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1.
Pharm Res ; 37(10): 194, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32918191

RESUMO

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Neoplasias/metabolismo , Farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B 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 , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Dibenzocicloeptenos/farmacologia , Dicetopiperazinas/farmacologia , Cães , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Madin Darby de Rim Canino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prazosina/farmacocinética , Quinidina/farmacocinética , Quinolinas/farmacologia , Especificidade por Substrato , Transfecção
2.
PLoS One ; 15(8): e0237150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760111

RESUMO

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.


Assuntos
Antinematódeos/farmacologia , Dirofilaria immitis/efeitos dos fármacos , Ivermectina/farmacologia , Macrolídeos/farmacologia , Fenótipo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Células Cultivadas , Dirofilaria immitis/metabolismo , Dirofilaria immitis/patogenicidade , Dirofilariose/parasitologia , Cães , Resistência a Medicamentos , Proteínas de Helminto/metabolismo
3.
PLoS Comput Biol ; 16(7): e1008104, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32735589

RESUMO

High levels of heterozygosity present a unique genome assembly challenge and can adversely impact downstream analyses, yet is common in sequencing datasets obtained from non-model organisms. Here we show that by re-assembling a heterozygous dataset with variant parameters and different assembly algorithms, we are able to generate assemblies whose protein annotations are statistically enriched for specific gene ontology categories. While total assembly length was not significantly affected by assembly methodologies tested, the assemblies generated varied widely in fragmentation level and we show local assembly collapse or expansion underlying the enrichment or depletion of specific protein functional groups. We show that these statistically significant deviations in gene ontology groups can occur in seemingly high-quality assemblies, and result from difficult-to-detect local sequence expansion or contractions. Given the unpredictable interplay between assembly algorithm, parameter, and biological sequence data heterozygosity, we highlight the need for better measures of assembly quality than N50 value, including methods for assessing local expansion and collapse.


Assuntos
Mapeamento de Sequências Contíguas , Genoma Helmíntico , Heterozigoto , Anotação de Sequência Molecular/métodos , Nematoides/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Algoritmos , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Funções Verossimilhança , Proteoma , Análise de Sequência de DNA
4.
Nat Commun ; 11(1): 2151, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358512

RESUMO

One of the key challenges to overcome multidrug resistance (MDR) in cancer is the development of more effective and general strategies to discover bioactive scaffolds. Inspired by natural products, we describe a strategy to achieve this goal by modular biomimetic synthesis of scaffolds of (Z)-allylic-supported macrolides. Herein, an Rh(III)-catalyzed native carboxylic acid-directed and solvent-free C-H activation allylation with high stereoselectivity and chemoselectivity is achieved. The generated poly-substituted allylic alcohol as a multifunctional and biomimetic building block is crucial for the synthesis of (Z)-allylic-supported macrolides. Moreover, the unique allylic-supported macrolides significantly potentiate the sensitivity of tumor cells to cytotoxic agents such as vinorelbine and doxetaxel by reversing p170-glycoprotein-mediated MDR. Our findings will inspire the evolution of synthetic chemistry and open avenues for expedient and diversified synthesis of bioactive macrocyclic molecules.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Biomimética/métodos , Macrolídeos/química , Catálise , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos , Vinorelbina/química
5.
Zhonghua Zhong Liu Za Zhi ; 42(3): 216-221, 2020 Mar 23.
Artigo em Chinês | MEDLINE | ID: mdl-32252200

