Levetiracetam plus Oxcarbazepine Combination Treatment Downregulates Serum Multidrug Resistance Protein 1 Levels and Upregulates Neuropeptide Y Levels in Children with Epilepsy

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.

Influence of 6-shogaol potentiated on 5-fluorouracil treatment of liver cancer by promoting apoptosis and cell cycle arrest by regulating AKT/mTOR/MRP1 signalling / 中国天然药物

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.

Regulatory effects of Bmi-1 and MDR1 on the chemosensitivity of human non-small cell lung cancer cells to cisplatin / 医学研究生学报

Objective The purpose of this study was to investigate the mechanism of Bmi-1 regulating the sensitivity of non-small cell lung cancer (NSCLC) to chemotherapy by observing its effects on multidrug-resistance protein 1 (MDR1) and apoptosis-related proteins. Methods Small interfering RNAs (siRNA) targeting Bmi-1 were transfected into A549 and A549/DDP cells of NSCLC and the logarithmic-phase cells were randomly divided into a siRNA-Bmi-1, a siRNA-negative control and a blank control group. The A549 cells were treated with siRNA-Bmi-1, DDP or Bmi-1+DDP, or left untreated (the control). CCK8 assay was employed to measure the 50% inhibitory concentration (IC50) of cisplatin in the A549 and A549/DDP cells before and after treatment. The apoptosis of the cells was detected by flow cytometry, the mRNA expression of Bmi-1 determined by RT-PCR, and the relationship of Bmi-1 with MDR1 and cleaved caspase-3 proteins analyzed by Western blot. Results After transfection, the relative mRNA and protein expressions of Bmi-1 in the A549 and A549/DDP cells were significantly lower in the siRNA-Bmi-1 than in the blank control group (P < 0.05), but higher in the A549/DDP than in the A549 cells. The survival rates of the A549 and A549/DDP cells were decreased with the increased concentration of cisplatin (P < 0.05), even lower in the Bmi-1+DDP than in the DDP subgroup (P < 0.05). The apoptosis rate of the A549 cells was markedly higher in the Bmi-1+DDP than in the DDP, Bmi-1 and control groups ([39.65±3.41]% vs [23.11±1. 62] %, [2.05±1.56]% and [1.98±1.05]%, P < 0.05). After 24 hours of treatment with DDP, both the expressions of Bmi-1 and MDR1 were remarkably elevated, while the down-regulation of Bmi-1 significantly decreased the expression of MDR1 and increased that of cleaved caspase-3. Conclusion The expression of siRNA-Bmi-1 makes non-small lung cancer cells more sensitive to cisplatin, which might be associated with its inhibition of MDR1 expression and activation of apoptosis-related proteins.

Comparison of Effect of Fine Powder and Ultrafine Powder of Dendrobium nobile Stem on Intestinal Transporters of Rats / 中国实验方剂学杂志

Objective:To compare the contents of alkaloids from fine and ultrafine powder of Dendrobium nobile stem in rat plasma,and investigate the effect of D. nobile stem with different particle sizes on gene expression of intestinal transporters. Method:Rats were randomly divided into the blank group,fine powder group of D. nobile stem(0.25 g·kg-1) and ultrafine powder group of D. nobile stem(0.25 g·kg-1).The rats were gavaged every 6 h for 5 days.The samples of rat plasma and small intestine were collected.The plasma samples were detected with UPLC-MS.The chromatography separation was performed on a Hypersil Gold C18 column(2.1 mm×150 mm,1.9 μm) with acetonitrile-0.1%formic acid solution as mobile phase for gradient elution.Electrospray ionization (ESI) was applied and operated in positive ion mode.The mRNA expression of multidrug resistance protein 1(MDR1),oligopeptide transporter protein 1(PEPT1),organic cation transporter protein 2(OCT2),breast cancer resistance protein 1(BCRP1),monocarboxylate transport protein 1(MCT1) and multidrug resistance related protein 2(MRP2) in small intestine were quantified by real time fluorescence quantitative polymerase chain reaction. Result:After intragastric administration of fine and ultrafine powder of D. nobile stem,dendrobine,mubironine B and dendramine could be detected in rat plasma.The contents of dendrobine and dendramine in the ultrafine powder group were significantly higher than that in the fine powder group(PD. nobile stem(PPD. nobile stem(PConclusion:Compared with the fine powder group of D. nobile stem,the plasma concentrations of dendrobine and dendramine in the ultrafine powder group are significantly increased,it may be related to the intestinal transporters of MDR1 and BCRP1.These results can provide experimental basis for selecting particle size of D. nobile stem.

