RESUMO
OBJECTIVES@#Hypoxia can alter the oral bioavailability of drugs, including various substrates (drugs) of P-glycoprotein (P-gp), suggesting that hypoxia may affect the function of P-gp in intestinal epithelial cells. Currently, Caco-2 monolayer model is the classic model for studying the function of intestinal epithelial P-gp. This study combines the Caco-2 monolayer model with hypoxia to investigate the effects of hypoxia on the expression and function of P-gp in Caco-2 cells, which helps to elucidate the mechanism of changes in drug transport on intestinal epithelial cells in high-altitude hypoxia environment.@*METHODS@#Normally cultured Caco-2 cells were cultured in 1% oxygen concentration for 24, 48, and 72 h, respectively. After the extraction of the membrane proteins, the levels of P-gp were measured by Western blotting. The hypoxia time, with the most significant change of P-gp expression, was selected as the subsequent study condition. After culturing Caco-2 cells in transwell cells for 21 days and establishing a Caco-2 monolayer model, they were divided into a normoxic control group and a hypoxic group. The normoxic control group was continuously cultured in normal condition for 72 h, while the hypoxic group was incubated for 72 h in 1% oxygen concentration. The integrity and polarability of Caco-2 cells monolayer were evaluated by transepithelial electrical resistance (TEER), apparent permeability (Papp) of lucifer yellow, the activity of alkaline phosphatase (AKP), and microvilli morphology and tight junction structure under transmission electron microscope. Then, the Papp of rhodamine 123 (Rh123), a kind of P-gp specific substrate, was detected and the efflux rate was calculated. The Caco-2 cell monolayer, culturing at plastic flasks, was incubated for 72 h in 1% oxygen concentration, the expression level of P-gp was detected.@*RESULTS@#P-gp was decreased in Caco-2 cells with 1% oxygen concentration, especially the duration of 72 h (P<0.01). In hypoxic group, the TEER of monolayer was more than 400 Ω·cm2, the Papp of lucifer yellow was less than 5×10-7 cm/s, and the ratio of AKP activity between apical side and basal side was greater than 3. The establishment of Caco-2 monolayer model was successful, and hypoxia treatment did not affect the integrity and polarization state of the model. Compared with the normoxic control group, the efflux rate of Rh123 was significantly reduced in Caco-2 cell monolayer of the hypoxic group (P<0.01). Hypoxia reduced the expression of P-gp in Caco-2 cell monolayer (P<0.01).@*CONCLUSIONS@#Hypoxia inhibits P-gp function in Caco-2 cells, which may be related to the decreased P-gp level.
Assuntos
Humanos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Células CACO-2 , Subfamília B de Transportador de Cassetes de Ligação de ATP , Hipóxia , OxigênioRESUMO
Abstract Paclitaxel (PTX) is one of the most effective drugs used in the treatment of breast cancer. Nonetheless, the appearance of MDR1 (multidrug resistance 1) in tumor cells has become a significant hindrance for efficacious chemotherapy. In this study, we show that the expression level of Egr-1 (early growth response gene-1) in cancer tissues (from paclitaxel chemotherapy failure patients) and MCF-7/PTX cells (the breast cancer cell line that was resistant to paclitaxel) was increased. Cell proliferation assay and apoptosis assay revealed that Egr-1 could promote cell growth and inhibit apoptosis in MCF-7/PTX. Mechanistic studies indicated that Egr-1 could bind to the proximal MDR1 promoter and enhance MDR1 transcription. These findings indicate that paclitaxel induced Egr-1 accumulation and upregulated the expression of MDR1, thereby inducing the drug resistance in MCF-7/PTX. Our results suggest a novel pathway by which paclitaxel induces MDR1 expression, possibly illuminating a potential target pathway for the prevention of MDR1-mediated drug resistance.
Assuntos
Neoplasias da Mama/patologia , Resistência a Medicamentos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Preparações Farmacêuticas/análiseRESUMO
Abstract The aim of the present study was to investigate the usefulness of multidrug resistance protein 1 (MDR1) and neuropeptide Y (NPY) levels in predicting the efficacy of levetiracetam (LEV) plus oxcarbazepine (OXC) treatment administered to children with epilepsy and to determine their prognosis. Overall, 193 children with epilepsy admitted to the hospital were enrolled and randomly divided into two groups according to different treatment methods: group A (n = 106, treated with LEV plus OXC combination) and group B (n = 87, treated with OXC only). After treatment, compared with group B, group A exhibited a remarkably higher total effective rate and a significantly lower total adverse reaction rate. Areas under the curve for MDR1 and NPY for predicting ineffective treatment were 0.867 and 0.834, whereas those for predicting epilepsy recurrence were 0.916 and 0.829, respectively. Electroencephalography abnormalities, intracranial hemorrhage, neonatal convulsion, premature delivery, and MDR1 and NPY levels were independent risk factors for poor prognosis in children with epilepsy. Serum MDR1 and NPY levels exhibited a high predictive value for early epilepsy diagnosis, treatment efficacy assessment, and prognostication in children with epilepsy treated with LEV plus OXC combination.
