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In this study, we investigated the role of two efflux transporters, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in the cytotoxicity and intracellular accumulation of the organophosphate pesticide chlorpyrifos (CPF) and its active metabolite, CPF-oxon (CPFO), in a human-derived liver cell line (HepG2) and kidney epithelial cell line (HK-2). The cytotoxicity to CPF and CPFO differed between cell lines where HK-2 had lower IC50 values which could be attributed to lower basal expression and inducibility of metabolizing enzymes, transporters, and nuclear receptors in HK-2 cells. In HepG2 cells, co-exposure of CPF with a specific inhibitor of either P-gp or BCRP enhanced the cytotoxicity of CPF while co-exposure of CPFO with VRP enhanced the cytotoxicity of CPFO, suggesting the role of these transporters in the elimination CPF and CPFO. Inhibition of efflux transporters did not affect the cytotoxicity of CPF and CPFO in HK-2 cells. Co-incubation of CPF with P-gp and BCRP inhibitors increased the intracellular concentration of CPF in HepG2 cells suggesting that both transporters play a role in limiting the cellular accumulation of CPF in HepG2 cells. Our results provide evidence that inhibition of efflux transporters can enhance CPF-induced toxicity through enhanced cellular accumulation and raises additional questions regarding how pesticide-transporter interactions may influence toxicity of mixtures containing pesticides and other environmental chemicals.
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Of the 450 cell membrane transporters responsible for shuttling substrates, nutrients, hormones, neurotransmitters, antioxidants, and signaling molecules, approximately nine are associated with clinically relevant drug-drug interactions (DDIs) due to their role in drug and metabolite transport. Therefore, a clinical study evaluating potential transporter DDIs is recommended if an investigational product is intestinally absorbed, undergoes renal or hepatic elimination, or is suspected to either be a transporter substrate or perpetrator. However, many of the transporter substrates and inhibitors administered during a DDI study also affect cytochrome P450 (CYP) activity, which can complicate data interpretation. To overcome these challenges, the assessment of endogenous biomarkers can help elucidate the mechanism of complex DDIs when multiple transporters or CYPs may be involved. This perspective article will highlight how creative study designs are currently being utilized to address complex transporter DDIs and the role of physiology-based -pharmacokinetic (PBPK) models can play.
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P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are important transporters causing drug-drug interaction (DDI). Here, we investigated the involvement of P-gp and BCRP in the oral absorption of ensitrelvir in non-clinical studies and estimated the DDI risk mediated by P-gp and BCRP inhibition in humans. Although ensitrelvir is an in vitro P-gp and BCRP substrate, it demonstrated high bioavailability in rats and monkeys after oral administration. Plasma exposures of ensitrelvir following oral administration were comparable in wild type (WT) and Bcrp (-/-) mice. On the other hand, the area under the plasma concentration-time curve (AUC) ratio of ensitrelvir in the Mdr1a/1b (-/-) mice to the WT mice was 1.92, indicating that P-gp, but not BCRP, was involved in the oral absorption of ensitrelvir. Based on our previous retrospective analyses, such a low AUC ratio (<3) in the Mdr1a/1b (-/-) mice indicates a minimal impact of P-gp on the oral absorption in humans. In conclusion, our studies demonstrate that the involvement of both P-gp and BCRP in the oral absorption of ensitrelvir is minimal, and suggest that ensitrelvir has a low risk for DDIs mediated by P-gp and BCRP inhibition in humans.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Ratones Noqueados , Animales , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Administración Oral , Humanos , Ratones , Ratas , Masculino , 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/antagonistas & inhibidores , Disponibilidad Biológica , Interacciones Farmacológicas , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacología , Células CACO-2 , Ratas Sprague-Dawley , Perros , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Tratamiento Farmacológico de COVID-19 , Macaca fascicularis , Células de Riñón Canino Madin Darby , Indazoles , Triazinas , TriazolesRESUMEN
P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) multidrug resistance (MDR) transporters are localized at the luminal surface of the blood-brain barrier (BBB). They confer fetal brain protection against harmful compounds that may be circulating in the peripheral blood. The fetus develops in low oxygen levels; however, some obstetric pathologies such as pre-eclampsia, placenta accreta/previa may result in even greater fetal hypoxic states. We investigated how hypoxia impacts MDR transporters in human fetal brain endothelial cells (hfBECs) derived from early and mid-stages of pregnancy. Hypoxia decreased BCRP protein and activity in hfBECs derived in early pregnancy. In contrast, in hfBECs derived in mid-pregnancy there was an increase in P-gp and BCRP activity following hypoxia. Results suggest a hypoxia-induced reduction in fetal brain protection in early pregnancy, but a potential increase in transporter-mediated protection at the BBB during mid-gestation. This would modify accumulation of various key physiological and pharmacological substrates of P-gp and BCRP in the developing fetal brain and potentially contribute to the pathogenesis of neurodevelopmental disorders commonly associated with in utero hypoxia.