RESUMO

Objective: To investigate the effect of compound matrine injection on morphine tolerance in mice with lung cancer in situ and the expressions of multidrug resistance gene 1 (MDR1) and P-glycoprotein (P-gp). Methods: A mouse model of lung cancer in situ and morphine tolerance mode was established. The mice were injected with gradient concentration of compound matrine. The pain thresholds under different conditions were measured by thermal radiation tail-flick method. The mRNA level of MDR1 was tested by reverse transcription polymerase chain reaction (RT-PCR) and the protein level of P-gp was detected by western blot. The DNA binding activity of cyclophosphoadenosine response element binding protein (CREB) to the promoter of MDR1 gene was detected by electrophoretic mobility shift assay (EMSA). Results: The maximum analgesic percentage (MPE) of the mice in the morphine group was (85.21±6.53)% on the 8th day, and decreased to (38.45±5.52)% and (28.14±4.52)% on the 10th and 12th day, respectively, which indicated the morphine tolerance of mice with lung cancer in situ.The MPE of the mice in the group treated with morphine and compound matrine injection (300 mg/kg) was (79.34±6.50)% on the 8th day, and decreased to (62.16±5.53)% and (40.20±4.50)% on the 10th and 12th day, respectively.The results of RT-PCR assay showed that the relative expression levels of MDR1 mRNA in the brain tissues of mice in the morphine group, saline group, morphine combined with compound matrine injection (300 mg/kg) group and compound matrine injection (200 mg/kg) group were 2.33±0.79, 1.04±0.38, 1.37±0.38, and 1.43±0.53, respectively. There were statistically significant differences between the morphine group and the normal saline group, the morphine group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). There was no significant difference between the normal saline group and the compound matrine injection (200 mg/kg) group (P=0.05). The results of western blot showed that the relative expression levels of P-gp protein in the brain tissue of mice in the morphine group, saline group, and morphine combined with compound matrine injection (300 mg/kg) group were 1.86±0.40, 1.00±0.23, and 1.27±0.27, respectively. The expression of P-gp protein in the morphine group was significantly higher than those of the normal saline group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). The DNA-binding activity of CREB in the saline group was (0.23±0.07) Pu, significantly lower than (0.89±0.23) Pu of morphine combined with naloxone group and (0.80±0.23) Pu of morphine group (P<0.05). While the CREB DNA binding activity of morphine combined with compound matrine injection (300 mg/kg) group was (0.79±0.21) Pu, implicated that compound matrine had marginal effect on the DNA-binding activity of CREB (P>0.05). Conclusion: Compound matrine injection can significantly improve morphine tolerance and drug resistance of lung cancer through inhibiting the upregulations of MDR1 and P-gp induced by morphine.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Alcaloides/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genes MDR , Neoplasias Pulmonares/fisiopatologia , Morfina/farmacologia , Quinolizinas/efeitos adversos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Alcaloides/administração & dosagem , Animais , Neoplasias Pulmonares/genética , Camundongos , Quinolizinas/administração & dosagem
6.
Xenobiotica ; 50(10): 1258-1264, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32302241

RESUMO

Aspirin (acetyl salicylic acid) is widely used co-medication in patients with cardiovascular and cerebrovascular diseases. Given the prevalence of acetyl salicylic acid's use as a co-medication and conflicting reports in the literature on it being a substrate of P-glycoprotein (P-gp). There is a potential risk for its interaction with compounds with P-gp liability, therefore, we have conducted a detailed investigation to determine substrate potential of acetyl salicylic acid towards P-gp. We observed significantly lower cellular uptake of acetyl salicylic acid in MDR1 transfected LLC-PK1 cells compared to LLC-PK1 wild-type (WT) cells, however, the in vitro efflux of acetyl salicylic acid in MDR1 transfected LLC-PK1 cells was not inhibited by known inhibitors under various conditions. Acetyl salicylic acid did not show active asymmetrical transport across MDR1 transfected LLC-PK1 cells compared to LLC-PK1-WT cells in transwell assay. Moreover, no difference in plasma and brain exposure of acetyl salicylic acid and its metabolite salicylic acid was observed between FVB-WT and Mdr1a/b knockout (KO) mice. Taken together, our findings indicate that acetyl salicylic acid is not a substrate of P-gp.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Aspirina/metabolismo , Animais , Transporte Biológico , Transporte Biológico Ativo , Encéfalo , Células LLC-PK1 , Suínos
7.
J Med Chem ; 63(7): 3701-3712, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32160459

RESUMO

Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor and a validated target for immune and inflammatory diseases. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified garcinoic acid as a selective and efficient PXR agonist. The properties of this natural molecule as a specific PXR agonist were demonstrated by the screening on a panel of nuclear receptors, the assessment of the physical and thermodynamic binding affinity, and the determination of the PXR-garcinoic acid complex crystal structure. Cytotoxicity, transcriptional, and functional properties were investigated in human liver cells, and compound activity and target engagement were confirmed in vivo in mouse liver and gut tissue. In conclusion, garcinoic acid is a selective natural agonist of PXR and a promising lead compound toward the development of new PXR-regulating modulators.


