Effects of hypoxia on the expression and function of P-gp in Caco-2 cells / 中南大学学报(医学版)

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.

Salmonella-mediated blood‒brain barrier penetration, tumor homing and tumor microenvironment regulation for enhanced chemo/bacterial glioma therapy

Chemotherapy is an important adjuvant treatment of glioma, while the efficacy is far from satisfactory, due not only to the biological barriers of blood‒brain barrier (BBB) and blood‒tumor barrier (BTB) but also to the intrinsic resistance of glioma cells via multiple survival mechanisms such as up-regulation of P-glycoprotein (P-gp). To address these limitations, we report a bacteria-based drug delivery strategy for BBB/BTB transportation, glioma targeting, and chemo-sensitization. Bacteria selectively colonized into hypoxic tumor region and modulated tumor microenvironment, including macrophages repolarization and neutrophils infiltration. Specifically, tumor migration of neutrophils was employed as hitchhiking delivery of doxorubicin (DOX)-loaded bacterial outer membrane vesicles (OMVs/DOX). By virtue of the surface pathogen-associated molecular patterns derived from native bacteria, OMVs/DOX could be selectively recognized by neutrophils, thus facilitating glioma targeted delivery of drug with significantly enhanced tumor accumulation by 18-fold as compared to the classical passive targeting effect. Moreover, the P-gp expression on tumor cells was silenced by bacteria type III secretion effector to sensitize the efficacy of DOX, resulting in complete tumor eradication with 100% survival of all treated mice. In addition, the colonized bacteria were finally cleared by anti-bacterial activity of DOX to minimize the potential infection risk, and cardiotoxicity of DOX was also avoided, achieving excellent compatibility. This work provides an efficient trans-BBB/BTB drug delivery strategy via cell hitchhiking for enhanced glioma therapy.

Effect of hypoxia on HIF -1 α/MDR1/VEGF expression in gastric cancer cells treated with 5 -fluorouracil / 中南大学学报(医学版)

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.

Sunitinib induced resistance of endothelial cells by up-regulating P-glycoprotein and PI3K/Akt pathway

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.

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

First report of nt230(del4) mutation in the MDR1 gene in German Shepherds in Southern Brazil / Primeiro relato da mutação nt230(del4) no gene MDR1 em Pastores Alemães no Sul do Brasil

ABSTRACT: The P-glycoprotein (P-gp) is a transmembrane protein encoded by the MDR1 gene that functions as a biological barrier by extruding toxins and xenobiotics out of cells. The MDR1 gene can carry a mutation called nt230(del4), which is a deletion of four base pairs resulting in the formation of a non-functional protein that may predispose to severe toxicosis, as observed in dogs with sensitivity to ivermectin. Several breeds have been described as carriers of the mutation, including German Shepherds (GS). However, the presence of the mutant allele in this breed has not been described in Brazil. This study aimed to determine the genotypic and allelic frequency of the nt230(del4) mutation in the MDR1 gene in GS from Southern Brazil. Blood samples were collected from 79 GS in the state of Rio Grande do Sul and genotype for the MDR1 gene was performed. Seventy-eight (98.7%) dogs were dominant homozygous genotype (wild) and one (1.3%) was heterozygous. This study showed that there is a low frequency (0.6%) of the mutant allele while the frequency of the wild allele is high (99.4%) in this specific population. This is the first report of the presence of the nt230(del4) mutation in the MDR1 gene in GS in Brazil. This information is important for breeders to prevent dissemination of the mutant allele in the national breeding population and international exchange of animals for breeding; for owners and veterinarians to be aware when dispensing and administering medications for GS dogs in Brazil.

