Effects of Hydrophobic Structure of Amphiphilic Polymers on the Activity of P-glycoprotein in MDCK-MDR1 Cell / 中国药学杂志

OBJECTIVE: To determine the effects of the amphiphilic block polymers, which have the same hydrophilic block with the different hydrophobic block, on the function of P-glycoprotein(P-gp). METHODS: The three different micelles were prepared by film dispersion method. The particle sizes and distributions were measured by dynamic light scattering. Critical micelle concentrations(CMC) were detected by fluorescence probe technique with the pyrene. Rhodamine 123, a specific probe substrate of P-gp, was applied to determine the effects of polymers on the function of P-gp using uptake and efflux method. RESULTS: The particle sizes of mPEG-PCL, mPEG-PDLLA, mPEG-PLGA were (55.9±0.2), (53.7±1.1) and (61.6±0.6)nm. The CMC values were 2.08, 5.42 and 26.4 μg·mL-1. R123 accumulation in Madin-Darby canine kidney/multidrug resistance 1(MDCK-MDR1)cell detected by uptake assay increased to a maximum in the presence of polymers at concentrations of 250 μg·mL-1 for mPEG-PCL, 1~25 μg·mL-1 for mPEG-PDLLA and mPEG-PLGA. In efflux assay, mPEG-PCL, mPEG-PDLLA, mPEG-PLGA decreased the percentage of efflux of R123 at concentrations above the CMC, below/at the CMC or below the CMC respectively, showed the similar RESULTS with uptake assay. CONCLUSION: The mPEG-PCL, mPEG-PDLLA, mPEG-PLGA polymers might have a potential to inhibit the efflux activity of P-gp, which was likely related to the structures of hydrophobic segments, concentrations and existing states of the polymers.

Inhibitory Effects of Deoxyschizandrin on P-glycoprotein in Vitro and in Vivo / 医药导报

Objective To investigate the influence of deoxyschizandrin (Deo) on P-glycoprotein (P-gp).Methods The effect of P-gp on Deo (20,40,80 μg·mL-1) was studied in the Caco-2 cell model in vitro,and the apparent permeability coefficient (Papp) of Deo (20-160 μg·mL-1) on a P-gp substrate,rhodamine123 or cyclosporine A,was calculated.Healthy male Sprague-Dawley rats were randomly divided into five groups:blank control group,verapamil group,low-,medium-and high-dose Deo group (8 rats in each group).Rats in the low-,medium-and high-dose Deo group were intragastrically administered once daily with Deo at 8,16 and 32 mg·kg-1 for 3 consecutive days,while rats similarly received gavagewith verapamil (4 mg·kg-1) in the verapamil group and equal volume of purified water in the blank control group.Thirty minutes after the rats were treated with their respective drugs,rhodamine123 (5 mg· kg-1) was orally administrated.Then the pharmacokinetic profiles of rhodamine 123 were analyzed to evaluate the inhibitory ability of Deo on P-gp in vivo.Results The bidirectional transport rates of Deo (20,40,80 μg·mL-1) were similar,with non-selectivity.Deo (20-160 pg·mL-1)significantly inhibited the basolateral→apical(BL→AP) directional transports of rhodamine 123 and cyclosporine A in Caco-2 cell model (P ＜ 0.05) in a concentration-dependent manner.And Deo (8-32 mg· kg-1) also dose-dependently decreased the peak concentrations (Cm.) and the area under the plasma concentration-time curve (AUC0-t) of Rho123.Conclusion Deo can inhibit P-gp in vitro and in vivo,but it is not a P-gp substrate.

Determination of Rhodamine 123 in Rat Plasma by High Performance Liquid Chromatography / 医药导报

Objective To establish a sensitive method for the determination of rhodamine 123 (Rh123) in rat plasma by high performance liquid chromatography (HPLC).Methods The plasma samples were extracted by acetonitrile,and then separated on a Hypersil BDS C18 colunm (4.6 mm×100 mm,5 μm) equipped with a guard column kept at 25 ℃.The mobile phase consisted of acetonitrile and phosphate buffer (0.02 mol·L-1,pH4.0) (60:40) and was pumped at a constant rate of 1.0 mL·min-1.The peak was detected using a fluorescence detector set at FLD1A:Ex=485 nm,Em=546 nm.Results In this study,the method was validated for the Rh123 range of 0.1 to 32.0 μg·L-1,and the lower limit of quantitation (LLO Q) was 0.1 μg·L-1.The intra-and inter-day precisions for Rh123 were less than 7%,and the mean recoveries of Rh123 were 87.93%,89.03%,86.11% at low,mid,and high concentrations,respectively.Conclusion A simple,rapid and reproducible HPLC method was developed for the determination of Rh123 in rat plasma,which was an applicable method in modeling and description of the possible pharmacological interactions between the medicines and P-glycolprotein transporter.