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In CNS drug discovery, the estimation of brain exposure to lead compounds is critical for their optimization. Compounds need to cross the blood-brain barrier (BBB) to reach the pharmacological targets in the CNS. The BBB is a complex system involving passive and active mechanisms of transport and efflux transporters such as P-glycoproteins (P-gp) and breast cancer resistance protein (BCRP), which play an essential role in CNS penetration of small molecules. Several in vivo, in vitro, and in silico methods are available to estimate human brain penetration. Preclinical species are used as in vivo models to understand unbound brain exposure by deriving the Kp,uu parameter and the brain/plasma ratio of exposure corrected with the plasma and brain free fraction. The MDCK-mdr1 (Madin Darby canine kidney cells transfected with the MDR1 gene encoding for the human P-gp) assay is the commonly used in vitro assay to estimate compound permeability and human efflux. The in silico methods to predict brain exposure, such as CNS MPO, CNS BBB scores, and various machine learning models, help save costs and speed up compound discovery and optimization at all stages. These methods enable the screening of virtual compounds, building of a CNS penetrable compounds library, and optimization of lead molecules for CNS penetration. Therefore, it is crucial to understand the reliability and ability of these methods to predict CNS penetration. We review the in silico, in vitro, and in vivo data and their correlation with each other, as well as assess published experimental and computational approaches to predict the BBB penetrability of compounds.
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Encéfalo , Proteínas de Neoplasias , Humanos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Reproducibilidad de los Resultados , Proteínas de Neoplasias/metabolismo , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Barrera Hematoencefálica/metabolismoRESUMEN
Drug transporters are among the factors that determine the pharmacokinetic profiles after drug administration. In this study, we investigated the roles of drug transporters involved in transport of SN-38, which is an active metabolite of irinotecan, in the intestine under inflammatory conditions in vitro and determined their functional consequences. The expression alterations of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 2B1 were determined at the mRNA and protein levels, and the subsequent functional alterations were evaluated via an accumulation study with the representative transporter substrates [prazosin and dibromofluorescein (DBF)] and SN-38. We also determined the cytotoxicity of SN-38 under inflammatory conditions. Decreased BCRP expression and increased OATP2B1 expression were observed under inflammatory conditions in vitro, which led to altered accumulation profiles of prazosin, DBF, and SN-38, and the subsequent cytotoxic profiles of SN-38. Treatment with rifampin or novobiocin supported the significant roles of BCRP and OATP2B1 in the transport and cytotoxic profile of SN-38. Collectively, these results suggest that BCRP and OATP2B1 are involved in the increased cytotoxicity of SN-38 under inflammatory conditions in vitro. Further comprehensive research is warranted to completely understand SN-38-induced gastrointestinal cytotoxicity and aid in the successful treatment of cancer with irinotecan.