Mechanisms of doxorubicin resistance of breast cancer MCF-7 cells mediated by exosomes / 药学学报

This study aimed to explore the roles of exosomes in doxorubicin-resistance in breast cancer cells. Using breast cancer parental cell line (MCF-7), doxorubicin-resistant cell line (MCF-7/ADR) and sensitive cell line co-cultured with doxorubicin-resistant supernatant (MCF-7/EXO) as models, the effects of doxorubicin on proliferation or apoptosis of MCF-7, MCF-7/EXO and MCF-7/ADR cells were detected by CCK8, and light or fluorescent microscopy. Exosomes in the supernatants of cell culture were extracted by ultracentrifugation, and the quantity of exosomes was determined by transmission electron microscopy, BCA and DiI labeling assay. Expression levels of exosome-specific biomarkers CD63 and Flotillin-1 were detected by Western blot. The uptake of MCF-7/ADR cell-derived exosomes by MCF-7 cells was observed by laser confocal microscopy. Western blot was used to detect the expression levels of multidrug resistance protein ATP-binding cassette subfamily B member 1 (ABCB1) in all three cell strains. Cell proliferation assays showed that IC50 of MCF-7/EXO cells to doxorubicin was 0.83 ± 0.09 μmol·L-1, which was significantly higher than 0.15 ± 0.05 μmol·L-1 (P<0.01) of MCF-7 cells, suggesting 5.5 times of increase in drug resistance. Apoptosis of MCF-7 cells was induced after doxorubicin treatment (P<0.001), but MCF-7/EXO cells were not significantly different (P>0.05). Exosome quantification and specific marker detection showed that MCF-7/EXO cells had significantly more exosomes than MCF-7 cells (P<0.05). PKH67 tracer markers indicated that MCF-7/ADR-derived exosomes could be taken up by MCF-7 cells. Western blot showed that the expression level of ABCB1 protein in MCF-7/EXO cells was significantly higher than that in MCF-7 cells. Taken together, these results indicate that exosomes of doxorubicin-resistant breast cancer cells can transmit drug resistance to sensitive cells, and the underlying mechanism may involve ABCB1 protein transport mediated by exosomes.

Effect of multidrug resistant associated protein 4 overexpression on lipopolysaccharide-induced vascular endothelial hyperpermeability and its mechanism / 中华危重病急救医学

Objective To investigate the effect of multidrug resistance protein 4 (MRP4) overexpression on lipopolysaccharide (LPS)-induced vascular endothelial hyperpermeability of rat pulmonary micro-vascular endothelial cells (PMVECs) and its molecule mechanism. Methods Three to six passages of PMVECs were cultured in vitro, and they were divided into three groups: the cells in LPS group were only challenged by LPS 10 μg/mL after being cultured in serum-free medium for 24 hours; the cells in Ad-shRNA and Ad-MRP4 groups were infected with the empty virus control or recombinant adenovirus expressing MRP4 for 2 hours, and then were cultured in serum-free medium for 24 hours followed by stimulation of LPS 10 μg/mL. Endothelial permeability was assayed by the Transwell chamber models at 2, 6, 12, and 24 hours after LPS stimulation. Intracellular cyclic adenosine monophosphate (cAMP) levels were detected by enzyme-linked immunosorbent assay (ELISA). The morphological characteristics and distribution of F-actin was determined by laser confocal fluorescence microscope. The protein expressions of MRP4,β-catenin, vascular endothelium-cadherin (VE-cad) and ZO-1 were measured by Western Blot. Results ① After LPS stimulation, endothelium permeability and intracellular cAMP levels in PMVECs were significantly increased, peaked at 12 hours, and then decreased after 24 hours. Compared with LPS group and Ad-shRNA group, PMVECs of Ad-MRP4 group were exhibited a significant increase in endothelial permeability [12-hour permeability (A value):1.88±0.06 vs. 1.12±0.17, 1.10±0.18] and a significant decrease in intracellular cAMP level [12-hour cAMP (μg/L):2.39±0.02 vs. 2.97±0.01, 3.00±0.02, all P 0.05).② Under laser confocal fluorescence microscope, after LPS stimulation, the stress fiber formation was induced in three groups. But there were pronounced irregular aggregation of fiber in PMVECs of Ad-MRP4 group. ③ Furthermore, compared with LPS group and Ad-shRNA group, protein expression of MRP4 in Ad-MRP4 group was dramatically increased (gray value: 0.76±0.03 vs. 0.44±0.02, 0.43±0.02, both P 0.05). Conclusion MRP4 overexpression can decrease intracellular cAMP levels, reduce intercellular junction protein expression, and then exaggerate LPS-induced vascular endothelial hyperpermeability.