Effect of Scutellarin on the Expression and Activity of P-gp in Caco-2 Cells / 中国药师

Objective:To investigate the effect of scutellarin on P-gp protein expression and activity in Caco-2 cells. Methods:Scutellarin(25,50 and 100 μmol·L-1 )was incubated with Caco-2 cells respectively for 24 h,48 h and 72 h. The expression of P-gp was determined by western blot assay and the activity of P-gp was determined by Rhodamine-123 assay. Results:P-gp protein ex-pression levels were significantly increased by scutelarin. After the incubation for 24 h with scutellarin,P-gp protein expression was up-regulated 2. 34-,2. 65-and 2. 00-fold in Caco-2 cells. After the incubation with scutellarin for 48 h,P-gp protein expression was up-regulated 2. 70-,4. 66-and 3. 13-fold. After the incubation with scutellarin for 72 h,P-gp protein expression was up-regulated 2. 82-, 2. 62-and 1. 84-fold. The intracellular accumulation of rhodamine-123 was significantly decreased by scutellarin,indicating that the ef-flux transport activity of P-gp was increased by scutellarin in Caco-2 cells. Conclusion:Scutellarin can significantly up-regulate P-gp protein expression and increase the efflux transport activity of P-gp in Caco-2 cells.

Effects of polyethylene glycols on intestinal efflux pump expression and activity in Caco-2 cells

The present study was planned to investigate the influence of polyethylene glycols (PEGs) on the activity and expression of P-glycoprotein (P-gp). Sub-toxic concentrations of PEGs in Caco-2 cells were determined using the MTT test assay. Then the measurement of Rhodamine-123 (Rho-123) uptake, a P-gp fluorescence substrate, in Caco-2 cells confronting PEG 400 (1% and 2% w/v), PEG 4000 (2% and 4% w/v), PEG 6000 (2% and 4% w/v), PEG 10000 (2% and 4% w/v), PEG 15000 (1% and 2% w/v), and PEG 35000 (2% and 4% w/v) overnight was taken to elucidate whether non-toxic concentrations of PEGs are able to impact P-gp activity. Furthermore, western blotting was carried out to investigate P-gp protein expression. The results showed that PEG 400 at concentrations of 1% (w/v) and 2% (w/v) and PEG 6000 at the concentration of 4% (w/v) are notably capable of blocking P-gp. Based on the obtained results it is concluded that the mentioned excipients could be used to obstruct P-gp efflux transporter in order to increase the bioavailability of co-administered substrate drug.

O presente estudo foi planejado para investigar a influência de polietileno glicóis sobre a atividade e expressão da P- glicoproteína (P-gp) . Concentrações sub-tóxicas de PGPs e em células Caco-2 foram determinadas por meio do ensaio de MTT. Em seguida, efetuou-se a a medida de captura de Rodamina-123 (Rho-123), um substrato fluorescente de P-gp, em células Caco-2, confrontando com PEG 400 (1% e 2% m/v), PEG 4000 (2% e 4% m/v) e PEG 6000 (2% e 4% m /v), PEG 10000 (2% e 4% w/v), PEG 15000 (1% e 2% m/v), e PEG 35000 (2% e 4% m/v). Essa medida foi efetuada durante a noite, para saber se as concentrações não tóxicas de excipientes são capazes de influenciar a actividade da P-gp. Além disso, realizou-se o western blotting para investigar a expressão da proteína P-gp. Os resultados mostraram que o PEG 400, nas concentrações de 1% (m/v) e 2% (m/v), e PEG 6000, na concentração de 4% (m/v) são capazes de bloquear P-gp. Com base nos resultados conclui-se que os excipientes mencionados poderiam ser utilizados para obstruir o efluxo por P-gp, a fim de aumentar a biodisponibilidade de do fármaco co-administrado.