Assuntos
Humanos , Masculino , Feminino , Neuropeptídeo Y/análise , Criança , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Epilepsia/patologia , Levetiracetam/antagonistas & inibidores , Oxcarbazepina/antagonistas & inibidores , Eficácia , Eletroencefalografia/métodosRESUMO
OBJECTIVES@#Nephrotic syndrome is a common disease of the urinary system. The aim of this study is to explore the effect of astragalus polysaccharides (APS) on multidrug resistance gene 1 (MDR1) and P-glycoprotein 170 (P-gp170) in adriamycin nephropathy rats and the underlying mechanisms.@*METHODS@#A total of 72 male Wistar rats were divided into a control group, a model group, an APS low-dose group, an APS high-dose group, an APS+micro RNA (miR)-16 antagomir group and an APS+miR-16 antagomir control group, with 12 rats in each group. Urine protein (UP) was detected by urine analyzer, and serum cholesterol (CHOL), albumin (ALB), blood urea nitrogen (BUN), and creatinine (SCr) were detected by automatic biochemical analyzer; serum interleukin-6 (IL-6), IL-1β, tumor necrosis factor α (TNF-α) levels were detected by ELISA kit; the morphological changes of kidney tissues were observed by HE staining; the levels of miR-16 and MDR1 mRNA in kidney tissues were detected by real-time RT-PCR; the expression levels of NF-κB p65, p-NF-κB p65, and P-gp170 protein in kidney tissues were detected by Western blotting; and dual luciferase was used to verify the relationship between miR-16 and NF-κB.@*RESULTS@#The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration. The renal glomeruli of rats in the model group were mildly congested, capillary stenosis or occlusion, and inflammatory cell infiltration was obvious. The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion, the proliferation of mesangial cells was significantly reduced, and the inflammatory cells were reduced. Compared with the high-dose APS group and the APS+miR-16 antagomir control group, there were more severe renal tissue structure damages in the APS + miR-16 antagomir group. Compared with the control group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased (all P<0.05); the levels of ALB and miR-16 were significantly decreased (both P<0.05). Compared with the model group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of pNF-κB p65 and P-gp170 in the low-dose and high-dose APS groups were significant decreased (all P<0.05); and the levels of ALB and miR-16 were significantly increased (both P<0.05). Compared with APS+miR-16 antagomir control group, the UP, CHOL, BUN, SCr, IL-6, IL-1β, and TNF-α levels, MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 were significantly increased (all P<0.05). The levels of ALB and miR-16 were significantly decreased in the APS+miR-16 antagomir group compared with the APS+miR-16 antagomir control group (both P<0.05).@*CONCLUSIONS@#APS can regulate the miR-16/NF-κB signaling pathway, thereby affecting the levels of MDR1 and P-gp170, and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.
Assuntos
Animais , Masculino , Ratos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antagomirs , Doxorrubicina/toxicidade , Genes MDR , Interleucina-6/metabolismo , Nefropatias/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Polissacarídeos/farmacologia , RNA Mensageiro , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Currently, chemoresistance seriously attenuates the curative outcome of liver cancer. The purpose of our work was to investigate the influence of 6-shogaol on the inhibition of 5-fluorouracil (5-FU) in liver cancer. The cell viability of cancer cells was determined by MTT assay. Liver cancer cell apoptosis and the cell cycle were examined utilizing flow cytometry. Moreover, qRT-PCR and western blotting was used to analyse the mRNA and protein expression levels, respectively. Immunohistochemistry assays were used to examine multidrug resistance protein 1 (MRP1) expression in tumour tissues. In liver cancer cells, we found that 6-shogaol-5-FU combination treatment inhibited cell viability, facilitated G0/G1 cell cycle arrest, and accelerated apoptosis compared with 6-shogaol or 5-FU treatment alone. In cancer cells cotreated with 6-shogaol and 5-FU, AKT/mTOR pathway- and cell cycle-related protein expression levels were inhibited, and MRP1 expression was downregulated. AKT activation or MRP1 increase reversed the influence of combination treatment on liver cancer cell viability, apoptosis and cell cycle arrest. The inhibition of AKT activation to the anticancer effect of 6-shogaol-5-FU could be reversed by MRP1 silencing. Moreover, our results showed that 6-shogaol-5-FU combination treatment notably inhibited tumour growth in vivo. In summary, our data demonstrated that 6-shogaol contributed to the curative outcome of 5-FU in liver cancer by inhibiting the AKT/mTOR/MRP1 signalling pathway.