RESUMO: A Glicoproteína-P é uma proteína transmembrana codificada pelo gene MDR1 que atua como uma barreira fisiológica através da extrusão de toxinas e xenobióticos para fora das células. O gene MDR1 pode carregar uma mutação chamada nt230(del4) que é uma deleção de quatro pares de bases, resultando na formação de uma proteína não-funcional que pode predispor à toxicoses graves, como as observadas em cães sensíveis à ivermectina. Diversas raças de cães foram descritas como portadoras da mutação nt230(del4), incluindo Pastores Alemães (PA). Entretanto, a presença do alelo mutante nessa raça não foi descrita em cães no Brasil. O objetivo desse estudo foi determinar a frequência genotípica e alélica da mutação nt230(del4) em PA no sul do Brasil. Amostras de sangue foram coletadas de 79 PA no estado do Rio Grande do Sul e o genótipo dos cães para o gene MDR1 realizado. Setenta e oito (98.7%) cães foram homozigotos dominantes (selvagem) e um (1.3%) tinha genótipo heterozigoto. A frequência do alelo mutante foi baixa (0.6%), enquanto a frequência do alelo selvagem foi alta (99.4%) nesta população. Este é o primeiro relato da presença desta mutação nt230(del4) no gene MDR1 em PA no Brasil. Esta informação é importante para criadores a fim de prevenir a disseminação do alelo mutante na população de criadores da raça no Brasil e programas internacionais de troca de animais para criação, para tutores e veterinários estarem conscientes quando prescreverem e administrarem medicações para cães PA no Brasil.

Progression on Multifunctional Nanoparticles Interfering with Cell Membrane Transporters-associated Drug Resistance / 中国医学科学院学报

Multi-drug resistance(MDR)refers to the loss of sensitivity of tumor cells to traditional chemotherapeutics agents under the mediation of various mechanisms,resulting in the reduction of chemotherapy efficacy.Current studies suggest that a variety of factors,including cell membrane transporter-mediated efflux of anti-tumor drugs,special microenvironment in tumor tissue,DNA self-repair and anti-apoptotic process,and epithelial-mesenchymal cell transformation,may contribute to the formation of MDR.Cell membrane transporter-mediated drug efflux refers to an increase in the amount of anti-tumor drug pumped out of the cell through the up-regulation of the ATP-binding cassette transporter on tumor cell membrane,which reduces the concentration of the drug in the cell,thus forming MDR.An effective method to inhibit the efflux pump caused by overexpression of membrane transporters plays an important role in overcoming MDR.As a promising drug delivery system,multifunctional nanoparticles have demonstrated many advantages in antitumor therapy.Meanwhile,nanoparticles with tailored design are capable of overcoming MDR when combined with a variety of strategies.This paper described in detail the studies relevant to the use of multifunctional nano-sized drug delivery system combined with different strategies,such as co-delivery of agents,external responsiveness or target modification for intervention with efflux pump in order to reverse MDR.This paper provides reference for the development of nano-sized drug delivery system and the formulation of reversal strategy in the future.

Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling / 中国实验方剂学杂志

Objective:To investigate the effect of notoginseng total saponins （TNS） on adriamycin （Adr） resistance in HepG2/Adr cells and the expression and activity of the mechanisms as the modulators of multi-drug resistance， so as to explore the possible mechanism of extracellular signal-regulated kinase （ERK） and protein kinase B （Akt） signaling pathways in reversing the resistance of HepG2/Adr cells mechanism. Method:Effect of TNS on HepG2/Adr cell proliferation was detected by thiazole blue （MTT） method. HepG2/Adr cells were treated with different concentrations （100， 50， 25， 0 mg·L^-1） of TNS and （20 μmol·L^-1） Adr respectively， and a blank group was set. The high-content screening platform was used to detect the accumulation of Adr in HepG2/Adr cells after 40 minutes， 3 hours and 6 hours. Western blot was used to detect the expression of P-glycoprotein /multidrug resistance/ATP binding cassette subfamily B member 1（P-gp/MDR1/ABCB1） and other drug resistance-related proteins and the main protein expression of ERK/Akt signaling pathway. The change of MDR1 on cell membranes was observed by laser confocal microscopy. Result:Compared with HepG2 cells， the expression of MDR1 in HepG2/Adr cells was significantly increased （<italic>P</italic><0.01）. Compared with the Adr group， the half-inhibitory concentration （IC₅₀） of TNS （25， 50， 100 mg·L^-1） and Adr （20 μmol·L^-1） co-administration group on HepG2/Adr cells <italic>in vitro</italic> significantly reduced （<italic>P</italic><0.01）， and the highest reversal multiple was 10 times. Compared with the Adr group， the co-administration group could significantly increase the accumulation of Adr in the cells （<italic>P</italic><0.05） in a dose-dependent manner. Compared with the blank group， the co-administration group could significantly reduce MDR1， ABC semitransporter （ABCG2）， multidrug resistance associated protein （MRP1）， ERK， phosphorylated extracellular regulatory protein kinase （p-ERK）， Akt， phosphorylated protein kinase B （p-Akt）， mammals， rapamycin target protein （mTOR） and phosphorylated mammalian rapamycin target protein （p-mTOR） （<italic>P</italic><0.05）， with the same results in the doxorubicin group. Compared with the blank group， there was no significant difference in the distribution and fluorescence intensity of MDR1 on the cell membrane between the Adr group and the notoginseng total saponins （25 mg·L^-1） group. Compared with the blank group and the doxorubicin group， TNS could significantly reduce the distribution of MDR1 on the cell membrane （<italic>P</italic><0.05）. Conclusion:TNS can inhibit the ERK/Akt pathway， reduce the expression of MDR1， and significantly increase the accumulation of doxorubicin in HepG2/Adr cells， which may be one of the mechanisms of notoginseng total saponins in reversing resistance.

P-glycoprotein associated with diabetes mellitus and survival of patients with pancreatic cancer: 8-year follow-up

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.

Effect of α-asarone on the Function and Expression of P-glycoprotein in Rat Brain Microvascular Endothelial Cells / 中国医学科学院学报

To investigate the effect of α-asarone on the function and expression of P-glycoprotein(P-gp)in rat brain microvascular endothelial cells(rBMECs). 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. The rBMECs,stimulated by glutamine,showed a high expression of P-gp(=1.924,=0.020)/Mdr1a(=1.788,=0.019) compared to the normal rBMECs.The treatment with 25.0(=1.924,=0.025;=1.788,=0.017) and 50.0 μg/μl(=1.924,=0.035;=1.788,=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. 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.

Kaempferol in reversing drug resistance of chronic myelogenous leukemia K562/ADM cells and its related mechanism / 白血病·淋巴瘤