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Antineoplásicos , Neoplasias de la Mama , Transportadores de Anión Orgánico , Humanos , Femenino , Irinotecán , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Membrana , Prazosina , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
Success of chemotherapy is often hampered by multidrug resistance. One mechanism for drug resistance is the elimination of anticancer drugs through drug transporters, such as breast cancer resistance protein (BCRP; also known as ABCG2), and causes a poor 5-year survival rate of human patients. Co-treatment of chemotherapeutics and natural compounds, such as baicalein, is used to prevent chemotherapeutic resistance but is limited by rapid metabolism. Boron-based clusters as meta-carborane are very promising phenyl mimetics to increase target affinity; we therefore investigated the replacement of a phenyl ring in baicalein by a meta-carborane to improve its affinity towards the human ABCG2 efflux transporter. Baicalein strongly inhibited the ABCG2-mediated efflux and caused a fivefold increase in mitoxantrone cytotoxicity. Whereas the baicalein derivative 5,6,7-trimethoxyflavone inhibited ABCG2 efflux activity in a concentration of 5 µm without reversing mitoxantrone resistance, its carborane analogue 5,6,7-trimethoxyborcalein significantly enhanced the inhibitory effects in nanomolar ranges (0.1 µm) and caused a stronger increase in mitoxantrone toxicity reaching similar values as Ko143, a potent ABCG2 inhibitor. Overall, in silico docking and in vitro studies demonstrated that the modification of baicalein with meta-carborane and three methoxy substituents leads to an enhanced reversal of ABCG2-mediated drug resistance. Thus, this seems to be a promising basis for the development of efficient ABCG2 inhibitors.
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Antineoplásicos , Flavanonas , Mitoxantrona , Humanos , Mitoxantrona/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Resistencia a Antineoplásicos , Proteínas de Neoplasias/metabolismo , Antineoplásicos/farmacologíaRESUMEN
ABC transporters are ubiquitous in the human body and are responsible for the efflux of drugs. They are present in the placenta, intestine, liver and kidney, which are the major organs that can affect the pharmacokinetic and pharmacologic properties of drugs. P-gp and BCRP transporters are the best-characterized transporters in the ABC superfamily, and they have a pivotal role in the barrier tissues due to their efflux mechanism. Moreover, during pregnancy, drug efflux is even more important because of the developing fetus. Recent studies have shown that placental and intestinal ABC transporters have great importance in drug absorption and distribution. Placental and intestinal P-gp and BCRP show gestational-age-dependent expression changes, which determine the drug concentration both in the mother and the fetus during pregnancy. They may have an impact on the efficacy of antibiotic, antiviral, antihistamine, antiemetic and oral antidiabetic therapies. In this review, we would like to provide an overview of the pharmacokinetically relevant expression alterations of placental and intestinal ABC transporters during pregnancy.
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Proteínas de Neoplasias , Placenta , Femenino , Humanos , Embarazo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportadoras de Casetes de Unión a ATP/genética , Intestinos , Proteínas de Transporte de MembranaRESUMEN
Multidrug resistance (MDR1) and breast cancer resistance protein (BCRP) play important roles in drug absorption and distribution. Computational prediction of substrates for both transporters can help reduce time in drug discovery. This study aimed to predict the efflux activity of MDR1 and BCRP using multiple machine learning approaches with molecular descriptors and graph convolutional networks (GCNs). In vitro efflux activity was determined using MDR1- and BCRP-expressing cells. Predictive performance was assessed using an in-house dataset with a chronological split and an external dataset. CatBoost and support vector regression showed the best predictive performance for MDR1 and BCRP efflux activities, respectively, of the 25 descriptor-based machine learning methods based on the coefficient of determination (R2). The single-task GCN showed a slightly lower performance than descriptor-based prediction in the in-house dataset. In both approaches, the percentage of compounds predicted within twofold of the observed values in the external dataset was lower than that in the in-house dataset. Multi-task GCN did not show any improvements, whereas multimodal GCN increased the predictive performance of BCRP efflux activity compared with single-task GCN. Furthermore, the ensemble approach of descriptor-based machine learning and GCN achieved the highest predictive performance with R2 values of 0.706 and 0.587 in MDR1 and BCRP, respectively, in time-split test sets. This result suggests that two different approaches to represent molecular structures complement each other in terms of molecular characteristics. Our study demonstrated that predictive models using advanced machine learning approaches are beneficial for identifying potential substrate liability of both MDR1 and BCRP.