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Humanos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Apoptose , Catecóis , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Neoplasias Hepáticas/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVES@#Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer. But the problem of drug resistance severely limits their clinical application. This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil (5-FU) and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition.@*METHODS@#Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment, and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group. The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression level of hypoxia inducible factor-1α (HIF-1α), multidrug resistance (MDR1) gene, P-glycoprotein (P-gp), and vascular endothelial growth factor (VEGF) which were related to 5-FU drug-resistance. We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU.@*RESULTS@#Compared with the Normoxic+5-FU group, the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced (P<0.05), and the expression of apoptosis promoter protein caspase 8 was also decreased. Compared with the the Normoxic+5-FU group, HIF-1α mRNA expression in the Hypoxia+5-FU group was significantly increased (P<0.05), and the mRNA and protein expression levels of MDR1, P-gp and VEGF were also significantly increased (all P<0.05). The increased expression of MDR1, P-gp and VEGF had the same trend with the expression of HIF-1α.@*CONCLUSIONS@#Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy. Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.
Assuntos
Humanos , Fluoruracila/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Resistência a Múltiplos Medicamentos , Fatores de Crescimento do Endotélio Vascular/metabolismo , Hipóxia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Hipóxia Celular , RNA Mensageiro/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Microambiente TumoralRESUMO
Abstract Drug resistance is a crucial obstacle to achieve satisfactory chemotherapeutic effects. Numerous studies have shown that the PI3K/Akt signaling pathway plays a significant role in various processes of cellular events and tumor progression, while few studies have focused on the PI3K/Akt signaling pathway in drug resistance of endothelial cells. The present study aims to explore the relationship of PI3K/Akt signaling and cellular resistance to anticancer drugs in human microvessel endothelial cells (HMEC-1). We established stable sunitinib-resiatant human microvessel endothelial cells (HMEC-su) after long-term exposure to sunitinib (a small-molecule tyrosine kinase receptor inhibitor) for 12 months. HMEC-su showed significant alternations of cell morphology and exhibited a 2.32-fold higher IC50 of sunitinib than parental HMEC-1 cells. Expression of P-glycoprotein (P-gp) and breast cancer-resistance protein (ABCG2) which mediates drug efflux, increased significantly in HMEC-su lines compared with HMEC-1 cells by western blots assay. Our study further demonstrates that LY294002 (blocking the PI3K/Akt pathway) enhances the sensibility of HMEC-su to suntinib and inhibits the gene transcription and protein expression of P-gp, ABCG2 in HMEC-su cells. In conclusion, these results indicate that LY294002 could reverse P-gp and ABCG2 mediated-drug resistance to sunitinib in HMEC-su cells by inhibiting PI3K/Akt signaling.
Assuntos
Resistência a Medicamentos , Células Endoteliais/classificação , Preparações Farmacêuticas/administração & dosagem , Western Blotting/instrumentação , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos adversos , Concentração Inibidora 50 , Células Endoteliais/patologia , Sunitinibe/agonistasRESUMO
Os parâmetros de permeabilidade e solubilidade são fundamentais à absorção oral de fármacos e a partir dessas características, foi criado o Sistema de Classificação Biofarmacêutica, através do qual os fármacos são divididos em quatro classes. Atualmente, para a determinação da solubilidade de um fármaco, existem diversos métodos padronizados por agências regulatórias, no entanto, para a determinação da permeabilidade, os ensaios são passíveis de diversas variações em sua execução, diminuindo a confiabilidade dos resultados obtidos e impossibilitando a comparação dos mesmos quando realizados com técnicas diferentes umas das outras. O objetivo do presente trabalho é avaliar as variáveis experimentais do modelo do saco intestinal que podem influenciar nos resultados de permeabilidade aparente de fármacos e na viabilidade do tecido. O presente estudo foi aprovado pelo Comitê de Ética no Uso de Animais da FCF-USP (109.2018-P574). Foram utilizados 33 Rattus norvegicus da linhagem Wistar, machos, jovens adultos, com peso entre 200 g e 300 g. Para realização do procedimento, cada animal permaneceu em jejum por cerca de quatro horas e após adequada anestesia a porção do jejuno do intestino delgado foi retirada e dividida em seis segmentos de aproximadamente 8,5cm cada. Foram realizados experimentos com e sem inversão do saco intestinal, submetidos a diferentes tempos de banho de gelo após sua ressecção, na presença ou ausência de inibidor da glicoproteína-P (verapamil). Os fármacos naproxeno e famotidina foram empregados como marcadores de alta e baixa permeabilidade, respectivamente. A