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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.
Toxicol Appl Pharmacol ; 401: 115080, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32497533

RESUMO

Upregulation of ABCB1/MDR1 (P-gp) and BIRC5/Survivin promotes multidrug resistance in a variety of human cancers. LCL161 is an anti-cancer DIABLO/SMAC mimetic currently being tested in patients with solid tumors, but the molecular mechanism of action of LCL161 in cancer cells is still incompletely understood. It is still unclear whether LCL161 is therapeutically applicable for patients with ABCB1-overexpressing multidrug resistant tumors. In this study, we found that the potency of LCL161 is not affected by the expression of ABCB1 in KB-TAX50, KB-VIN10, and NTU0.017 cancer cells. Besides, LCL161 is equally potent towards the parental MCF7 breast cancer cells and its BIRC5 overexpressing, hormone therapy resistance subline MCF7-TamC3 in vitro. Mechanistically, we found that LCL161 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multi-drug efflux activity at low cytotoxic concentrations (i.e. 0.5xIC50 or less). Further analysis revealed that LCL161 also decreases intracellular ATP levels in part through BIRC5 downregulation. Therapeutically, co-treatment with LCL161 at low cytotoxic concentrations restored the sensitivity to the known ABCB1 substrate, paclitaxel, in ABCB1-expressing cancer cells and increased the sensitivity to tamoxifen in MCF7-TamC3 cells. In conclusion, LCL161 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide important information to physicians for designing a more "patient-specific" LCL161 clinical trial program in the future.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Proteínas Mitocondriais/farmacologia , Survivina/antagonistas & inibidores , Tiazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Proteínas Reguladoras de Apoptose/química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Mitocondriais/química , Estrutura Secundária de Proteína , Survivina/biossíntese , Survivina/genética , Tiazóis/química
3.
Phytomedicine ; 71: 153239, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32447245

RESUMO

BACKGROUND AND PURPOSE: Multidrug resistance (MDR) remains the main obstacle in cancer treatment and overexpression of P-glycoprotein (P-gp) is one of the most common causes of chemoresistance. The development of novel P-gp inhibitors from natural products is a prospective strategy to combat MDR cancers. Among the natural sesquiterpene compounds, sesquiterpene pyridine alkaloids exhibit various biological properties. Therefore, in the present study, we evaluated the modulatory effects of wilforine on P-gp expression and function. The molecular mechanisms and kinetic models of wilforine-mediated P-gp inhibition were further investigated. METHODS: The human P-gp stable expression cells (ABCB1/Flp-InTM-293) and human cervical cancer cells (sensitive: HeLaS3; MDR: KBvin) were used. The cell viability was assessed by SRB assay. The inhibitory effect of wilforine on P-gp efflux and the underlying mechanism were evaluated by assays for calcein-AM uptake, rhodamine123 and doxorubicin efflux, ATPase activity, real-time quantitative RT-PCR, apoptosis, and cell cycle analysis. Molecular docking was performed by the docking software CDOCKER with BIOVIA Discovery Studio 4.5 (D.S. 4.5). RESULTS: We found that wilforine significantly inhibited the efflux activity of P-gp in a concentration-dependent manner. Further kinetic analysis demonstrated that wilforine significantly inhibited P-gp efflux function by competitive inhibition and stimulated the basal P-gp ATPase activity. In addition, wilforine re-sensitized MDR cancer cells to chemotherapeutic drugs. The docking model indicated that wilforine was bound to residues of P-gp such as LEU884, LYS887, THR176 and ASN172. CONCLUSION: These results suggest a novel future therapeutic strategy for MDR cancer using wilforine as an adjuvant treatment with chemotherapy.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lactonas/farmacologia , Piridinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/fisiologia , Células HeLa , Humanos , Cinética , Lactonas/química , Simulação de Acoplamento Molecular , Estudos Prospectivos , Piridinas/química
4.
Am J Pathol ; 190(6): 1284-1297, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32240619

RESUMO

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr-/-) mice were crossbred with ATP binding cassette subfamily B member 4-deficient (Abcb4-/-, alias Mdr2-/-) mice. Male and female wild-type, Tr-/-, Mdr2-/-, and Tr-/-Mdr2-/- mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2-/- and Tr-/-Mdr2-/- mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2-/- mice, compared with extensive bridging fibrosis in Tr-/-Mdr2-/- mice. DR cell population was greatly expanded in the Tr-/-Mdr2-/- versus Mdr2-/- mice. The expanded DR cell population in Tr-/-Mdr2-/- mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr-/-Mdr2-/- versus Mdr2-/- mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2-/- mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2-/- model of cholestasis.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Cirrose Hepática/genética , Fígado/metabolismo , Macrófagos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Colestase/genética , Colestase/metabolismo , Colestase/patologia , Modelos Animais de Doenças , Feminino , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/fisiologia
5.
Chem Biol Interact ; 324: 109097, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32305507