Correlation Analysis of the expression of HIF-1α and MRP in hepatocellular carcinoma / 国际检验医学杂志

Objective To analyze the correlation between liver tissue hypoxia inducible factor‐1α(HIF‐1α) protein expression and multidrug resistance protein (MRP) expression between .Methods Our hospital from March 2012 to March 2013 the Depart‐ment of Pathology of the liver paraffin‐embedded specimens of a total of 83 cases of specimen processing ,production of tissue sec‐tions ,HIF‐1αexpression was observed ,the positive expression of MRP .Results 83 cases of specimens in HIF‐1α 50 cases were positive ,the positive rate was 60 .2% ;of which the well‐differentiated tumor samples of HIF‐1α positive rate was 74 .1% ,signifi‐cantly higher than the 28 .0% poorly differentiated (P0 .05) .In 83 cases of samples ,58 samples were MRP positive ,the positive rate was 70 .0% ;MRP positive rate among the high degree of 77 .6% ,significantly higher than the 52 .0% poorly differentiated (P0 .05) .MRP expression in the same samples were positive ,the HIF‐1α expression was also significantly increased .Conclusion HCC HIF‐1αprotein expression and MRP expression has some relevance .

Effects of the concentrations of glutathione and the related mechanisms of arsenic trioxide (As2 O3) on Multidrug Resist-ance-associated Pr otein / 中国医师杂志

Objective To investigate the apoptosis-inducing effect , inhibiting MRP-1 and mRNA expression of arsenic trioxide (As2O3) and buthionine sulfoximine (BSO) on multidrug-resistant cell-K562A/DM cell .To compare the effect of As2O3 and the com-bined group .To determine the effect of intracellular GSH content on the arsenic effect .Methods To investigate the effect of the arse-nic group (0.5,2.0,5.0μmol/L)and/or BSO (100μmol/L) on multidrug-resistant cell -K562/ADM cell.To detect the change of the correlated index .⑴Intracellular GSH contents were measured using Glutathione Assay Kit by spectrophotometry .⑵MRP-1 expression were determined by flow cytometry .⑶MRP-1 mRNA expression were directed by semi-quantitative RT-PCR.Results After the GSH contents were degraded by the combination of clinic dose arsenic group (0.5,2μmol/L) and BSO(100μmol/L), in 48 hours and 72 hours, the effect of the combination group ( clinic dose arsenic group ) was obviously stronger than the clinic dose arsenic group and the high dose arsenic group .In 48 hours, the MRP-1 mRNA depressive effect of the combination group ( clinic dose arsenic group ) was obviously stronger than high dose arsenic group .In 72 hours, the MRP-1 depressive effect of the combination group ( clinic dose arsenic group ) was obviously stronger than high dose arsenic group .Conclusions The intracellular GSH contents closely correlated with the arsenic effect .The combination of clinic dose arsenic and BSO inhibit obviously MRP-1 expression and MRP-1 mRNA expres-sion in K562/ADM cell.

Hepatocyte growth factor (HGF), heat shock proteins (HSPs) and multidrug resistance protein (MRP) expression in co-culture of colon tumor spheroids with normal cells after incubation with interleukin-1β (IL-1 β) and/or camptothecin (CPT-11).

Tumor chemoresistance and metastasis are some of the most important problems in colon cancer therapy. In the present study, co-cultures of human colon carcinoma cell spheroids, obtained from different grades of tumor, with human colon epithelium, myofibroblast and endothelial cell monolayers were performed. The purpose of these co-cultures was to reflect, in in vitro conditions, different stages of colon tumor development. In order to investigate the invasive capacities of the tumor cells and their resistance to chemotherapy, HGF, HSP27, HSP72 and MRP levels were analyzed after incubation of the co-cultures with IL-1 and irinotecan (CPT-11) added as single agents or in combination. Myofibroblasts produced significantly higher amounts of HGF than epithelial cells. Tumor cells released trace amounts of this molecule. In co-cultures, IL-1 induced HGF release, while CPT-11 alone or combined with IL-1decreased HGF secretion. An immunoblotting analysis followed by densitometry revealed that the combination of IL-1 plus CPT-11 added to the co-cultures led to a decrease in HSPs and MRP levels. In conclusion, direct and paracrine interactions of colon tumor cell spheroids with normal cells and exogenously added CPT-11 change HSP27, HSP72 and MRP expression in comparison to monocultures. IL-1 and CPT-11, dependent on whether they are added separately or jointly, differentially modulate HGF expression in monocultures of colon tumor spheroids or normal cells and their co-cultures.