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Proteínas de Transporte de Membrana , Proteínas de Neoplasias , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Aprendizaje Automático , Resistencia a Múltiples MedicamentosRESUMEN
The study of transporter proteins is key to understanding the mechanism behind multi-drug resistance and drug-drug interactions causing severe side effects. While ATP-binding transporters are well-studied, solute carriers illustrate an understudied family with a high number of orphan proteins. To study these transporters, in silico methods can be used to shed light on the basic molecular machinery by studying protein-ligand interactions. Nowadays, computational methods are an integral part of the drug discovery and development process. In this short review, computational approaches, such as machine learning, are discussed, which try to tackle interactions between transport proteins and certain compounds to locate target proteins. Furthermore, a few cases of selected members of the ATP binding transporter and solute carrier family are covered, which are of high interest in clinical drug interaction studies, especially for regulatory agencies. The strengths and limitations of ligand-based and structure-based methods are discussed to highlight their applicability for different studies. Furthermore, the combination of multiple approaches can improve the information obtained to find crucial amino acids that explain important interactions of protein-ligand complexes in more detail. This allows the design of drug candidates with increased activity towards a target protein, which further helps to support future synthetic efforts.
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Background and purpose: Canine mammary tumors are the most common tumor disease of female dogs, and adjuvant chemotherapy often results in multi-drug resistance. Currently, the mechanisms underlying the development of tumor multi-drug resistance are unclear. The translation of research applications that can be used to effectively overcome tumor resistance is similarly hampered. Therefore, it is urgent to construct multi-drug resistance models of canine mammary tumors that can be used for research, to explore the mechanisms and means of overcoming resistance. Materials and methods: In this study, the canine triple negative breast cancer cell line CMT-7364 was induced to develop multidrug resistance using doxorubicin by high-dose drug pulse method. The drug resistance and the expression of drug transport pumps of the cells was verified by CCK8 assay, immunoblotting, qPCR and immunofluorescence. Next, we used scratch assay and Transwell invasion assay to compare the migration and invasion abilities of the two cell lines and examined the expression of EMT-related proteins in both using immunoblotting. The differences of transcriptome between parental and drug-resistant cell lines were detected by RNA-seq sequencing. Finally, mouse xenograft models of drug-resistant and parental cell lines were constructed to evaluate the tumorigenic ability. Results: After more than 50 generations of continuous passages stimulated by high-dose drug pulse method, the morphology of drug-resistant cell line CMT-7364/R tended to be mesenchymal-like and heterogeneous under light microscopy compared with the parental cell line CMT-7364/S, and developed resistance to doxorubicin and other commonly used chemotherapeutic drugs. In CMT-7364/R, BCRP was expressed at higher levels at both transcriptional and protein levels, while P-glycoprotein was not significantly different. Secondly, the migration and invasion ability of CMT-7364/R was significantly enhanced, with decreased expression of E-cadherin and increased expression of vimentin and mucin 1-N terminus. Finally, mouse xenograft models were constructed, while there was no significant difference in the volume of masses formed at 21 days. Conclusion: In summary, by using the canine mammary tumor cell line CMT-7364/S as the parental cell line, we successfully constructed a multidrug-resistant CMT-7364/R with high-dose drug pulse methods. Compared to its parental cell line, CMT-7364/R has decreased growth rate, overexpression of BCRP and increased migration and invasion ability due to EMT. The results of this study showed that CMT-7364/R might serve as a model for future studies on tumor drug resistance.