Objective@#To investigate the drug resistance of kaempferol reversed adriamycin (ADM)-resistant K562/ADM cells in chronic myelogenous leukemia (CML) and its related mechanism.@*Methods@#Methyl thiazolyl tetrazolium (MTT) method was used to detect the toxicity of ADM on K562 and K562/ADM cells for 24 h. The half inhibitory concentration (IC50) of ADM and the drug resistance multiple for 24 h were calculated. MTT method was used to detect the toxicity of kaempferol on K562/ADM cells for 24 h. The 5% inhibitory concentration (IC5) and 10% inhibitory concentration (IC10) of kaempferol for 24 h were calculated to determine the concentration of kaempferol in the subsequent experiments. And the cells untreated by the kaempferol were selected as the control group. The cell inhibition after the treatment of ADM for 24 h of the blank control group and kaempferol intervention group was detected by using MTT method. And then the cell inhibition for 24 h and ADM IC50 for 24 h in the above groups were calculated. The ratio of IC50 in the blank control group and kaempferol group was the reversal drug resistance multiple of kaempferol. The fluorescence intensity of ADM in K562/ADM cells treated by kaempferol was detected by using flow cytometry. Western blotting was used to detect the expressions of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), phosphorylated p38 (p-p38), and total p38 (t-p38) protein in K562/ADM cells after the treatment of kaempferol, the specific inhibitor of p38-MAPK signaling pathway SB202190, and the combination of kaempferol and SB202190.@*Results@#After the treatment of ADM for 24 h, the IC50 value of K562 and K562/ADM cells was (0.9±0.6), (28.1 ±3.5) μg/ml, respectively. The drug resistance multiple of K562/ADM cells on the treatment of ADM for 24 h was 31.16 compared with the K562 cells. MTT method showed that kaempferol inhibited the proliferation of K562/ADM cells in a dose-dependent manner. According to the IC5 and IC10, 0.5 μmol/L and 1.0 μmol/L kaempferol were determined to do the subsequent experiments. After the combined interaction of kaempferol and ADM for 24 h, the ADM IC50 of K562/ADM cells in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was (33.7±5.7), (21.4±0.6), (15.9±1.8) μg/ml, respectively (F = 30.85, P < 0.05), and there was a statistical difference of pairwise comparison (both P < 0.05). The reversal drug resistance multiple of K562/ADM cells for 24 h in 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 1.58 and 2.12, respectively. Flow cytometry results showed that the mean fluorescence intensity (MFI) of ADM in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 138.4±8.9, 154.3±2.2, 165.7±4.8, respectively, and the difference was statistically significant (F = 161.48, P < 0.05). Compared with the blank control group, after treatment of K562/ADM cells with 0.5 μmol/L and 1.0 μmol/L kaempferol for 24 h, the relative expressions of P-gp, MRP1 and p-p38 protein were decreased in K562/ADM cells (all P < 0.05), but there was no statistical difference in the expression of t-p38 protein (P > 0.05); SB202190 could reduce the relative expressions of P-gp, MRP1 and p-p38 protein (all P < 0.05); after the treatment of SB202190 combined with different concentration of kaempferol, the relative expressions of P-gp, MRP1 and p-p38 protein in K562/ADM cells did not decrease (P > 0.05).@*Conclusions@#Kaempferol can decrease the relative expressions of P-gp and MRP1 in K562/ADM cells by inhibiting p38-MAPK pathway, so as to increase the concentrations of ADM and to reverse the drug resistance of K562/ADM cells.

Potential synergic mechanism of Wutou-Gancao herb-pair by inhibiting efflux transporter P-glycoprotein / 药物分析学报

Wutou-Gancao herb-pair is extensively used to attenuate the toxicity and enhance the efficacy of aconite. In this study, potential synergic mechanism of the herb pair was investigated by utilizing multiple ap-proaches. In silico and in vitro Caco-2 cell models were applied to study the potential binding mode of bioactive ingredients existing in liquorice with P-glycoprotein (P-gp), as well as the inhibition effects on P-gp. Additionally, anti-inflammatory activity of aconitine (AC) combined with active ingredients of liquorice, as well as pharmacokinetic patterns of AC after co-administration was investigated. Anti-inflammatory effect of AC (1 mg/kg) in rats was enhanced in combination with bioactive ingredients of liquorice (10 mg/kg). In the meanwhile, the exposure of AC in vivo was altered, in terms of Cmax and AUC. For instance, the Cmax and AUC were increased to 1.9 and 1.3 folds, respectively, when used in combination with liquiritigenin. The in silico study revealed the potential binding mode with outward facing conformation of P-gp. The resulting data obtained from transport of rhodamine-123 (Rh-123) across Caco-2 cell monolayer further indicated that the function of P-gp was inhibited by chemicals in liquorice. The synergic effect was therefore proposed to be attributed to inhibition of P-gp by liquorice since AC has been demonstrated to be the substrate of P-gp. The resuls revealed that potential synergic mechanism of Wutou-Gancao herb-pair by inhibiting function of key efflux transporter P-gp to enhance the exposure of AC in systematic circulation, and further the anti-inflammatory effect, which helps clarify the compatibility rationale of these two herbs.