losartana foi utilizada como substrato da glicoproteína P. Cada um dos sacos intestinais foi colocado em um tubo de ensaio contendo tampão Krebs, a 37°C, saturado com gás carbogênio. Para avaliação da integridade e viabilidade dos segmentos intestinais, observou-se a presença de movimentos peristálticos e coletaram-se amostras do meio de incubação nos tempos 0, 30, 45, 60, 90 e 120 minutos para quantificação dos fármacos e de glicose, uma vez que esta é ativamente transportada para a serosa do intestino delgado. Determinou-se a permeabilidade aparente de cada fármaco e as concentrações de glicose nas diferentes condições experimentais, realizou-se planejamento fatorial multinível e os resultados foram analisados por análise variância (ANOVA), seguida de pós-teste de Tukey. Observou-se que as variáveis experimentais interferiram de forma significativa na viabilidade tecidual e na permeabilidade aparente dos fármacos. Não foram observadas diferenças significativas da permeabilidade de fármacos nos diferentes segmentos do jejuno. A glicose mostrou-se um bom marcador de viabilidade tecidual e foi constatado que a presença ou ausência de movimentos peristálticos não está relacionada diretamente com a viabilidade do tecido. Uma vez que foram constatadas tantas interferências nos resultados, é imprescindível que os procedimentos experimentais sejam padronizados, para que os resultados apresentem menor variabilidade e possam ser comparados entre si
The permeability and solubility parameters are fundamental to the oral absorption of drugs and from these characteristics, the Biopharmaceutical Classification System was created, through which drugs are divided into four classes. Currently, for the determination of the solubility of a drug, there are several methods standardized by regulatory agencies, however, for the determination of permeability, the tests are subject to several variations in their execution, reducing the reliability of the results obtained and making it impossible to compare the results obtained. same when performed with different techniques. The aim of this study is to evaluate if different experimental conditions can influence the results of apparent drug permeability and tissue viability on gut sac model. The present study was approved by the Ethics Committee for the Use of Animals of FCF-USP (109.2018-P574). Thirty-three male, young adult Rattus norvegicus were used, weighing between 200 g and 300 g. To perform the procedure, each animal fasted for about four hours and after adequate anesthesia, the portion of the jejunum of the small intestine was removed and divided into six segments of approximately 8.5 cm each. Experiments were performed with and without inversion of the gut sac, submitted to different times of ice bath after its resection, in the presence or absence of a P-glycoprotein inhibitor (verapamil). The drugs naproxen and famotidine were used as markers of high and low permeability, respectively. Losartan was used as a substrate for P-glycoprotein. Each of the gut sacs was placed in a test tube containing Krebs buffer, at 37°C, saturated with carbogen gas. To evaluate the integrity and viability of the intestinal segments, the presence of peristaltic movements was observed and samples of the incubation medium were collected at 0, 30, 45, 60, 90 and 120 minutes for quantification of drugs and glucose, as it is actively transported to the serosa of the small intestine. The apparent permeability of each drug and the glucose concentrations were determined under different experimental conditions, multilevel factorial design was performed and the results were analyzed by analysis of variance (ANOVA), followed by Tukey's post-test. It was observed that the experimental variables significantly interfered in the tissue viability and in the apparent permeability of the drugs. No significant differences in drug permeability were observed in the different segments of the jejunum. Glucose proved to be a good marker of tissue viability and it was found that the presence or absence of peristaltic movements is not directly related to tissue viability. Since so many interferences were found in the results, it is essential that the experimental procedures be standardized, so that the results show less variability and can be compared between different authors
Assuntos
Animais , Masculino , Ratos , Permeabilidade , Solubilidade , Biofarmácia/instrumentação , Preparações Farmacêuticas/análise , Intestino Delgado/metabolismo , Métodos , Padrões de Referência , Análise de Variância , Jejum/efeitos adversos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos adversos , Absorção , Jejuno/anormalidadesRESUMO