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Breast cancer resistance protein (BCRP,ABCG2) is an efflux transporter protein that transports various substrates from the cell to the extracellular space or organ cavities. The aim of this study was a complex assessment of the amount of BCRP during pregnancy in rabbits. The amount of BCRP in samples of the rabbit jejunum, liver, kidney, cerebral cortex, and placenta was determined by enzyme immunoassay, and in human hepatocellular carcinoma (HepG2) cells by the Western blot. To study the mechanisms involved in control of the dynamic BCRP levels during pregnancy, serum concentrations of sex hormones were investigated by radioimmunoassay and relative amounts of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in these organs were evaluated using the Western blot method. The putative role of CAR and PXR in regulation of the BCRP level by progesterone was evaluated in vitro experiments on HepG2 cells. It was found that amount of BCRP in the jejunum of pregnant rabbits was higher than in the placenta, liver, kidneys, and cerebral cortex. An increase in the amount of BCRP in the liver of rabbits was noted on the 21st day of pregnancy and a tendency to the increase was also detected on the 28th day; in the kidney and cerebral cortex increased BCRP levels were detected on the 28th day and 14th day of pregnancy, respectively, as compared with non-pregnant females. In vitro experiments with HepG2 cells have shown that the increase in the BCRP level is determined by the activating effect of progesterone on PXR.
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Neoplasias de la Mama , Proteínas de Neoplasias , Femenino , Humanos , Embarazo , Animales , Conejos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Progesterona , RiñónRESUMEN
BACKGROUND: The multidrug resistance (MDR) transporters, P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2) contribute to the blood-brain barrier (BBB), protecting the brain from drug exposure. The impact of infection on MDR in the developing human BBB remains to be determined. We hypothesized that exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs) modify MDR expression and activity in human fetal brain endothelial cells (hfBECs) isolated from early and mid-gestation brain microvessels. METHODS: We modelled infection (4 h and 24 h) using the bacterial PAMP, lipopolysaccharide (LPS; a toll-like receptor [TLR]-4 ligand) or the viral PAMPs, polyinosinic polycytidylic acid (Poly I:C; TLR-3 ligand) and single-stranded RNA (ssRNA; TLR-7/8 ligand). mRNA expression was assessed by qPCR, whereas protein expression was assessed by Western blot or immunofluorescence. P-gp and BCRP activity was evaluated by Calcein-AM and Chlorin-6 assays. RESULTS: TLRs-3,4 and 8 were expressed by the isolated hfBECs. Infection mimics induced specific pro-inflammatory responses as well as changes in P-gp/ABCB1 or BCRP/ABCG2 expression (P < 0.05). LPS and ssRNA significantly decreased P-gp activity at 4 and 24 h in early and mid-gestation (P < 0.03-P < 0.001), but significantly increased BCRP activity in hfBECs in a dose-dependent pattern (P < 0.05-P < 0.002). In contrast, Poly-IC significantly decreased P-gp activity after 4 h in early (P < 0.01) and mid gestation (P < 0.04), but not 24 h, and had no overall effect on BCRP activity, though BCRP activity was increased with the highest dose at 24 h in mid-gestation (P < 0.05). CONCLUSIONS: Infectious PAMPs significantly modify the expression and function of MDR transporters in hfBECs, though effects are PAMP-, time- and dose-specific. In conclusion, bacterial and viral infections during pregnancy likely have profound effects on exposure of the fetal brain to physiological and pharmacological substrates of P-gp and BCRP, potentially leading to altered trajectories of fetal brain development.