Effect of glycyrrhetinic acid C-18 epimerides on membrane transport of P-gp substrate rhodamine 123 in Caco-2 cell monolayers / 中国药学杂志

OBJECTIVE: To study the effects of C-18 epimers of glycyrrhetinic acid, 18α-glycyrrhetinic acid(α-GA) and 18β-glycyrrhetinic acid(β-GA), on membrane transport of P-gp substrate rhodamine 123 in Caco-2 cell monolayers. METHODS: MTT assay was applied to determine the maximum non-cytotoxic dose of α-GA, β-GA and verapamil to ensure the activity of the cells during the test and to consult the maximum test concentration of each drug; appropriate Caco-2 monolayers were established in Transwell™ plates. ELISA Reader was applied to detect the concentration of Rho-123 in transfer fluid. The bi-directional transport of Rho-123 after being treated by instantaneous action and incubation with α-GA and β-GA for 72 h was studied. RESULTS: High concentration (10 μmol · L-1) of α-GA induced the efflux of Rho-123 both in the instantaneous action test and the incubation test. High concentration (10 μmol · L-1) of β-GA induced the efflux of Rho-123 only in the incubation test. The transport from AP to BL was not affected by any test drugs. CONCLUSION: The results of the transport experiments of P-gp substrates in Caco-2 monolayers indicated that α-GA and β-GA can induce the efflux function of P-gp and accelerate the excretion of P-gp substrates. It may be one of the mechanisms of that glycyrrhizin preparation enhances liver detoxification. Copyright 2012 by the Chinese Pharmaceutical Association.

Modest isolation and enrichment of intestinal epithelial stem cells by rhodamine 123 Staining / 国际外科学杂志

Objective To discuss the technology of modest isolating and enriching the intestinal epithelial stem cells. Methods Mouse intestinal epithelial cells were stained by Rhodamine 123 (Rho), sorting the Rhodamine 123 low staining cell population ( Rholow ) and Rhodamine 123 strong straining cell population ( Rhobri ) by fluorescence activated cell sorting(FACS) in flow cytometer; Detecting the musaashi-1 and p-glycoprotein 1 (p-g-p1) mRNA expression of two groups by RT-PCR; Analyzing the cell cycle and the percentage of the musashi-1 positive cells by flow cytometry. Results The intestinal epithelial cells were divided into three groups, Rhodamine 123 low staining cells( 12. 34% of total cells), Rhodamine 123 middle staining cells (45.26% of total cells) and Rhodamine 123 strong staining cells ( 41. 40% of total cells). The Rholow cell fraction and Rhobri cell fraction were isolated successfully. Both of musashi-1 and p-g-p1 mRNA were strongly expressed in Rho1ow cell fraction, and Rhobri cell fraction little expressed p-g-p1 mRNA. Most of Rho1ow cells were in G0/G1 phase, and the musashi-1 positive cells were about 10.37% of total cells in this fraction. Conclusion The intestinal epithelial stem cells can be modestly isolated and enriched by Rhodamine 123 staining.

Lomerizine inhibited the function of P-glycoprotein(P-gp) without decreasing the expression of mdr1 gene and P-gp in primarily cultured rat brain microvessel endothelial cells / 中国临床药理学与治疗学

AIM: To study the effect of Lomerizine on the activity of P-glycorprotein (P-gp) in primary cultured rat brain microvessel endothelial cells (RBMECs). METHODS: Flow cytometry was used to study the efflux of rhodamine123 (Rh123) and expression of P-gp in RBMECs. RT-PCR was used to measure the expression in mRNA level of mdr1 gene in RBMECs. Transwell model was used to detect the influence of Lomerizine on the transport of Rh123 through RBMECs monolayer. RESULTS: Lomerizine inhibited the efflux of Rh123 in RBMECs. No changes of P-gp and mdr1 gene mRNA expression were detected in RBMECs after the treatment with 30 μmol·L-1 Lomerizine for 72 h. In the study of Transwell model, Lomerizine increased significantly the transport of Rh123 through RBMECs monolayer from upper compartments to lower compartments, and inhibited obviously the transport in reverse direction. CONCLUTION: The effect of Lomerizine on the activity of P-gp was mainly via its direct inhibitory effect on the function of P-gp in RBMECs and the transport of P-gp substrates in BBB may be affected by lomerizine.