OBJECTIVE@#To investigate the effect of ursane triterpenoids 3β,19α-dihydroxyursu-12-ene-23,28-dicarboxylic acid (Rotundioic acid, RA) on the sensitivity of adriamycin-resistant K562 cells (K562/ADM Cell) anti-tumor drug, and to explore the effect and mechanism of RA on the multidrug resistance of K562/ADM cells.@*METHODS@#CCK-8 method was used to detect the effect of RA on the sensitivity of K562 cells and K562/ADM cells to anti-tumor drug. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression level of mRNA and the protein in K562 and K562/ADM cells, and the effect of RA on the expression of MDR1 mRNA and P-gp in K562/ADM cells was also detected; Western blot was used to detect the expression of p-JNK, p-p38 and p-ERK1/2 in K562/ADM cells.@*RESULTS@#RA could increased the sensitivity of K562/ADM cells to adriamycin(the reversal factor was 1.61 times), the difference showed statistically significantly (P<0.05); the resistance factor of K562/ADM to ADM was 41.76 times. The expression of MDR1 mRNA in K562 cells was extremely low, and the protein product P-glycoprotein (P-gp) was almost not expressed; MDR1 mRNA and P-gp in K562/ADM cells were highly expressed; RA could down-regulate the expression levels of MDR1 and P-gp in K562/ADM cells. In addition, RA could upregulate the phosphorylation levels of p38 and ERK1/2 in K562/ADM cells, but it has no effect on the expression of p-JNK.@*CONCLUSION@#RA may participate in the regulation of MAPK signaling pathway by upregulating the expression levels of p-p38 and p-ERK1/2 in K562/ADM cells, and thus inhibit the transcription and translation levels of MDR1, and finally reverse the multidrug resistance of leukemia cells.
Assuntos
Humanos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Células K562RESUMO
OBJECTIVES@#To investigate the effect of adriamycin (ADM), idelalisib or ADM and their combination on cell proliferation and intracellular concentration of ADM, and to explore the reversal effect of idelalisib on drug resistance to ADM.@*METHODS@#The K562 and K562/ADM cells were respectively treated with ADM and idelalisib at different concentrations. The 50% inhibitory concentration (IC@*RESULTS@#The cell survival rates were significantly decreased in a dose-dependent manner when the cells were treated with different doses of ADM (0.001-10.000 mg/L ). The IC@*CONCLUSIONS@#Idelalisib exerts effect on inhibition of the proliferation in myeloid leukemia K562 and K562/ADM cells, which may partially reverse the drug resistance of K562/ADM cells to ADM. The mechanisms for the effect of idelalisib may be related to increasing the accumulation of ADM and inducing the cell apoptosis in the K562 and K562/ADM cells.
Assuntos
Humanos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Proliferação de Células , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Células K562 , Leucemia Mieloide , Purinas , QuinazolinonasRESUMO
Diabetes mellitus (DM) has a high prevalence in patients with pancreatic cancer (PaC), but the prognostic value of DM in PaC remains controversial. Alterations of P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4) contribute to multidrug resistance and intestinal metabolism in a variety of cancer types, which may be implicated in DM development. This study aimed to explore the potential prognostic value of P-gp and CYP3A4 in PaC patients in the context of DM through long-term follow-up. We retrospectively reviewed the medical records of patients with PaC admitted at The First People's Hospital of Changzhou, Jiangsu, China, from January 2011 to November 2019 and identified two cohorts of adult patients with PaC, including 24 with DM and 24 without DM (non-DM). The baseline clinical characteristics and outcomes were compared. Immunohistochemistry showed that protein expression of P-gp, but not CYP3A, in duodenum tissues was significantly upregulated in PaC patients with DM compared with those without DM. Kaplan-Meier analysis and log-rank test showed that the survival of patients with PaC and DM/high expression of P-gp was not significantly reduced compared with that of patients without DM/low expression of P-gp. These findings suggested that P-gp expression levels were different in the DM and non-DM groups of patients with PaC, but DM and duodenal P-gp levels were not associated with the long-term survival of patients with PaC. It appears that the presence of DM or P-gp expression levels may not serve as effective prognostic markers for PaC.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Neoplasias Pancreáticas , Diabetes Mellitus , China/epidemiologia , Estudos Retrospectivos , Seguimentos , Membro 1 da Subfamília B de Cassetes de Ligação de ATPRESUMO
Clitoria ternateaL. is a vital ayurvedic herbfeatured with a wide spectrum of mental health benefits. The present study investigates the competence of the plant against depression and to inhibit the membrane efflux protein P-glycoprotein (P-gp) that can regulate and restrict drug permeation into the brain. Antidepressant competence of the aqueous plant extract was assessed by animal despair studies on depression induced female mice models. The P-glycoprotein inhibitory potential of active phytocompounds was evaluated by molecular docking assay and substantiated by a cell line study. The in vivostudies exhibited a significant difference in the immobility time thereby establishing antidepressant activity. The histopathological sections from cortex region of treated brain showed decreased degenerative changes. Ten imperative phytocompounds facilitated docking complexes against P-glycoprotein among which Kaempferol 3-O-(2״,6״-di-O-rhamnopyranosyl) glucopyranoside exhibited a finest docking score of -12.569 kcal mol-1. Conversely it was attested by the rhodamine transport assay that demonstrated the inhibitory potential to surpass blood brain barrier. The outcome of the study endorses the efficacy of Clitoria ternateaL. as an idyllic brain drug and facilitates brain permeability.