RESUMO

Oroxylin A is a flavonoid monomer extracted from Scutellaria baicalensis Georgi with neuroprotective, anti-tumor activity and many other biological functions. However, the interaction between Oroxylin A and the drug transporters has not been clearly reported. The purpose of this study is to investigate the interaction between Oroxylin A and the solute carrier transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2K), and ATP-binding cassette transporters (BCRP, MDR1). The HEK293 cell lines (HEK293-OATP1B1, HEK293-OATP1B3, HEK293-OAT1, HEK293-OAT3, HEK293-OCT2, HEK293-MATE1, and HEK293-MATE2K) that stably expressing previous listed human-derived transporters were employed to evaluate the solute carrier transporters. Vesicles expressing human BCRP and MDR1 transporters was employed to research ATP-binding cassette transporters. Our work suggested that Oroxylin A was a substrate of OATP1B1, OATP1B3, but not a substrate of the other transporters in the concentration range of our study. Oroxylin A shows concentration-dependent inhibition of OATP1B1, OAT1, OAT3 and BCRP transportation with the half-inhibitory concentration (IC50) of 7.03, 0.961, 0.112 µM, and 0.477 µM, respectively. No inhibitory effects on the transport activities of other transporters were observed for Oroxylin A. Drug transporters profile of Oroxylin A was first confirmed by our work, which provides important information for its pharmacokinetics, pharmacodynamics, and drug-drug interactions studies.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Flavonoides/metabolismo , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores
6.
Bull Cancer ; 107(5): 574-585, 2020 May.
Artigo em Francês | MEDLINE | ID: mdl-32252973

RESUMO

Direct oral anticoagulants, anti-IIa or anti-Xa, are widely used in the treatment and prevention of venous thromboembolic disease as well as in nonvalvular atrial fibrillation. Direct oral anticoagulants are characterized by a rapid onset of activity, a predictable response and a relatively wide therapeutic window. Nevertheless, theoretical drug interactions exist since direct oral anticoagulants are substrates of the transport protein P-glycoprotein and/or of isoforms of cytochromes P450 pathway. Direct oral anticoagulants do not have a marketing authorization for the treatment of cancer-associated thrombosis unlike low-molecular-weight heparins which remain the gold standard treatment today. However, recent studies have compared low-molecular-weight heparins to direct oral anticoagulants in the treatment of cancer-associated thrombosis. Results of these studies showed a non-inferiority of direct oral anticoagulants in the prevention of recurrent thromboembolic events but at the cost of an increased hemorrhagic risk, in particular for patients with gastrointestinal and urogenital cancers. Thus, international guidelines, unlike French guidelines, integrate them in first line of the therapeutic strategy of cancer patients. We are certainly entering an era of personalized therapy for cancer-associated thrombosis, considering cancer type and also the theoretical risk of drug interactions with anti-cancer treatments or supportive care.


Assuntos
Inibidores do Fator Xa/uso terapêutico , Neoplasias/complicações , Trombose/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Anticoagulantes/uso terapêutico , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Sistema Enzimático do Citocromo P-450/metabolismo , Dabigatrana/metabolismo , Dabigatrana/uso terapêutico , Interações Medicamentosas , Inibidores do Fator Xa/efeitos adversos , Inibidores do Fator Xa/metabolismo , Hemorragia/induzido quimicamente , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Piridinas/metabolismo , Piridinas/uso terapêutico , Piridonas/metabolismo , Piridonas/uso terapêutico , Recidiva , Rivaroxabana/metabolismo , Rivaroxabana/uso terapêutico , Prevenção Secundária , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Trombose/etiologia , Tromboembolia Venosa/prevenção & controle
7.
PLoS One ; 15(4): e0231423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302323

RESUMO

Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches.