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Barrera Hematoencefálica , Células Endoteliales , Femenino , Embarazo , Humanos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Moléculas de Patrón Molecular Asociado a Patógenos , Ligandos , Lipopolisacáridos , Proteínas de Neoplasias , Encéfalo , Proteínas de Transporte de Membrana , Resistencia a Múltiples MedicamentosRESUMEN
P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP/ABCG2) are efflux multidrug resistance (MDR) transporters localized at the syncytiotrophoblast barrier of the placenta and protect the conceptus from drug and toxin exposure throughout pregnancy. Infection is an important modulator of MDR expression and function. This review comprehensively examines the effect of infection on the MDR transporters, P-gp and BCRP in the placenta. Infection PAMPs such as bacterial lipopolysaccharide (LPS) and viral polyinosinic-polycytidylic acid (poly I:C) and single-stranded (ss)RNA, as well as infection with Zika virus (ZIKV), Plasmodium berghei ANKA (modeling malaria in pregnancy - MiP) and polymicrobial infection of intrauterine tissues (chorioamnionitis) all modulate placental P-gp and BCRP at the levels of mRNA, protein and or function; with specific responses varying according to gestational age, trophoblast type and species (human vs. mice). Furthermore, we describe the expression and localization profile of Toll-like receptor (TLR) proteins of the innate immune system at the maternal-fetal interface, aiming to better understand how infective agents modulate placental MDR. We also highlight important gaps in the field and propose future research directions. We conclude that alterations in placental MDR expression and function induced by infective agents may not only alter the intrauterine biodistribution of important MDR substrates such as drugs, toxins, hormones, cytokines, chemokines and waste metabolites, but also impact normal placentation and adversely affect pregnancy outcome and maternal/neonatal health.
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Infección por el Virus Zika , Virus Zika , Embarazo , Femenino , Humanos , Ratones , Animales , Placenta/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Distribución Tisular , Proteínas de Neoplasias/genética , Resistencia a Múltiples Medicamentos , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Many mothers need to take some medications during breastfeeding, which may carry a risk to breastfed infants. Thus, determining the amount of a drug transferred into breast milk is critical for risk-benefit analysis of breastfeeding. Breast cancer resistance protein (BCRP), an efflux transporter which usually protects the body from environmental and dietary toxins, was reported to be highly expressed in lactating mammary glands. In this study, we developed a mechanistic lactation physiologically based pharmacokinetic (PBPK) modeling approach incorporating BCRP mediated transport kinetics to simulate the concentration-time profiles of five BCRP drug substrates (acyclovir, bupropion, cimetidine, ciprofloxacin, and nitrofurantoin) in nursing women's plasma and milk. Due to the lack of certain physiological parameters and scaling factors in nursing women, we combine the bottom up and top down PBPK modeling approaches together with literature reported data to optimize and determine a set of parameters that are applicable for all five drugs. The predictive performance of the PBPK models was assessed by comparing predicted pharmacokinetic profiles and the milk-to-plasma (M/P) ratio with clinically reported data. The predicted M/P ratios for acyclovir, bupropion, cimetidine, ciprofloxacin, and nitrofurantoin were 2.48, 3.70, 3.55, 1.21, and 5.78, which were all within 1.5-fold of the observed values. These PBPK models are useful to predict the PK profiles of those five drugs in the milk for different dosing regimens. Furthermore, the approach proposed in this study will be applicable to predict pharmacokinetics of other transporter substrates in the milk.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Lactancia , Leche Humana , Femenino , Humanos , Lactante , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Bupropión/farmacocinética , Cimetidina/farmacocinética , Ciprofloxacina/farmacocinética , Lactancia/metabolismo , Leche Humana/química , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Nitrofurantoína/farmacocinética , Aciclovir/farmacocinéticaRESUMEN
Lenvatinib is the first-line treatment for hepatocellular carcinoma (HCC), the most common type of primary liver cancer; however, some patients become refractory to lenvatinib. The underlying mechanism of lenvatinib resistance (LR) in patients with advanced HCC remains unclear. We focused on exploring the potential mechanism of LR and novel treatments of lenvatinib-resistant HCC. In particular, we established a Huh7 LR cell line and performed in vitro, bioinformatic, and biochemical assays. Additionally, we used a Huh7-LR cell-derived xenograft mouse model to confirm the results in vivo. Following LR induction, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) transporters were markedly upregulated, and the epidermal growth factor receptor (EGFR), MEK/ERK, and PI3K/AKT pathways were activated. In vitro, the co-administration of elacridar, a dual MDR1 and BCRP inhibitor, with lenvatinib inhibited proliferation and induced apoptosis of LR cells. These effects might be due to inhibiting cancer stem-like cells (CSCs) properties, by decreasing colony formation and downregulating CD133, EpCAM, SOX-9, and c-Myc expression. Moreover, the co-administration of gefitinib, an EGFR inhibitor, with lenvatinib retarded proliferation and induced apoptosis of LR cells. These similar effects might be caused by the inhibition of EGFR-mediated MEK/ERK and PI3K/AKT pathway activation. In vivo, co-administration of lenvatinib with elacridar or gefitinib suppressed tumour growth and angiogenesis. Therefore, inhibiting MDR1 and BCRP transporters or targeting the EGFR/PI3K pathway might overcome LR in HCC. Notably, lenvatinib should be used to treat HCC after LR induction owing to its role in inhibiting tumour proliferation and angiogenesis. Our findings could help develop novel and effective treatment strategies for HCC.
