively correlated with intestinal Aadac protein expression levels but did not vary with short-term inhibition of P-gp efflux activity by elacridar.P-gp-deficient mice, rather than elacridar-treated mice, exhibited significant upregulation of intestinal Aadac and Ces2 and thus, enhanced metabolic activation of and platelet response to vicagrel, suggesting that the metabolic activation of vicagrel may vary with P-gp deficiency, not P-gp inhibition, in mice.
RESUMO
Multidrug resistance (MDR) is the phenomenon in which cancer cells simultaneously develop resistance to a broad spectrum of structurally and mechanistically unrelated drugs. MDR severely hinders the effective treatment of cancer and is the major cause of chemotherapy failure. ATP-binding cassette (ABC) transporters are extensively expressed in various body tissues, and actively transport endogenous and exogenous substrates through biological membranes. Overexpression of ABC transporters is frequently observed in MDR cancer cells, which promotes efflux of chemotherapeutic drugs and reduces their intracellular accumulation. Increasing evidence suggests that ABC transporters regulate tumor immune microenvironment (TIME) by transporting various cytokines, thus controlling anti-tumor immunity and sensitivity to anticancer drugs. On the other hand, the expression of various ABC transporters is regulated by cytokines and other immune signaling molecules. Targeted inhibition of ABC transporter expression or function can enhance the efficacy of immune checkpoint inhibitors by promoting anticancer immune microenvironment. This review provides an update on the recent research progress in this field.