Assuntos
Benzopiranos/farmacologia , Receptor de Pregnano X/agonistas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Benzopiranos/metabolismo , Benzopiranos/toxicidade , Linhagem Celular Tumoral , Cristalografia por Raios X , Citocromo P-450 CYP3A/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X/metabolismo
8.
Chem Biol Interact ; 323: 109074, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32217108

RESUMO

Non-small-cell lung cancer (NSCLC) is one of the common malignant tumors, and multidrug resistance (MDR) and tumor metastasis limit the anticancer effect of NSCLC. Therefore, it is necessary to develop new anticancer drug that can inhibit MDR and metastasis of NSCLC. In the present study, we found that 5-(2-chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H-1,2,3- triazole (MAY) displayed strong cytotoxic effect on A549 and taxol-resistant A549 cells (A549/Taxol cells). We further discovered that MAY led to G2/M phase arrest by inhibiting microtubule polymerization in both cells. Then MAY caused apoptosis by the mitochondrial pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. Interestingly, MAY was not a substrate for P-glycoprotein (P-gp), which was highly expressed in A549/Taxol cells, and MAY inhibited the expression and efflux function of P-gp. Furthermore, MAY inhibited epithelial-mesenchymal transition (EMT) by targeting Twist1 in A549/Taxol cells. In summary, our results suggest that MAY induces apoptosis in A549 and A549/Taxol cells and inhibits EMT in A549/Taxol cells. These findings suggest that MAY could provide a promising method for the treatment of NSCLC, especially for the treatment of resistant NSCLC.


Assuntos
Apoptose/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Paclitaxel/farmacologia , Triazóis/farmacologia , Moduladores de Tubulina/farmacologia , Células A549 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Paclitaxel/química , Polimerização , Transdução de Sinais/efeitos dos fármacos , Triazóis/química , Moduladores de Tubulina/química , Proteína 1 Relacionada a Twist/metabolismo
9.
J Photochem Photobiol B ; 204: 111811, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32028187

RESUMO

The development of multidrug resistance is often associated with the over-expression of P-glycoprotein (P-gp). This protein prevents drug accumulation and extrudes them out of the cell before they reach the intended target. The aim of this study was to develop an in vitro MCF-7 cell line with increased expression of P-gp and test the phototoxicity of a novel photoactivated zinc phthalocyanine tetrasulfonic acid (ZnPcS4) on these cells. The over-expressed P-gp MCF-7 cells (MCF-7/DOX) were developed from wildtype (WT) MCF-7 cells by a stepwise continuous exposure of the WT cells to different concentrations of Doxorubicin (DOX) (0.1 - 1 µM) over a period of 4 months. The P-gp expression was measured using flow cytometry, immunofluorescence and enzyme immunoassay. To verify whether zinc phthalocyanine-mediated photodynamic therapy (ZnPcS4 - PDT) is effective in MCF-7/DOX, we studied the subcellular localization, phototoxicity and nuclear damage. The flow cytometry result showed two distinct peaks of P-gp positive and negative expression in MCF-7/DOX cell population, which correlates with the ELISA-based assay (p˂0.001). The ME16C (Normal breast cells) was used as control. The localization studies showed that ZnPcS4 have greater affinity for lysosome than mitochondria. Phototoxicity results indicated that photoactivated zinc phthalocyanine decreased the cell proliferation and viability as the drug and laser light dosages increased to 16 µM and 20 J/cm2 respectively. PDT-induced cytotoxicity using lactose dehydrogenase (LDH) enzyme leakage as measure did not increase likewise. The ZnPcS4-induced PDT was less effective for MCF-7/DOX cells which could be attributed to decreased retention of ZnPcS4 in major cellular organelles due to the presence of increased drug efflux P-gp. The current findings suggest that, increased P-gp expression, a characteristic of multidrug resistance together with other related intrinsic mechanisms might contribute to render MCF-7/DOX cells less sensitive to ZnPcS4-induced phototoxicity.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Lasers Semicondutores , Compostos Organometálicos/farmacologia , Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Feminino , Humanos , Indóis/química , Células MCF-7 , Compostos Organometálicos/química , Fotoquimioterapia , Rodamina 123/química , Rodamina 123/metabolismo
10.
Molecules ; 25(2)2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31936160