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There is little information about the functional expression of the multidrug resistance (MDR) transporters P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in the developing blood−brain barrier (BBB). We isolated and cultured primary human fetal brain endothelial cells (hfBECs) from early and mid-gestation brains and assessed P-gp/ABCB1 and BCRP/ABCG2 expression and function, as well as tube formation capability. Immunolocalization of the von Willebrand factor (marker of endothelial cells), zonula occludens-1 and claudin-5 (tight junctions) was detected in early and mid-gestation-derived hfBECs, which also formed capillary-like tube structures, confirming their BEC phenotype. P-gp and BCRP immunostaining was detected in capillary-like tubes and in the cytoplasm and nucleus of hfBECs. P-gp protein levels in the plasma membrane and nuclear protein fractions, as well as P-gp protein/ABCB1 mRNA and BCRP protein levels decreased (p < 0.05) in hfBECs, from early to mid-gestation. No differences in P-gp or BCRP activity in hfBECs were observed between the two age groups. The hfBECs from early and mid-gestation express functionally competent P-gp and BCRP drug transporters and may thus contribute to the BBB protective phenotype in the conceptus from early stages of pregnancy.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Encéfalo/metabolismo , Resistencia a Múltiples Medicamentos , Células Endoteliales/metabolismo , Femenino , Humanos , Proteínas de Neoplasias/metabolismo , EmbarazoRESUMEN
Dextran is a promising candidate as a nanocarrier of chemotherapeutic drugs due to its biocompatibility, biodegradability, and ability to accumulate in tumors. Furthermore, dextran derivatives interact with P-glycoprotein (P-gp), so we hypothesized that they may be available as tumor-specific drug delivery systems with the ability to reverse multidrug resistance. Here, to test this idea, we investigated whether dextran and its derivatives inhibit breast cancer resistance protein (BCRP), multidrug resistance associated protein 1 (MRP1), and P-gp in vitro. First, we examined their effect on the uptake of specific fluorescent substrates by inside-out Sf-9 membrane vesicles overexpressing BCRP, MRP1, and P-gp. BCRP and MRP1 were significantly inhibited by 2-hydroxypropyl-trimethylammonium-dextran of 4 and 70 kDa (Q-D4 and Q-D70) at a concentration near the clinically used concentration of dextran; however, P-gp was not inhibited. A structure-activity study showed that Q-D4, Q-D70, and 40 kDa diethylaminoethyl-dextran (DEAE-D40) significantly inhibited BCRP, while 4, 40, and 70 kDa dextrans (D4, D40, and D70), dextran sulfate (Sul-D40), and the individual saccharide components of dextran did not. These results suggest that the cationic side chains, but not the saccharides, are important for BCRP inhibition. Finally, cell-based efflux assay was conducted. Q-D4, Q-D70, and DEAE-D40 did not specifically increase the retention of Hoechst33342 in BCRP-overexpressing KB cells. Similarly, Q-D4 and Q-D70 did not affect the intracellular retention of specific fluorescent substrates in MRP1- and P-gp-overexpressing KB cells. The ineffectiveness in cellular systems is presumably due to inability of the dextran derivatives to access transporters located on the cytoplasmic side of the cell membrane.