Assuntos
Antidepressivos , Ayurveda , Barreira Hematoencefálica , Biotecnologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análiseRESUMO
PURPOSE: The DNA damage response (DDR) is a multi-complex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints, and apoptosis. In the case of biliary tract cancer (BTC), the strategy of DDR targeting has not been evaluated, even though many patients have DNA repair pathway alterations. The purpose of this study was to test the DDR-targeting strategy in BTC using an ataxia-telangiectasia and Rad3-related (ATR) inhibitor. MATERIALS AND METHODS: A total of nine human BTC cell lines were used for evaluating anti-tumor effect of AZD6738 (ATR inhibitor) alone or combination with cytotoxic chemotherapeutic agents through MTT assay, colony-forming assays, cell cycle analyses, and comet assays. We established SNU478-mouse model for in vivo experiments to confirm our findings. RESULTS: Among nine human BTC cell lines, SNU478 and SNU869 were the most sensitive to AZD6738, and showed low expression of both ataxia-telangiectasia mutated (ATM) and p53. AZD6738 blocked p-Chk1 and p-glycoprotein and increased γH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis, G2/M arrest and p21, and decreased CDC2. Combinations of AZD6738 and cytotoxic chemotherapeutic agents exerted synergistic effects in colony-forming assays, cell cycle analyses, and comet assays. In our mouse models, AZD6738 monotherapy decreased tumor growth and the combination with cisplatin showed more potent effects on growth inhibition, decreased Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling than monotherapy with each drug. CONCLUSION: In BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC.
Assuntos
Animais , Humanos , Camundongos , Apoptose , Ataxia Telangiectasia , Neoplasias do Sistema Biliar , Sistema Biliar , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Cisplatino , Ensaio Cometa , Dano ao DNA , Reparo do DNA , DNA , Membro 1 da Subfamília B de Cassetes de Ligação de ATPRESUMO
Canine MDR1 gene mutations produce translated P-glycoprotein, an active drug efflux transporter, resulting in dysfunction or over-expression. The 4-base deletion at exon 4 of MDR1 at nucleotide position 230 (nt230[del4]) in exon 4 makes P-glycoprotein lose function, leading to drug accumulation and toxicity. The G allele of the c.-6-180T>G variation in intron 1 of MDR1 (single nucleotide polymorphism [SNP] 180) causes P-glycoprotein over-expression, making epileptic dogs resistant to phenobarbital treatment. Both of these mutations are reported to be common in collies. This study develops a more efficient method to detect these two mutations simultaneously, and clarifies the genotype association with the side effects of chemotherapy. Genotype distribution in Taiwan was also investigated. An oligonucleotide microarray was successfully developed for the detection of both genotypes and was applied to clinical samples. No 4-base deletion mutant allele was detected in dogs in Taiwan. However, the G allele variation of SNP 180 was spread across all dog breeds, not only in collies. The chemotherapy adverse effect percentages of the SNP 180 T/T, T/G, and G/G genotypes were 16.7%, 6.3%, and 0%, respectively. This study describes an efficient way for MDR1 gene mutation detection, clarifying genotype distribution, and the association with chemotherapy.