RESUMO

: Multidrug resistance (MDR) is a complicated ever-changing problem in cancer treatment, and P-glycoprotein (P-gp), a drug efflux pump, is regarded as the major cause. In the way of developing P-gp inhibitors, natural products such as phenolic acids have gotten a lot of attention recently. The aim of the present study was to investigate the modulating effects and mechanisms of caffeic acid on human P-gp, as well as the attenuating ability on cancer MDR. Calcein-AM, rhodamine123, and doxorubicin were used to analyze the interaction between caffeic acid and P-gp, and the ATPase activity of P-gp was evaluated as well. Resistance reversing effects were revealed by SRB and cell cycle assay. The results indicated that caffeic acid uncompetitively inhibited rhodamine123 efflux and competitively inhibited doxorubicin efflux. In terms of P-gp ATPase activity, caffeic acid exhibited stimulation in both basal and verapamil-stimulated activity. The combination of chemo drugs and caffeic acid resulted in decreased IC50 in ABCB1/Flp-InTM-293 and KB/VIN, indicating that the resistance was reversed. Results of molecular docking suggested that caffeic acid bound to P-gp through GLU74 and TRY117 residues. The present study demonstrated that caffeic acid is a promising candidate for P-gp inhibition and cancer MDR attenuation.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Ácidos Cafeicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Ácidos Cafeicos/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Quimioterapia Combinada , Fluoresceínas/farmacologia , Humanos , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/farmacologia , Verapamil/farmacologia
11.
Chemistry ; 26(11): 2470-2477, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-31912555

RESUMO

Multidrug resistance (MDR) is regarded as a main obstacle for effective chemotherapy, and P-glycoprotein (P-gp)-mediated drug efflux has been demonstrated to be the key factor responsible for MDR. In this study, a novel pH-responsive hybrid drug delivery system was developed by conjugating d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a kind of P-gp inhibitor, on the surface of laponite nanodisks to overcome MDR. The prepared LM-TPGS display excellent colloidal stability, a high encapsulation efficiency of doxorubicin (DOX), and a pH-responsive drug release profile. In vitro experiments verified that LM-TPGS/DOX could exhibit significantly enhanced therapeutic efficacy in treating DOX-resistant breast cancer cells (MCF-7/ADR) through inhibiting the activity of P-gp-mediated drug efflux and effectively accumulating DOX within cancer cells. In vivo results revealed that LM-TPGS/DOX outstandingly suppressed MCF-7/ADR tumors with low side effects. Therefore, the high drug payload, enhanced inhibition efficacy to drug-resistant cells, and low side effects make the LM-TPGS/DOX a promising nanoplatform to reverse MDR for effective chemotherapy.


Assuntos
Antibióticos Antineoplásicos/química , Doxorrubicina/química , Nanocápsulas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Composição de Medicamentos/métodos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Vitamina E/química , Vitamina E/metabolismo
12.
J Agric Food Chem ; 68(5): 1257-1265, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31927919

RESUMO

Bedaquiline (TMC-207) is a recently approved drug for the treatment of multidrug-resistant tuberculosis (MDR-TB). Moreover, there is a present and growing concern for natural-product-mediated drug interaction, as these are inadvertently taken by patients as a dietary supplement, food additive, and medicine. In the present study, we investigated the impact of 20 plant-based natural products, typically phenolics, on in vivo oral bedaquiline pharmacokinetics, as previous studies are lacking. Three natural phenolics were identified that can significantly enhance the oral exposure of bedaquiline upon coadministration. We further investigated the possible role of all of the phytochemicals on in vitro P-glycoprotein (P-gp) induction and inhibition and CYP3A4 inhibition in a single platform as bedaquiline is the substrate for both P-gp and CYP3A4. In conclusion, curcumin, CC-I (3',5-dihydroxyflavone-7-O-ß-d-galacturonide-4'-O-ß-d-glucopyranoside), and 6-gingerol should not be coadministered with bedaquiline to avoid untoward drug interactions and, subsequently, its dose-dependent adverse effects.


Assuntos
Antituberculosos/farmacocinética , Diarilquinolinas/farmacocinética , Suplementos Nutricionais/efeitos adversos , Interações Alimento-Droga , Fenóis/efeitos adversos , Extratos Vegetais/efeitos adversos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antituberculosos/administração & dosagem , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Diarilquinolinas/administração & dosagem , Suplementos Nutricionais/análise , Feminino , Humanos , Fenóis/administração & dosagem , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Resistente a Múltiplos Medicamentos/metabolismo
13.
J Neuropathol Exp Neurol ; 79(3): 266-276, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999342