Assuntos
Colo/citologia , Células Epiteliais/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Desmossomos/metabolismo , Cães , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Membranas Artificiais , Microvilosidades/fisiologia , Mucinas/metabolismo , Nanoporos , Células-Tronco/citologia , Células-Tronco/metabolismo , Junções Íntimas/metabolismo
8.
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
9.
J Med Chem ; 63(10): 5458-5476, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32329342

RESUMO

SIS3 is a specific inhibitor of Smad3 that inhibits the TGFß1-induced phosphorylation of Smad3. In this article, a variety of SIS3 derivatives were designed and synthesized to discover potential inhibitors against P-glycoprotein-mediated multidrug resistance aided by late-stage functionalization of a 2-(4-(pyridin-2-yl)phenoxy)pyridine analogue. A novel class of potent P-gp reversal agents were investigated, and a lead compound 37 was identified as a potent P-gp reversal agent with strong bioactivity and outstanding affinity for P-gp.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Descoberta de Drogas/métodos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Isoquinolinas/química , Isoquinolinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Resistência a Múltiplos Medicamentos/fisiologia , Humanos , Isoquinolinas/metabolismo , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular/métodos , Piridinas/metabolismo , Pirróis/metabolismo , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 42(1): 1-6, 2020 Feb 28.
Artigo em Chinês | MEDLINE | ID: mdl-32131933

RESUMO

Objective To investigate the effect of α-asarone on the function and expression of P-glycoprotein(P-gp)in rat brain microvascular endothelial cells(rBMECs). Methods rBMECs were exposed to L-glutamate(100 µmol/L) for 30 mins to induce the overexpression of P-gp/multidrug resistance gene 1a(Mdr1a)on the cell membranes,which mimicked the overexpression of P-gp/Mdr1a in blood brain barrier(BBB) when drug-resistant epilepsy attacked.MTT assay was used to detect the safe range of α-asarone concentration.The model cells were intervened with different concentrations of α-asarone at 12.5,25.0,and 50.0 µg/µl for 24 hours.After the treatment of α-asarone,the expression and the function of P-gp/Mdr1 were measured by Western blotting,real-time PCR,and intracellular rhodamine 123 accumulation assays. Results The rBMECs,stimulated by glutamine,showed a high expression of P-gp(F=1.924,P=0.020)/Mdr1a(F=1.788,P=0.019) compared to the normal rBMECs.The treatment with 25.0(F=1.924,P=0.025;F=1.788,P=0.017) and 50.0 µg/µl(F=1.924,P=0.035;F=1.788,P=0.026) α-asarone significantly depressed the expression of P-gp/Mdr1a.The treatment with 25.0 and 50.0 µg/µl α-asarone significantly increased intracellular accumulation of Rhodamine 123 by 40% and 60% respectively. Conclusions α-asarone down-regulates the high expressions of P-gp and Mdr1a mRNA in rBMECs induced by L-glutamate.Moreover and increases intracellular accumulation of rhodamine-123.Thus,α-asarone may reverse drug resistance in P-gp-mediated drug-resistant epilepsy.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Anisóis/farmacologia , Encéfalo/citologia , Células Endoteliais/efeitos dos fármacos , Animais , Células Cultivadas , Epilepsia Resistente a Medicamentos , Células Endoteliais/metabolismo , Ácido Glutâmico , Ratos
11.
J Exp Clin Cancer Res ; 39(1): 50, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164732

RESUMO

BACKGROUND: Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. METHODS: The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. RESULTS: Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. CONCLUSIONS: Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.


Assuntos
Benzodiazepinas/administração & dosagem , Doxorrubicina/administração & dosagem , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Células HL-60 , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Neoplasias , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
BMC Complement Med Ther ; 20(1): 84, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171300

RESUMO

BACKGROUND: Current chemotherapies for Burkitt lymphoma (BL) have dramatically improved its clinical outcome. However, chemoresistance can lead to chemotherapy failure and very poor prognosis; thus, novel strategies are urgently required for patients with drug-resistant BL. To investigate the mechanisms underlying drug resistance in BL, we established drug-resistant BL cell lines: HS-Sultan/ADM (adriamycin-resistant), HS-Sultan/VCR (vincristine-resistant), HS-Sultan/DEX (dexamethasone-resistant), and HS-Sultan/L-PAM (melphalan-resistant). METHODS: Drug transporter and survival factor expression were investigated the using western blotting and real time polymerase chain reaction. Cell survival was analyzed by trypan blue dye exclusion method. RESULTS: The established cell lines acquired cross-resistance to adriamycin, vincristine, dexamethasone, and melphalan and exhibited 50% inhibitory concentration values 106-, 40-, 81-, and 45-fold higher than the parental cell lines, respectively. We found that protein and mRNA expression of MDR1 and Survivin were higher in drug-resistant BL cells than in the parent cells. Treatment with verapamil, an MDR1 inhibitor, or Survivin siRNA alongside each anti-cancer drug suppressed the proliferation of all drug-resistant BL cells. Src kinase activity was higher in all resistant cell lines than the parental cells; suppressing Src with dasatinib restored drug sensitivity by reducing MDR1 and Survivin expression. CONCLUSIONS: MDR1 and Survivin upregulation are responsible for resistance to conventional drugs and dasatinib can restore drug sensitivity by reducing MDR1 and Survivin expression in drug-resistant BL cells. Src inhibitors could therefore be a novel treatment strategy for patients with drug resistant BL.