Assuntos
Animais , Cães , Alelos , Tratamento Farmacológico , Éxons , Genótipo , Íntrons , Métodos , Análise de Sequência com Séries de Oligonucleotídeos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Fenobarbital , TaiwanRESUMO
BACKGROUND: P-glycoprotein (P-gp) transports many chemicals that vary greatly in their structure and function. It is normally expressed in renal proximal tubular cells. We hypothesized that P-gp expression influences light chain excretion. Therefore, we investigated whether renal tubular P-gp expression is altered in patients with plasma cell disorders. METHODS: We evaluated renal biopsy specimens from patients with plasma cell disorders (n = 16) and primary focal segmental glomerulosclerosis (the control group, n = 17). Biopsies were stained with an anti-P-gp antibody. Loss of P-gp expression was determined semi-quantitatively. Groups were compared for loss of P-gp expression, and clinical variables. RESULTS: P-gp expression loss was more severe in patients with plasma cell disorders than it was in those with glomerulonephritis (P = 0.021). In contrast, clinical and histological parameters including serum creatinine, level of urinary protein excretion, and interstitial fibrosis/tubular atrophy grade were not significantly different between the groups. P-gp expression loss increased with age in patients with plasma cell disorders (P = 0.071). This expression loss was not associated with serum creatinine, the level of urinary protein excretion or the interstitial fibrosis/tubular atrophy grade. There was no significant association between the severity of P-gp expression loss with the types and serum levels of light chains, isotypes and serum immunoglobulin levels. CONCLUSION: Renal tubular P-gp expression is significantly down-regulated in patients with plasma cell disorders characterized by nephrotic range proteinuria. Additional studies are needed to determine whether reintroduction of renal tubular P-gp expression would mitigate the proximal tubular injury that is caused by free-light chains.
Assuntos
Humanos , Amiloidose , Atrofia , Biópsia , Creatinina , Glomerulonefrite , Glomerulosclerose Segmentar e Focal , Cadeias Leves de Imunoglobulina , Imunoglobulinas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Plasmócitos , Plasma , ProteinúriaRESUMO
ABSTRACT BACKGROUND: Right ventricular (RV) dysfunction may develop over the course of chronic obstructive pulmonary disease (COPD) and is an important predictor of morbidity and mortality. Polymorphism of the multidrug resistance-1 (MDR-1) gene has been correlated with worse clinical findings among patients with COPD. Our aim here was to investigate the relationship between MDR-1 C3435T gene polymorphism and RV dysfunction in COPD patients. DESIGN AND SETTING: This was a cross-sectional study investigating the relationship between RV dysfunction and genetic defects in COPD patients. METHODS: Forty-one consecutive patients diagnosed with COPD and hospitalized due to acute exacerbation were enrolled. Polymorphism was analyzed using the strip assay technique. RV parameters were evaluated, and RV dysfunction was identified via transthoracic echocardiography. Patients were categorized into three groups according to gene polymorphism: MDR-1 CC (wild type, n = 9), MDR-1 CT (heterozygote mutant, n = 21) or MDR-1 TT (homozygote mutant, n = 11). RESULTS: The study included 14 males and 27 females (mean age 65 ± 11 years). The mean systolic pulmonary artery pressure was 31.4 ± 8 mmHg in the wild-type group, 42.2 ± 12 mmHg in the heterozygote mutant group and 46.5±14 mmHg in the homozygote mutant group (P = 0.027). Presence of RV dilatation was significantly different among the three groups (33%, 71%, and 100%, respectively; P = 0.005). In multiple logistic regression analysis, MDR-1 C3435T gene polymorphism (OR = 9.000, P = 0.019) was an independent predictor of RV dysfunction after adjustment for potential confounders. CONCLUSION: MDR-1 C3435T gene polymorphism was associated with RV dysfunction in patients with COPD.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Disfunção Ventricular Direita/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Doença Pulmonar Obstrutiva Crônica/complicações , Ecocardiografia , Estudos Transversais , Disfunção Ventricular Direita/complicações , Subfamília B de Transportador de Cassetes de Ligação de ATP/genéticaRESUMO
<p><b>OBJECTIVE</b>To establish the adriamycin(ADR)-resistant ALL cell lines and to investigate their drug-resistan mechanisms.</p><p><b>METHODS</b>The drug-resistant cell lines SUP-B15/ADR and RS4;11/ADR were derived by exposing the parental cells [SUP-B15(Ph) and RS4;11(Ph)] to the ascending concentrations of ADR. The cell viability was detected by CCK-8 method. The expression of P-gp was examined by Western blot, and RT-qPCR was performed to detect the expression of MDR1.</p><p><b>RESULTS</b>The drug-resistant cell lines SUP-B15/ADR and RS4;11/ADR were successfully established, their resistance indexes were 14.088±0.763 and 10.473±1.024, respectively. After the cryopreserved SUP-B15/ADR and RS4;11/ADR cells were resuscitated, their survival rates were 88.4±1.2% and 89.3±1.6% respectively, while their resistance indexes were 13.976±0.967 and 10.342±0.846 respectively (P>0.05). When the drug-resistant cells were cultured in the medium without ADR for 1 month, their drug-resistance indexes dropped down to 12.893±1.255 and 9.327±0.321 respectively(P<0.05). Drug-resistant cell lines had the cross-resistance to cytarabine and etoposide. The expression of P-gp and MDR1 in drug-resistant cells was significantly higher than that in wild-type cells.</p><p><b>CONCLUSION</b>Two drug-resistant ALL cell lines have been successfully established by exposing to the ascending concentration of ADR. The over-expression of MDR1 and P-gp in drug-resistant cells may be one of the mechanisms underlying the drug resistance.</p>