RESUMO

For amyotrophic lateral sclerosis (ALS), achieving and maintaining effective drug levels in the brain is challenging due to the activity of ATP-binding cassette (ABC) transporters which efflux drugs that affect drug exposure and response in the brain. We investigated the expression and cellular distribution of the ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) using immunohistochemistry in spinal cord (SC), motor cortex, and cerebellum from a large cohort of genetically well characterized ALS patients (n = 25) and controls (n = 14). The ALS group included 17 sporadic (sALS) and 8 familial (fALS) patients. Strong P-gp expression was observed in endothelial cells in both control and ALS specimens. Immunohistochemical analysis showed higher P-gp expression in reactive astroglial cells in both gray (ventral horn) and white matter of the SC, as well as in the motor cortex of all ALS patients, as compared with controls. BCRP expression was higher in glia in the SC and in blood vessels and glia in the motor cortex of ALS patients, as compared with controls. P-gp and BCRP immunoreactivity did not differ between sALS and fALS cases. The upregulation of both ABC transporters in the brain may explain multidrug resistance in ALS patients and has implications for the use of both approved and experimental therapeutics.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Cerebelo/metabolismo , Córtex Motor/metabolismo , Proteínas de Neoplasias/metabolismo , Medula Espinal/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/análise , Adulto , Idoso , Astrócitos/metabolismo , Astrócitos/patologia , Cerebelo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Motor/patologia , Proteínas de Neoplasias/análise , Medula Espinal/patologia
14.
Biochem Pharmacol ; 174: 113813, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31954717

RESUMO

P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a "closed" conformation, while DOX shifts Pgp to an "intermediate" conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Doxorrubicina/farmacologia , Animais , Relação Dose-Resposta a Droga , Camundongos , Conformação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia
15.
Clin Exp Rheumatol ; 38(2): 299-305, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31376257

RESUMO

OBJECTIVES: P-glycoprotein (P-gp) mediated drug efflux is the most essential mechanism of multi-drug resistance (MDR) in rheumatoid arthritis (RA). The study was undertaken to clarify the mechanism whereby IL-17 regulate the P-gp efflux function in peripheral blood lymphocytes of patients with RA. METHODS: Lymphocytes from RA patients and healthy individuals were cultured with IL-17A (0, 10, 100 ng/ml), IL-17A+(5Z)-7-Oxozeaenol (TAK1 inhibitor), and IL-17A+PD98059 (ERK inhibitor), respectively. 24h later, the level of P-gp mRNA expression in peripheral blood lymphocytes was detected by RT-PCR. Meanwhile, the efflux potential of P-gp was assessed by flow cytometry using the fluorescent dye Rhodamine 123, a substrate of P-gp. In order to confirm whether the inhibitors had worked, ERK1/2 and p65, as well as their phosphorylation were detected utilising Western blot analysis. RESULTS: With the exception of the expression of P-gp mRNA between control and IL-17A group, the mRNA expression, as well as the function of P-gp in the different group of healthy individuals was similar, and there was no significant difference (p>0.05). However, as for the RA patients, increased expressions of P-gp mRNA and efflux function were detected in IL-17A group compared with control. Moreover, IL-17A upregulated mRNA level and function of P-gp in a concentrate dependent manner. Upregulated expression of P-gp mRNA and efflux potential of P-gp were inhibited by TAK1 or ERK inhibitors in RA peripheral blood lymphocytes. Among them, TAK1 inhibitor, (5Z) -7-Oxozeaenol, showed a significant difference (p<0.05). Also, the decreased phosphorylation levels of ERK1/2 and p65 were detected with PD98059 and (5Z) -7-Oxozeaenol addition, respectively. CONCLUSIONS: This study showed that inflammatory cytokines IL-17A can upregulate the mRNA expression level and drug efflux function of P-gp on lymphocytes in RA patients through TAK1, in a concentrate dependent manner, contributing to RA drug resistance. Therefore, this may represent a new target for improving the therapeutic reactivity of DMARDs in the long term for RA patients.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antirreumáticos , Artrite Reumatoide , Interleucina-17/metabolismo , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Resistência a Múltiplos Medicamentos , Humanos , Linfócitos/metabolismo , Regulação para Cima
16.
Chem Biol Interact ; 315: 108865, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31628941

