RESUMO
Preparations of Echinacea purpurea (E. purpurea) are widely used for the management of upper respiratory infections, influenza, and common cold, often in combination with other conventional drugs. However, the potential of phytochemical constituents of E. purpurea to cause herb-drug interactions via ABCB1 and ABCG2 efflux transporters remains elusive. The purpose of this study was to investigate the impact of E. purpurea-derived caffeic acid derivatives (cichoric acid and echinacoside) and tetraenes on the mRNA and protein expression levels as well as on transport activity of ABCB1 and ABCG2 in intestinal (Caco-2) and liver (HepG2) cell line models. The safety of these compounds was investigated by estimating EC20 values of cell viability assays in both cell lines. Regulation of ABCB1 and ABCG2 protein in these cell lines were analyzed after 24 h exposure to the compounds at 1, 10, and 50 µg/mL. Bidirectional transport of 0.5 µg/mL Hoechst 33342 and 5 µM rhodamine across Caco-2 monolayer and profiling for intracellular concentrations of the fluorophores in both cell lines were conducted to ascertain inhibition effects of the compounds. Cichoric acid showed no cytotoxic effect, while the EC20 values of tetraenes and echinacoside were 45.0 ± 3.0 and 52.0 ± 4.0 µg/mL in Caco-2 cells and 28.0 ± 4.3 and 62.0 ± 9.9 µg/mL in HepG2 cells, respectively. In general, the compounds showed heterogeneous induction of ABCB1 with the strongest 3.6 ± 1.2-fold increase observed for 10 µg/mL tetraenes in Caco-2 cells (p < 0.001). However, the compounds did not induce ABCG2. None of the phytocompounds inhibited significantly net flux of the fluorophores across Caco-2 monolayers. Overall, tetraenes moderately induced ABCB1 but not ABCG2 in Caco-2 and HepG2 cells while no compound significantly inhibited activity of these transporters at clinically relevant concentration to cause herb-drug interactions.
Assuntos
Ácidos Cafeicos/farmacologia , Echinacea/química , Glicosídeos/farmacologia , Interações Ervas-Drogas , Succinatos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/agonistas , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/agonistas , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Células Hep G2 , Eliminação Hepatobiliar , Humanos , Eliminação Intestinal , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismoRESUMO
Multidrug resistance (MDR) is the main cause of diminished success in cancer chemotherapy. ABC transport proteins are considered to be one important factor of MDR. Besides P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance Protein (BCRP, ABCG2), Multidrug Resistance-associated Protein 1 (MRP1, ABCC1) is associated with non-response to chemotherapy in different cancers. While considerable effort was spent in overcoming MDR during the last two decades, almost nothing is known with respect to activators of transport proteins. In this work we present certain pyrrolo[3,2-d]pyrimidine derivatives with variations at positions 4 and 5 and purine analogs with variations at position 6 as novel activators of MRP1-mediated transport of the MRP1 substrate calcein AM and the anticancer drug daunorubicin in low nanomolar concentration range. Two different MRP1 overexpressing cell lines were used, the doxorubicin-selected human lung cancer cell line H69 AR and the transfected Madin-Darby Canine Kidney cell line MDCK II MRP1. No effect was observed in the sensitive counterparts H69 and MDCK II wild type (wt). Derivatives with higher molecular weight possessed also inhibitory properties at low micromolar concentrations, although most compounds were rather poor MRP1 inhibitors. Purine analogs derived from potent MRP1 inhibitors of the pyrrolopyrimidine class showed equal activating, but no inhibiting effects at all. All tested compounds were non-toxic and had only minor impact on P-gp or BCRP, showing no inhibition or activation.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética , Purinas/síntese química , Pirimidinas/síntese química , Pirróis/síntese química , Subfamília B de Transportador de Cassetes de Ligação de ATP/agonistas , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/agonistas , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Daunorrubicina/farmacologia , Cães , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Expressão Gênica , Humanos , Cinética , Células Madin Darby de Rim Canino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/agonistas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Purinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Relação Estrutura-AtividadeRESUMO
Increasing evidence shows that the intestinal tract plays an important role in maintaining urate homeostasis and might be a potential therapeutic target for hyperuricaemia. However, uric acid-lowering drugs available in the clinic do not target intestinal excretion as a therapeutic strategy. We previously reported that mangiferin had potent hypouricaemic effects in hyperuricaemic animals. However, the underlying mechanisms are not completely clear. Here, we investigated the effects of mangiferin on the intestinal excretion of urate and its underlying mechanisms. The data revealed that mangiferin concentration-dependently promoted the intestinal secretion of endogenous urate in in situ intestinal closed loops in normal and hyperuricaemic mice, as well as inhibited the absorption of exogenous uric acid perfused into the intestinal loops in rats. Administration of mangiferin not only decreased the serum urate levels in the hyperuricaemic mice but also increased the protein expression of ATP-binding cassette transporter, subfamily G, member 2 (ABCG2) and inhibited the protein expression of glucose transporter 9 (GLUT 9) in the intestine. These findings suggested that intestinal ABCG2 and GLUT9 might be pivotal and possible action sites for the observed hypouricaemic effects. Moreover, no significant changes in intestinal xanthine oxidoreductase activities were observed, suggesting that mangiferin did not affect intestinal uric acid generation in the hyperuricaemic mice. Overall, promoting intestinal elimination of urate by upregulating ABCG2 expression and downregulating GLUT9 expression might be an important mechanism underlying mangiferin lowering serum uric acid levels. Mangiferin supplementation might be beneficial for the prevention and treatment of hyperuricaemia.