Assuntos
Antineoplásicos/farmacologia , Linfoma de Burkitt/metabolismo , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Survivina/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Linfoma de Burkitt/tratamento farmacológico , Linhagem Celular Tumoral , Regulação para Baixo , Humanos
13.
J Cancer Res Clin Oncol ; 146(4): 843-858, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32056006

RESUMO

PURPOSE: Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1. METHODS: CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU). RESULTS: We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU. CONCLUSIONS: Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Compostos de Anilina , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Fluoresceínas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HT29 , Humanos , Microscopia Confocal , Substrato Quinase C Rico em Alanina Miristoilada/deficiência , Nitrilos , Fosforilação , Quinolinas
14.
PLoS One ; 15(2): e0228848, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32050000

RESUMO

We investigated the relation of 99mTc-MIBI uptake to mitochondrial membrane potential (MMP) in cancer cell lines and patient-derived tumor cells (PDCs). In T47D and HT29 cells with low MDR1 expression, FCCP dose-dependently reduced MMP and 99mTc-MIBI accumulation in similar patterns with nearly perfect linear relationships. T47D and HT29 cells with high MDR1 expression had low 99mTc-MIBI accumulation that was minimally affected by FCCP dose. In these cells, verapamil markedly increased 99mTc-MIBI accumulation to magnitudes that were excessive compared to MMP increase. Decreased plasma membrane potential by verapamil and its recovery by FCCP suggested that enhanced 99mTc-MIBI transport through modified plasma membranes contributed to the excess accumulation. Evaluation of three different colon cancer PDCs with low to modest MDR1 expression verified that FCCP significantly suppressed MMP and similarly reduced 99mTc-MIBI accumulation. Verapamil partially recovered both MMP and 99mTc-MIBI accumulation that was lowered by FCCP. Importantly, a high linear correlation was found (r = 0.865) between 99mTc-MIBI accumulation and MMP in these cells. These findings indicate that low baseline 99mTc-MIBI uptake that is markedly increased by verapamil represents cancer cells with high levels of MDR1 expression. However, in cancer cells with low or modest levels of MDR1 expression that do not markedly increase 99mTc-MIBI uptake by verapamil, the magnitude of uptake is largely dependent on cellular MMP.


Assuntos
Transporte Biológico/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tecnécio Tc 99m Sestamibi/metabolismo , Verapamil/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Células HT29 , Humanos , Compostos Radiofarmacêuticos/metabolismo , Células Tumorais Cultivadas
15.
Cancer Sci ; 111(5): 1619-1630, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32058643

RESUMO

Recent studies have shown that MDR could be induced by the high stemness of cancer cells. In a previous study, we found bufalin could reverse MDR and inhibit cancer cell stemness in colorectal cancer, but the relationship between them was unclear. Here we identified overexpressing CD133 increases levels of Akt/nuclear factor-κB signaling mediators and MDR1, while increasing cell chemoresistance. Furthermore, bufalin reverses colorectal cancer MDR by regulating cancer cell stemness through the CD133/nuclear factor-κB/MDR1 pathway in vitro and in vivo. Taken together, our results suggest that bufalin could be developed as a novel 2-pronged drug that targets CD133 and MDR1 to eradicate MDR cells and could ultimately be combined with conventional chemotherapeutic agents to improve treatment outcomes for patients with colorectal cancer.


Assuntos
Antígeno AC133/metabolismo , Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Antígeno AC133/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/uso terapêutico , Bufanolídeos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Quimioterapia Combinada , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Fator de Transcrição RelA/genética
16.
J Exp Clin Cancer Res ; 39(1): 33, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041631