Effectiveness of malabaricone-A in P-glycoprotein over-expressing cancer cell lines

Background: A major impediment in treatment for cancers is resistance to chemotherapy and is primarily attributed to over-expression of efflux pumps. This study aimed to establish the cytotoxicity of malabaricone-A (MAL-A) in P-glycoprotein/multidrug resistance (P-gp/MDR) over-expressing hematopoietic cancer cell lines.Methods: Leukemia and multiple myeloma cell lines were indirectly evaluated for their P-gp/MDR status by examining Calcein-AM fluorescence and cell viability was assessed by the MTS-PMS assay.Results: The fluorescence of calcein was significantly decreased in three cell lines LP-1, RPMI-8226 and CEM-ADR 5000 and reversal with verapamil endorsed their P-gp/MDR activity. The mean IC50 of MAL-A in these MDR+ cell lines (5.40±1.41 to 12.33±0.78 µg/ml) was comparable with the MDR- leukemic (9.72±1.08 to 19.26±0.75 µg/ml) and multiple myeloma cell lines (9.65±0.39 to 18.05±0.17 ?g/ml).Conclusions: Irrespective of their P-gp activity, the cytotoxicity of MAL-A was comparable, making it worthy of future pharmacological consideration in multidrug resistance.

The experimental research of dexamethasone reducing paclitaxel chemotherapy on lung cancer cells / 中国药理学通报

Aim: To investigate the effect of dexamethasone on paclitaxel chemotherapy, when given before chemotherapy. Methods: MTT method was used to investigate the effects of paclitaxel at different concentrations and dexamethasone (10 μ, mol · L-1) combined with paclitaxel at indicate dosages on the cell viability of lung cancer cells A549 and Calu-1. The cell cycle was detected by flow cytometry. The effect of dexamethasone on protein expression of glucocorticoid receptor (GR), P-glycoprotein (P-gP) and multidrug resistance protein-1 (MRP-1) expression were detected by Western blot. The effect of dexamethasone on lung cancer resistance of paclitaxel xenografts in nude mice was studied as well. Results paclitaxel at indicate dosages with dexamethasone (10 μmol · L-1) showed significantly increased IC50 value (A549: from 0.58 to 1.35 μmol · L-1; Calu-1 from 1. 10 to 2. 10 μmol · L-1). Dexamethasone activated GR, increased GR expression, and up-regulated the expression of P-gP and MRP-1. Dexamethasone combined with paclitaxel significantly decreased the antitumor effect of paclitaxel on nude mice transplanted tumor. Conclusions Giving dexamethasone before chemotherapy may cause resistance to cancer cells and reduce the effect of paclitaxel chemotherapy.

Study on transport mechanism of brucine and strychnine in MDCK-MDR1 cell monolayer model / 中草药

Objective: To study the bi-direction transport behavior of brucine and strychnine in the MDCK-MDR1 cell monolayer model. Methods: MTT method was employed to confirm the safe concentration of brucine and strychnine towards MDCK-MDR1 cells. The effects of transport time, drug concentration, and P-glycoprotein inhibitor verapamil on cumulative absorption concentration (Ccum) and apparent permeability coefficient (Papp) of brucine and strychnine in MDCK-MDR1 monolayer cells were studied. Results: The Papp value of brucine and strychnine was larger than 1 × 10-5 cm/s and the ratio of Papp(BL→AP) vs Papp(AP→BL) was less than 2. Brucine/ strychnine combined with verapamil decreased the ratio of Papp(BL→AP) vs Papp(AP→BL). Conclusion: The absorption of brucine and strychnine in MDCK-MDR1 cell monolayer model was well and the passive transference was its main intestinal absorption mechanism. The P-gp inhibitor verapamil has a significant inhibitory effect on brucine and strychnine absorption. Brucine and strychnine may be a substrate of P-glycoprotein.