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Linhagem Celular , Citarabina , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Etoposídeo , Leucemia-Linfoma Linfoblástico de Células PrecursorasRESUMO
Paclitaxel (PTX) is one of the most frequently used anticancer agent for treating refractory ovarian cancer, metastatic breast cancer and non-small cell lung cancer. However, its oral administration is impeded by very low bioavailability (<5%) due to the P-glycopprotein (P-gp) efflux pump effect. This study investigated in vitro and in vivo P-gp inhibitory effects of adamantyl derivatives AC-603 and AC-786 in rats. Two adamantyl derivatives tested in this study increased the cytotoxicity of daunomycin (DNM) in P-gp overexpressed cell line by inhibiting P-gp efflux function. Pharmacokinetics of PTX with orally co-administered P-gp inhibitors were assessed in rats to improve PTX absorption. The pharmacokinetic parameters of PTX were determined in rats after intravenous (2 mg/kg) or oral (25 mg/kg) administration in the presence or absence of verapamil (a positive control), AC-603 or AC-786 (0.5 mg/kg or 5 mg/kg). Compared to control group (PTX alone), experimental groups (PTX with AC-603 or AC-786) significantly increased the area under the plasma concentration-time curve of PTX following oral administration by 1.7–2.2 fold. The volume of distribution and total clearance of PTX were decreased, while other parameters were not significantly changed. In conclusion, co-administration of AC-603 or AC-786 enhanced the relative bioavailability of orally administered PTX as compared to control.
Assuntos
Animais , Ratos , Absorção , Administração Oral , Disponibilidade Biológica , Neoplasias da Mama , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular , Daunorrubicina , Técnicas In Vitro , Neoplasias Ovarianas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Paclitaxel , Farmacocinética , Plasma , VerapamilRESUMO
Oseltamivir is a substrate of P-glycoprotein, an efflux drug transporter encoded by ABCB1. The objective of this study was to assess the role of ABCB1 (c.1236C>T, c.2677G>T/A, and c.3435C>T) polymorphisms in the pharmacokinetics of oseltamivir and its active metabolite, oseltamivir carboxylate in humans. Nineteen healthy male subjects were enrolled, and their ABCB1 polymorphisms were evaluated. After the oral administration of 75 mg oseltamivir, the plasma concentrations of oseltamivir and oseltamivir carboxylate were measured. Pharmacokinetic analysis was carried out. Systemic exposure to oseltamivir and oseltamivir carboxylate was higher in the mutant group than in the wild-type and heterozygous groups. We suggest that ABCB1 polymorphisms affect the pharmacokinetics of oseltamivir in humans. Further studies in a large population are necessary to validate the results of this preliminary study (Clinical Trial Registration Information [CRIS] registry: http://cris.nih.go.kr, No. KCT0001903).
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Humanos , Masculino , Administração Oral , Oseltamivir , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Farmacocinética , PlasmaRESUMO
<p><b>OBJECTIVE</b>To predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin (L-OHP) in human colorectal cancer cells.</p><p><b>METHODS</b>L-OHP-resistant human colorectal cancer cell line (HCT116/L-OHP) was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control (NC-miRNA) were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPover-expressing miR-145 and HCT116/L-OHP. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98(GPR98) as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPover-expressing GPR98 and HCT116/L-OHP. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHP) were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation (A value) and sensitivity to L-OHP (the lower the IC50, the stronger the sensitivity) in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein (gp), multiple drug-resistance protein 1(MRP1), cancer-inhibition gene PTEN.</p><p><b>RESULTS</b>HCT116/L-OHP cell line was successfully established with ICof (42.34±1.05) mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than (9.81±0.95) mg/L (t=39.784, P=0.000) and lower than 1.00±0.09 (t=13.021, P=0.000) in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPcells were established successfully, with relative miR-145 expression of 10.01±1.05, which was higher than 1.06±0.14 in HCT116/L-OHPand 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHPcells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHP(mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHP(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHPcells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHPcells, which were higher than those in HCT116/L-OHPcells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHPcells was lower as compared to HCT116/L-OHPcells (1.41±0.16 vs. 1.98±0.13, P<0.05).</p><p><b>CONCLUSION</b>MiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.</p>