RESUMO

BACKGROUND: Renal cell carcinoma (RCC) is a highly vascular tumor and patients with low risk metastatic RCC of clear-cell histological sub-type (mccRCC) are treated with tyrosine-kinase inhibitors (TKIs), sunitinib, as the first-line of treatment. Unfortunately, TKI resistance eventually develops, and the underlying molecular mechanism is not well understood. METHODS: RCC cell-line with metastatic clear-cell histology (Caki-1), and patient samples were analysed to identify the role of Y-box binding protein 1 (YB-1) and ATP-binding cassette sub-family B member 1 (ABCB-1) in acquired sunitinib-resistance development. Caki-1 was conditioned with increasing sunitinib doses to recapitulate acquired resistance development in clinics. Sunitinib-conditioned and wild-type Caki-1 were subjected to cell viability assay, scratch assay, chicken embryo chorioallantoic membrane engraftment and proteomics analysis. Classical biochemical assays like flow cytometry, immunofluorescent staining, immunohistochemical staining, optical coherence tomography imaging, Western Blot and RT-PCR assays were applied to determine the possible mechanism of sunitinib-resistance development and the effect of drug treatments. Publicly available data was also used to determine the role of YB-1 upregulation in ccRCC and the patients' overall survival. RESULTS: We demonstrate that YB-1 and ABCB-1 are upregulated in sunitinib-resistant in vitro, ex vivo, in vivo and patient samples compared to the sensitive samples. This provides evidence to a mechanism of acquired sunitinib-resistance development in mccRCC. Furthermore, our results establish that inhibiting ABCB-1 with elacridar, in addition to sunitinib, has a positive impact on reverting sunitinib-resistance development in in vitro, ex vivo and in vivo models. CONCLUSION: This work proposes a targeted therapy (elacridar and sunitinib) to re-sensitize sunitinib-resistant mccRCC and, possibly, slow disease progression.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Proteína 1 de Ligação a Y-Box/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Masculino , Camundongos , Modelos Biológicos , Metástase Neoplásica , Estadiamento de Neoplasias , Fenótipo , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína 1 de Ligação a Y-Box/metabolismo
17.
J Cardiovasc Pharmacol ; 75(2): 174-179, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32023226

RESUMO

Clopidogrel is still widely used in acute coronary syndrome despite the development of more potent P2Y12 inhibitors. Previously, we conducted a trial that evaluated serial clopidogrel dose adjustment based on platelet function testing in acute coronary syndrome patients with initial high on-treatment platelet reactivity (HTPR). In this substudy, we performed post hoc analysis of the effect of ABCB1 genetic variants C3435T and G2677T/A on platelet inhibition and outcomes. There were no differences in the proportion of HTPR patients among C3435T carriers and noncarriers in both interventional and control group. G2677T carriers expressed significantly higher proportion of HTPR pattern throughout 12-month follow-up in the control group with no difference in the interventional group. There was no difference in ischemic outcomes between C3435T and G2677T carriers and noncarriers in both groups of patients. The results indicate that ABCB1 genotyping is not useful to guide clopidogrel therapy tailoring to improve high-risk patient management.


Assuntos
Síndrome Coronariana Aguda/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Clopidogrel/administração & dosagem , Absorção Gastrointestinal/genética , Variantes Farmacogenômicos , Inibidores da Agregação de Plaquetas/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/diagnóstico , Idoso , Plaquetas/metabolismo , Clopidogrel/metabolismo , Monitoramento de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação de Plaquetas/metabolismo , Antagonistas do Receptor Purinérgico P2Y/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores Purinérgicos P2Y12/sangue , Resultado do Tratamento
18.
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
19.
Cancer Res ; 80(4): 663-674, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31888888

RESUMO

Clinical evidence shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and, eventually, most tumors become resistant to all available therapies. We compiled a focused library consisting of >500 commercially available or newly synthetized 8-hydroxyquinoline (8OHQ) derivatives whose toxicity is paradoxically increased rather than decreased by the activity of P-glycoprotein (Pgp), a transporter conferring multidrug resistance (MDR). Here, we deciphered the mechanism of action of NSC297366 that shows exceptionally strong Pgp-potentiated toxicity. Treatment of cells with NSC297366 resulted in changes associated with the activity of potent anticancer iron chelators. Strikingly, iron depletion was more pronounced in MDR cells due to the Pgp-mediated efflux of NSC297366-iron complexes. Our results indicate that iron homeostasis can be targeted by MDR-selective compounds for the selective elimination of multidrug resistant cancer cells, setting the stage for a therapeutic approach to fight transporter-mediated drug resistance. SIGNIFICANCE: Modulation of the MDR phenotype has the potential to increase the efficacy of anticancer therapies. These findings show that the MDR transporter is a "double-edged sword" that can be turned against resistant cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Neoplasias/tratamento farmacológico , Oxiquinolina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Quelantes de Ferro/uso terapêutico , Neoplasias/patologia , Oxiquinolina/análogos & derivados , Oxiquinolina/uso terapêutico
20.
Parasite ; 27: 3, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31934848

RESUMO

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MßCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Exoesqueleto/química , Membrana Celular/química , Colesterol/química , Resistência a Medicamentos , Haemonchus/química , Haemonchus/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Membrana Celular/efeitos dos fármacos , Fluidez de Membrana , Xenobióticos/farmacologia
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