Study on silencing of endogenetic mdr-1 gene expression by RNA interference in ovarian cancer resistant strain SKOV3/TAXOL / 肿瘤研究与临床

Objective To construct the small hairpinRNA recombinant plasmids targeting mdr-1 gene which expresses highly in ovarian cancer resistance strain SKOV3/TAXOL to silence endogenetic mdr-1 gene expression and investigate the role of mdr-1 gene in the development of resistant ovarian cancer. Methods The pPGPU6/GFP/Neo-mdr-1 were constructed by gene clone technology. The influence on proliferation and apoptosis were investigated by CCK-8 in SKOV3/TAXOL after transfected. pPGPU6/GFP/Neo-mdr-1. Results The expression against mdr-1 proteins were inhibited by pPGPU6/GFP/Neo-mdr-1. The cell proliferation were inhibited after transfected pPGPU6/GFP/Neo-mdr-1 by CCK-8. The apoptosis were observed in DAB experiments and the apoptosis rate incised. Conclusion mdr-1 plays an important role in proliferation of resistant ovarian cancer and the short hairpinRNA of mdr-1 can efficiently suppress mdr-1expression and enhance the apoptosis in SKOV3/TAXOL, which provides a novel method for chemotherapy resistant tumors.

Genetics Research of Multidrug Resistance Protein 3,Farnesoid X Receptor Gene in Intrahepatic Cholestasis / 实用儿科临床杂志

The etiology and pathogenesis of intrahepatic cholestasis are complex,and those are not still very clear in current.Studies suggest that genetic factors play an important role in the pathogenesis of the disease.Some familial cholestasis have been confirmed by gene mutation causing.Bile secretion process regulated by a number of bile relation gene at the molecular level.Farnesoid X receptor (FXR) gene is related to intrahepatic bile secretion process.Bile secretion is indirect control by FXR which formats a complex network,becoming more attention to researcher in recent years.

Prediction of Histologic Grade and Multidrug Resistance of Brain Tumors Using Brain SPECT

OBJECTIVE: Computerized tomography(CT) and magnetic resonance imaging(MRI) are very useful in detection of structural change in the brain including tumors. However, they can not inform functional and biological behavior of such lesions. 99m Tc-Methoxyisobutyl isonitrile(MIBI) is considered as a substrate for MDR1 gene-encoded permeability glycoprotein(P-gp) and it has been used in the evaluation of multidrug resistance(MDR) in various tumors. The purpose of the study is to demonstrate the presence of MDR in brain tumor and brain tumor grading by an external imaging with 201Thallium and 99m Tc-MIBI SPECT. METHODS: 201Thallium and 99m Tc-MIBI SPECT were performed in 18 patients with malignant tumors and in six patients with benign tumors. Immunohistochemical staining(IHC) of the tumor specimen for P-gp was also performed. The histologic grading of the tumors and immunohistochemical staining for P-gp were compared to the dual brain SPECT findings of 201Thallium and 99m Tc-MIBI SPECT studies. Brain tumor with positive 201Thallium SPECT and negative 99m Tc-MIBI SPECT is considered to be multidrug resistance. An uptake index obtained from brain SPECT was used for tumor grading. MDR1 gene-encoded P-gp was assessed by immunohistochemical staining using a monoclonal antibody for P-gp. RESULTS: The malignant group showed significantly higher uptake indices in the 201Thallium and 99m Tc-MIBI SPECT than benign group. The uptake index of 99m Tc-MIBI SPECT was inversely correlated with P-gp immunohistochemical staining without statistical significance. CONCLUSION: 201Thallium and 99m Tc-MIBI SPECT are useful for predicting histologic grade of brain tumors, and 99m Tc-MIBI SPECT might be useful for predicting the presence of MDR protein.

Molecular aspects of organic ion transporters in the kidney

A function of the kidney is elimination of a variety of xenobiotics ingested and wasted endogenous compounds from the body. Organic anion and cation transport systems play important roles to protect the body from harmful substances. The renal proximal tubule is the primary site of carrier-mediated transport from blood into urine. During the last decade, molecular cloning has identified several families of multispecific organic anion and cation transporters, such as organic anion transporter (OAT), organic cation transporter (OCT), and organic anion-transporting polypeptide (oatp). Additional findings also suggested ATP-dependent organic ion transporters such as MDR1/P-glycoprotein and the multidrug resistance-associated protein (MRP) as efflux pump. The substrate specificity of these transporters is multispecific. These transporters also play an important role as drug transporters. Studies on their functional properties and localization provide information in renal handling of drugs. This review summarizes the latest knowledge on molecular properties and pharmacological significance of renal organic ion transporters.