Effects of clinical P-glycoprotein inhibitors on oral bioavailability and brain penetration of gefitinib / 中国药科大学学报

@#To investigate the effects of clinical P-glycoprotein inhibitors on oral bioavailability and brain penetration of gefitinib, 16 inhibitors and gefitinib were co-administered orally to ICR mice. The suspension of gefitinib and CMC-Na were co-administered to the control group. The suspension of gefitinib and clinical P-glycoprotein inhibitors were co-administered to the control group. Blood samples and brain homogenate samples were extracted by protein precipitation with acetonitrile and determinated by LC-MS/MS. It was found that ritonavir can significantly increase the oral bioavailability of gefitinib, and the area under the plasma concentration-time curves(AUC)of gefitinib was increased by 2 times; while brain exposure was increased, there was no increment in brain penetration. Some other drugs can also increase the plasma AUC of gefitinib, but can not enhance the brain penetration; After we corrected brain concentration with fraction of unbound drug in brain, it was found that the brain concentration of gefitinib in both control group and ritonavir group did not achieve the in vitro IC50 of inhibiting non-small cell lung cancer(NSCLC)cell growth. Our results suggest that clinical doses of the 16 clinical P-glycoprotein inhibitors can not specifically increase brain tissue exposure, more specific and safer P-gp inhibitors are required. After we corrected brain concentrations with fraction of unbound drug in brain, our preclinical studies found that insufficient brain exposure may be one of the reasons for the unsatisfactory efficacy of gefitinib in the treatment of brain metastases.

Effect of Acori Tatarinowii Rhizoma on Intestinal Absorption of Ginsenosides in Dingzhi Xiaowan / 中国实验方剂学杂志

Objective: To explore the effect of Acori Tatarinowii Rhizoma on intestinal absorption of ginsenosides in Dingzhi Xiaowan,and reveal the mechanism of Acori Tatarinowii Rhizoma acting as " adjuvant drug" in this formula. Method: The contents of ginsenoside Rg1,Re and Rb1 were measured by UPLC-MS/MS and the absorption of three ginsenosides in different intestine segments was investigated by rat single pass intestinal perfusion in situ,including absorption rate constant(Ka) and apparent permeability coefficient(Papp).Everted intestinal sac model was used to investigate the absorption dosage of three ginsenosides affected by volatile oil from Acori Tatarinowii Rhizoma and verapamil[Ver,a P-glycoprotein(P-gp) inhibitor]. Result:Papp values of three ginsenosides were ≤ 0.191×10-3 cm·min-1 in Dingzhi Xiaowan when lack of Acori Tatarinowii Rhizoma.Compared with lack of Acori Tatarinowii Rhizoma in Dingzhi Xiaowan group,the Ka and Papp values of lack of volatile oil from Acori Tatarinowii Rhizoma in Dingzhi Xiaowan group slightly increased without significant difference in the four intestinal segments,but when the prescription had Acori Tatarinowii Rhizoma,the Ka increased by 3.97-8.35 fold and the Papp increased by 3.99-8.49 fold.The results of everted intestinal sac test showed that volatile oil of Acori Tatarinowii Rhizoma could significantly promote the intestinal absorption of ginsenoside Rg1,Re and Rb1,but there was no dose-dependent. Conclusion:Volatile oil of Acori Tatarinowii Rhizoma can promote the intestinal absorption of three ginsenosides in Dingzhi Xiaowan,and the mechanism may be related to the inhibiting function on P-gp.

Effect of Berbamine Hydrochloride on Intestinal Absorption Characteristics of Berberine Hydrochloride by Rat Everted Intestinal Sac Model / 中国实验方剂学杂志

Objective: To investigate the effect of berbamine hydrochloride on the absorption characteristics of berberine hydrochloride in different intestinal segments of rats in normal environment and high calcium environment. Method: Taking rat everted intestinal sac model,the content of berberine hydrochloride in absorbent solution of everted intestinal sac from different compatibility groups was determined by HPLC,and the uptake per unit area in different groups was analyzed by One-way ANOVA. Result: Compared with the normal J70 group(in normal environment,the concentration of berberine hydrochloride was 70 mg·L-1) at the same time point,the uptake per unit area of the normal J70+Ver100 group(in normal environment,the concentration of berberine hydrochloride was 70 mg·L-1,adding verapamil hydrochloride to a concentration of 100 mg·L-1) was significantly increased in the ileum(P-1,adding berbamine hydrochloride to a concentration of 35 mg·L-1) were significantly increased in the duodenum(P-1,adding berbamine hydrochloride to a concentration of 70 mg·L-1) were significantly increased in the ileum(PConclusion: Berbamine hydrochloride can promote the absorption of berberine hydrochloride in intestine to a certain extent,especially in the high calcium environment.