Asunto(s)
Dextranos , Neoplasias , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Dextranos/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológicoRESUMEN
Patient and providers' fear of fetal exposure to medications may lead to discontinuation of treatment, disease relapse, and maternal morbidity. Placental drug transporters play a critical role in fetal exposure through active transport but the majority of data are limited to the 3rd trimester, when the majority of organogenesis has already occurred. Our objective was to define gestational age (GA) dependent changes in protein activity, expression and modifications of five major placental drug transporters: SERT, P-gp, NET, BCRP and MRP3. Apical brush border membrane fractions were prepared from fresh 1st, 2nd and 3rd trimester human placentas collected following elective pregnancy termination or planned cesarean delivery. A structured maternal questionnaire was used to identify maternal drug use and exclude exposed subjects. Changes in placental transporter activity and expression relative to housekeeping proteins were quantified. There was evidence for strong developmental regulation of SERT, NET, P-gp, BCRP and MRP3. P-gp and BCRP decreased with gestation (r = -0.72, p < 0.001 and r = -0.77, p < 0.001, respectively). Total SERT increased with gestation but this increase was due to a decrease in SERT cleavage products across trimesters. Uncleaved SERT increased with GA (r = 0.89, p < 0.001) while cleaved SERT decreased with GA (r = -0.94, p < 0.001). Apical membrane NET overall did not appear to be developmentally regulated (r = -0.08, p = 0.53). Two forms of MRP3 were identified; the 50 kD form did not change across GA; the 160 kD form was steady in the 1st and 2nd trimester and increased in the 3rd trimester (r = 0.24, p = 0.02). The 50 kD form was expressed at higher levels. The observed patterns of SERT, NET P-gp, BCRP and MRP3 expression and activity may be associated with transporter activity or decreased placental permeability in the 1st trimester to transporter specific substrates including commonly used psychoactive medications such as anti-depressants, anti-psychotics, and amphetamines, while transport of nutrients and serotonin is important in the 1st trimester. Overall these observations are consistent with a strong protective effect during organogenesis. 3rd trimester estimates of fetal exposure obtained from cord blood likely significantly overestimate early fetal exposure to these medications at any fixed maternal dose.
RESUMEN
Cannabichromene (CBC) and cannabichromenic acid (CBCA) are cannabis constituents currently under evaluation for their therapeutic potential, but their pharmacological properties have not been thoroughly investigated. The most studied ATP-binding cassette (ABC) transporters, ABC subfamily G member 2 (ABCG2) and ABC subfamily B member 1 (ABCB1) limit absorption of substrate drugs in the gut and brain. Moreover, inhibitors of these proteins can lead to clinically significant drug-drug interactions (DDIs). The current study sought to examine whether CBC and CBCA affect ABCB1 and ABCG2 to advance their basic pharmacological characterisation. The plant cannabinoids CBC and CBCA were screened in vitro in a bidirectional transport assay to determine whether they were substrates and/or inhibitors of ABCB1 and ABCG2. Transwell assays with polarized epithelial Madin-Darby Canine Kidney II (MDCK) cells expressing ABCB1 or ABCG2 were used. Samples were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). CBCA was found to be an ABCB1 substrate, but not an ABCG2 substrate. CBC was not a substrate of either transporter. Neither CBCA nor CBC inhibited ABCB1 transport of prazosin or ABCG2 transport of digoxin. In silico molecular docking suggested CBCA binds ABCB1 in the access tunnel and the central binding pocket. CBC, an agent with anticonvulsant, anti-inflammatory and anti-depressant properties, is not a substrate or inhibitor of ABCB1 or ABCG2, which is favourable to its therapeutic development. CBCA is an ABCB1 substrate in vitro which might contribute to its poor absorption. These findings provide important basic pharmacological data to assist the therapeutic development of these cannabis constituents.