RESUMO

Treatment of breast cancer by paclitaxel (PAX) often encounters therapeutic failure most likely caused by innate/acquired resistance. Cancer stem cells (CSCs) and multidrug resistance complex (MDR-1 or P-glycoprotein) overexpression are main mechanisms implicated in chemoresistance. Increased aldehyde dehrogenase-1 (ALDH-1) was previously correlated with the stemness features of CSCs and hence is used as a marker for identification and CSCs targeting. The present study, therefore, aimed at investigating the effect of both curcumin (CUR) and vitamin D3 (D3) on MDR-1 and ALDH-1 expression and consequently the resistance to PAX both in vitro and in vivo. CUR was isolated from Turmeric rhizomes and identified using UPLC-ESI-MS/MS. For in vitro studies, the antiproliferative effect of PAX, CUR, 1,25(OH)2D3 (the active form of D3, also known as calcitriol) was determined, each alone and combined (PAX+CUR, PAX+1,25(OH)2D3, and PAX+CUR+1,25(OH)2D3) on MCF-7 breast cancer cells. Ehrlich ascites carcinoma solid tumor animal model was also used for in vivo studies. Combining CUR and/or 1,25(OH)2D3 to PAX showed synergistic cytotoxic interaction on MCF-7 cells. The apoptotic potential was also enhanced, as evidenced by a significant increase in caspase-7 and -9 as well as the pro-apoptotic Bax whereas a decrease in Bcl-2 levels was reported. Combining CUR and 1,25(OH)2D3 to PAX caused a downregulation in both MDR-1 and ALDH-1 gene expression in MCF-7 besides a decrease in their protein levels. In vivo, the triple therapy group (PAX+CUR+D3) showed the least tumor size. It also showed the lowest levels of MDR-1 and ALDH-1. PAX alone, however, showed increased levels of MDR-1 and ALDH-1 compared to control. Overall, the present study showed that PAX, as a monotherapy, demonstrated acquired resistance possibly by increasing MDR-1 expression and enriching CSCs population, as evidenced by increased ALDH-1. However, using CUR and D3 enhanced tumor response to PAX.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aldeído Desidrogenase 1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Colecalciferol/farmacologia , Curcumina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Paclitaxel/farmacologia , Retinal Desidrogenase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Camundongos , Receptores de Calcitriol/metabolismo
17.
Chem Biol Interact ; 315: 108892, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31704064

RESUMO

Oleanolic acid (OA) is a triterpenoid, widely found in plants and possesses antitumor activity in many cancer lines. However, cancer cells develop multidrug resistance (mdr) hindering the effect of anticancer drugs. P-glycoprotein (P-gp) is a major cause of mdr. Therefore, the cytotoxic effect of OA was evaluated on human breast cancer MDA-MB-231 and human liver cancer HepG2 with absence and presence of P-gp, respectively. OA reduced MDA-MB-231 viability in a dose dependent manner, whereas no remarkable effect was observed on HepG2 in the same range of concentrations (1-60 µM). Moreover, cytotoxicity studies were conducted in the presence of verapamil (20 mg/L), a P-gp inhibitor. OA exhibited the same effect on MDA-MB-231 in the absence and presence of verapamil. However, the cytotoxicity was greatly enhanced for HepG2 cells in the presence of verapamil (cell viability dropped from 63.7% to 25% after 72 h at 60 µM). The results were then confirmed in vivo on zebrafish embryos. Increased mortality and malformations were observed in verapamil pretreated group between 5 and 15 µM of OA compared to control; also, all embryos died at 20 µΜ OA and above. These results demonstrate that inhibiting P-gp enhances the chemotherapeutic activity of OA.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Ácido Oleanólico/farmacologia , Peixe-Zebra/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Feminino , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Verapamil/farmacologia
18.
Expert Opin Drug Metab Toxicol ; 16(1): 59-78, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31821048