Effect of Chaibei Zhixian Decoction and Its Absorption Component Peimine on Content of Carbamazepine in Rat Brain and Investigation of Its Mechanism / 中国实验方剂学杂志

Objective:To observe the effect of Chaibei Zhixian decoction and peimine on Carbamazepine (CBZ) concentration, P-glycoprotein (P-gp) and multi drug resistance 1(MDR1) expression in the brain tissues of rats with refractory epilepsy, and to understand the contribution of Peimine in the compound prescription to treat the refractory epilepsy. Method:Epilepsy rat models were established by injecting kainic acid (KA) in the lateral ventricle. The successfully modeled rats were randomly divided into model group, CBZ group(0.12 g·kg-1),Chaibei Zhixian decoction+CBZ group(8.39 g·kg-1+0.12 g·kg-1), peimine+CBZ group(0.01 g·kg-1+0.12 g·kg-1) and sham operation group. After 60 days of intervention, the expression levels of P-glycoprotein (P-gp) and MDR1b mRNA in the brain cortex were detected by Western blot and quantitative real\|time fluorescence polymerase chain reaction(Real-time PCR),the contents of CBZ and 10,11-epoxidation of carbamazepine (CBZE) were measured by liquid chromatography-mass spectrometry (LC-MS). Result:Compared with sham group, the expression of P-gp/MDR1 in the cortex of model group was significantly increased (PPPPPPPConclusion:Chaibei Zhixian decoction and peimine may increase the content of CBZ and CBZE in the brain tissues in rats with intractable epilepsy by reducing the expression of MDR1/P-gp in the cortex.

Analysis of Transport Mechanism of Cyperotundone in Caco-2 Cell Model / 中国实验方剂学杂志

Objective: To investigate transport mechanism of cyperotundone in Caco-2 cell model and provide experimental basis for clinical application of Cyperi Rhizoma. Method: The toxicity of cyperotundone with different concentrations to Caco-2 cells was investigated by methyl thiazolyl tetrazolium (MTT) colorimetry, in order to determine the concentration of administration in transport test. The content of cyperotundone was determined by liquid chromatography-mass spectrometry (LC-MS) with apparent permeability coefficient (Papp) and cumulative transport capacity as indexes. The chromatographic conditions were as following:mobile phase of acetonitrile (A)-water (B) for gradient elution (0-1.5 min, 35%A; 1.5-2 min, 35%-90%A; 2-4 min, 90%A; 4-4.1, 90%-35%A; 4.1-8 min, 35%A), the flow rate at 0.3 mL · min-1, injection volume of 1 μL, and temperature of column at 30℃. The mass spectrometric conditions was electrospray ionization (ESI) and positive ion mode, the detection ions of cyperotundone and osthole (internal standard substance) were m/z 219.2-110.9 and m/z 245.0-189.0, respectively. Effect of concentration of cyperotundone, administration time, ethylenediamine tetraacetic acid (EDTA) and P-glycoprotein (P-gp) inhibitor on the transmembrane transport of cyperotundone on in vitro cell model were investigated. Result: Cyperotundone didn't have significant toxicity to Caco-2 cells at 3-90 mg · L-1 after incubation for 4 h. The transportion of cyperotundone in Caco-2 cell model was related to the concentration and time to a certain extent, its Papp was higher than 1×10-6 cm · s-1, which indicated that absorption of cyperotundone was good, the efflux rate (ER) of cyperotundone was 0.5-1.5.There was no significant difference in bidirectional Papp of cyperotundone after the addition of cell bypass transport inhibitor (EDTA) and P-gp transport inhibitor (verapamil). Conclusion: The transport mechanism of cyperotundone in Caco-2 cell model is mainly passive diffusion, and cell bypass transport and P-gp are not involved in its transport.