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/biossíntese , Eliminação Intestinal/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/biossíntese , Ácido Úrico/metabolismo , Xantonas/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/agonistas , Animais , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Eliminação Intestinal/fisiologia , Masculino , Camundongos , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Xantonas/uso terapêuticoRESUMO
In bovine mammary glands, the ABCG2 transporter actively secretes xenobiotics into dairy milk. This can have significant implications when cattle are exposed to pesticide residues in feed. Recent studies indicate that the fungicide prochloraz activates the aryl hydrocarbon receptor (AhR) pathway, increasing bovine ABCG2 (bABCG2) gene expression and efflux activity. This could enhance the accumulation of bABCG2 substrates in dairy milk, impacting pesticide risk assessment. We therefore investigated whether 13 commonly used pesticides in Europe are inducers of AhR and bABCG2 activity. MDCKII cells expressing mammary bABCG2 were incubated with pesticides for up to 72 h. To reflect an in vivo situation, applied pesticide concentrations corresponded to the maximum residue levels (MRLs) permitted in bovine fat or muscle. AhR activation was ascertained through CYP1A mRNA expression and enzyme activity, measured by qPCR and 7-ethoxyresorufin-Ο-deethylase (EROD) assay, respectively. Pesticide-mediated increase of bABCG2 efflux activity was assessed using the Hoechst 33342 accumulation assay. For all assays, the known AhR-activating pesticide prochloraz served as a positive control, while the non-activating tolclofos-methyl provided the negative control. At 10-fold MRL concentrations, chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron, and tebuconazole significantly increased CYP1A1 mRNA levels, CYP1A activity, and bABCG2 efflux activity compared to the vehicle control. In contrast, dimethoate, dimethomorph, glyphosate, iprodione, methiocarb and thiacloprid had no impact on AhR-mediated CYP1A1 mRNA levels, CYP1A activity or bABCG2 efflux. In conclusion, the MDCKII-bABCG2 cell model proved an appropriate tool for identifying AhR- and bABCG2-inducing pesticides. This provides an in vitro approach that could reduce the number of animals required in pesticide approval studies.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Alternativas aos Testes com Animais/métodos , Fungicidas Industriais/toxicidade , Receptores de Hidrocarboneto Arílico/agonistas , Testes de Toxicidade Crônica/métodos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/agonistas , Animais , Bovinos , Cães , Alemanha , Lactação/efeitos dos fármacos , Células Madin Darby de Rim Canino , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Overexpression of breast cancer resistance protein (BCRP) has been shown to produce multidrug resistance (MDR) in colon cancer, leading to major obstacles for chemotherapy. In this study, we evaluated the effect of regorafenib, an oral multi-kinase inhibitor, in inhibiting BCRP-mediated MDR in silico, in vitro and in vivo. We found that regorafenib significantly sensitized MDR colon cancer cells to BCRP substrates by increasing their intracellular accumulation. There are no significant changes in the expression level or the subcellular distribution of BCRP in the cells exposed to regorafenib. Investigation of the mechanism revealed that regorafenib stimulated BCRP ATPase activity. Our induced-fit docking and molecular dynamics simulations suggested the existence of a strong and stable interaction between regorafenib and the transmembrane domain of human crystalized BCRP. In vivo tumor xenograft study revealed that the combination of regorafenib and topotecan exhibited synergistic effects on mitoxantrone-resistant S1-M1-80 xenograft tumors. In conclusion, our studies indicate that regorafenib would be beneficial in combating MDR in colon cancer.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/agonistas , Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/agonistas , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Masculino , Camundongos Nus , Mitoxantrona/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Compostos de Fenilureia/química , Ligação Proteica , Conformação Proteica , Piridinas/química , Topotecan/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ATP binding cassette (ABC) transporters can transfer a variety of antiviral agents from the cytoplasm to body fluid, which results in a reduced intracellular concentration of the drugs. Proteins of HIV-1, e.g., Tat and gp120, altered some types of ABC transporter expression in brain microvascular endothelial cells and astrocytes. However, the effect of Tat on ABC transporters in T lymphocytes is unclear. In this study the status of breast cancer resistance protein (BCRP) in Tat expressing cell lines was examined with real-time PCR and flow cytometry. It was found that HIV-1 Tat protein upregulated BCRP expression and enhanced efflux mediated by BCRP significantly, which could inhibit antiviral drugs from entering infected cells and interfere with the therapeutic effect of HAART.