RESUMO

Introduction: ATP-binding cassette (ABC) transporters, especially P-glycoprotein (P-gp), and various metabolic enzymes, in particular, CYP3A, expressed in the small intestine cooperatively limit the absorption of orally administered P-gp substrate drugs. The expression and/or function of intestinal P-gp, however, is easily modulated by various factors.Areas covered: Through extensive literature searches primarily utilizing PubMed, the authors reviewed factors that may cause inter- or intra-individual variations of the pharmacokinetics of orally administered P-gp substrate drugs due to the modulation of intestinal P-gp expression/function. The information on P-gp modulating factors can help to develop safer and more reliable oral formulations and pharmacotherapy.Expert opinion: In clinical pharmacotherapy with orally administered P-gp substrate drugs, the pharmacological action may exhibit a large interindividual variation among patients. Factors modulating intestinal P-gp expression/function listed here include: circadian rhythm (or drug dosing time), drug-drug interactions, formulation/excipients (vehicle, nonionic surfactants), food/supplements, gene polymorphism, obesity, colorectal carcinomas, diarrhea, hepatic failure, inflammation, inflammatory bowel disease, ischemia/reperfusion, organ transplant, renal failure, and others. We will discuss the methods for reducing the effect of modulated intestinal P-gp function on the pharmacokinetics of orally administered P-gp substrate drugs to achieve safer and more reliable oral formulations and pharmacotherapy.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Absorção Intestinal , Preparações Farmacêuticas/metabolismo , Administração Oral , Animais , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Humanos , Mucosa Intestinal/metabolismo , Preparações Farmacêuticas/administração & dosagem
19.
Xenobiotica ; 50(3): 346-353, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31023123

RESUMO

P-glycoprotein (P-gp), encoded by ABCB1 gene, participants in the transmembrane transport of multiple anticancer drugs. The aim of the current research was to observe in vitro the impacts of ABCB1 (1236 C > T, 2677G > T, and 3435C > T) polymorphisms on the efflux activity of P-gp-mediated sunitinib.Stable recombinant colonic adenocarcinoma cell (Caco-2) systems transfected with ABCB1 wild-type allele and variant alleles (1236 T, 2677T and 3435T) were constructed. The resistance of each cell line to sunitinib was assessed by cell counting kit-8 (CCK8) assay. The effects of ABCB1 (1236 C > T, 2677G > T and 3435C > T) polymorphisms on the intracellular accumulation and transepithelial permeability of sunitinib were also investigated.The recombinant cell lines transfected with ABCB1 variant alleles (1236 T, 2677T, and 3435T) showed higher resistance to sunitinib compared to cells transfecting with ABCB1 wild-type allele (p < .05). The intracellular accumulation of sunitinib was significantly decreased in the three types of recombinant cell lines overexpressing ABCB1 variant alleles in comparison of their wild-type cell lines (p < .05). The transepithelial permeability of sunitinib in recombinant cell systems in transfected with variant alleles was significantly increased compared with cells overexpressing ABCB1 wild-type allele. The P-gp activity in recombinant variant cells is stronger when mediated transport of sunitinib than wild-type counterpart. P-gp encoded by ABCB1 (1236 T, 2677T, and 3435T) variant alleles may be more efficient to transport sunitinib than wild-type allele. Our observation suggests that ABCB1 (1236 C > T, 2677G > T, and 3435C > T) polymorphisms affect the transport ability of P-gp-mediated sunitinib.Collectively, ABCB1 polymorphisms may alter the P-gp-mediated sunitinib sensitivity via regulating drug transport.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sunitinibe/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Humanos , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único
20.
Xenobiotica ; 50(3): 354-362, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31132291

RESUMO

1. More than 30% of epilepsy patients suffer pharmacoresistance. Transport of antileptic drugs by P-glycoprotein (P-gp) and MRP2 plays an important role in drug-resistant epilepsy. Huperzine A (Hup-A) is a natural compound, which might have potential in treating neurological disorders including epilepsy and Alzheimer's disease. In this study, we investigated whether human P-gp and MRP2 transport Hup-A.2. LLC-PK1 and MDCKII cells transfected with human P-gp or MRP2 were used to establish concentration equilibrium transport assays (CETAs) and determine the transport profile of Hup-A. The expression of P-gp and MRP2 was detected by qPCR and western blotting. The transport function of P-gp and MRP2 was measured by Rho123 and CDFDA cell uptake assay.3. In CETAs, Hup-A at concentrations of 10 ng/mL or 2 µg/mL was transported by MDR1 and MRP2 from basolateral to apical sides of the cell monolayers. P-gp and MRP2 inhibitors completely blocked the efflux of Hup-A. There was no efflux of Hup-A in LLC-PK1 or MDCKII wild-type (WT) cells.4. We demonstrate that Hup-A is a substrate of P-gp and MRP2. These results imply the efflux of Hup-A across the blood-brain barrier (BBB) in vivo, suggesting potential drug resistance of Hup-A.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Alcaloides/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Sesquiterpenos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Transporte Biológico , Barreira Hematoencefálica/metabolismo
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