Significance of Multidrug Resistance (MDR) Activity in Remission Induction & Survival of Acute Myeloid Leukemia / 대한혈액학회지

BACKGROUND: The significant improvement in the treatment of adults with acute myeloid leukemia (AML) has been achieved in recent years. However, many patients still fail to achieve a complete remission and long term survival because of either toxic death during aplasia periods of induction chemotherapy or resistance to induction chemotherapy. The P-glycoprotein (Pgp) associated with Multidrug Resisitance (MDR) gene is the best characterized mechanism of resistance to induction chemotherapy. In this study, the authors effort to examine the functional activity of Pgp using the rhodamine123 functional efflux assay and discuss for the predictive value of MDR functional assay for treatment outcomes of AML. METHODS: Between January 1996 and June 2003, 45 patients with AML were enrolled in this study. For evaluation of functional MDR activity using the rhodamine123 functional efflux assay, mononuclear cells isolated from bone marrow aspirates of 45 patients were used. All patients were received induction chemotherapy and consolidation therapy with high dose chemotherapy or stem cell transplantation. RESULTS: Among the 45 AML patients, 30 (66.7%) patients showed positive functional MDR activity and 15 (33.3%) patients negative functional MDR activity. Complete remission rate was lower in the group with positive functional MDR activity than negative, but no statistical significance was observed (P=0.453). Survival time in both groups was investigated. Leukemia free survival was 40.9 months in negative group and 18.7 months in positive group (P=0.336). Overall survival was 48.5 months and 26.6 months respectively (P=0.513). CONCLUSION: The functional MDR activity using the rhodamine123 functional efflux assay does not significantly affect induction rate and survival rate of AML patients.

Effect of Cyclosporin A and Tetrandrine on rhodamine 123 accumulationin in bovine brain capillary endothelial cells / 中国药理学通报

Aim To study the effect of cyclosporin A and tetrand ri ne on P-glycoprotein (P-gp)of bovine brain capillary endothelial cell. M ethods The fluorescent dye, rhodamine-123 (Rh-123) was used to evaluate t he functional activity of the P-glycoprotein (P-gp) efflux transport system in primary cultured bovine brain capillary endothelial cell (BCEC) monolayer. Results Rhodamine-123 accumulation was increased significantly in monola yer treated with the P-gp modifying agent, cyclosporin A and tetrandrine. Conclusion The observation suggests that this Rh-123 method is sens itive, stable to evaluate the function of P-gp of blood-brain barrier (BBB). R h-123 accumulation is also increased by tetrandrine in dose-dependent manner.

DYNAMIC ANALYSIS OF RHODAMINE 123 STAINING OF HUMAN BRONCHIAL EPITHELIUM DURING THE WOUND REPAIR PROCESS INDUCED BY FLUOROURACIL / 解剖学报

Objective To analyze the condition of Rhodamine 123 staining of human bronchial epithelium in vitro during the wound-repair process induced by fluorouracil(5-FU). Methods The bronchial epithelial cells in normal and 5-FU injurious group were obtained by enzymatic digestion and analyzed by flow cytometry.1.PI staining to identify the percentage of cells in different cell phase;2.Rhodamine 123 staining in live cells was to contrast the percentage of negative cells in two groups. Results 1.Analysis of cell cycle:apoptotic cells in injurious group increased,cells in S+G-2/M phase almost disappeared,majority of the remain live cells of injurious group were in G-0/G-1 phase;with the recover of bronchial epithelium,cells in S+G-2/M phase increased;2.The percentage of Rhodamine 123 negative staining live cells in injurious group was higher than that of normal group,and with the recover of bronchial epithelium,this percentage lowered to nearly normal.Conclusion 5-FU can make the cycling cells apoptosis and reserve quiescent phase cells.Among them there were bronchial stem cells which had the capacity to efflux Rhodamine 123.Just the proliferation and differentiation of these stem cells regenerated bronchial epithelium.Rhodamine 123 staining to sort the remain cells after 5-FU treatment by FACS can be used to enrich and purify bronchial stem cells.

Implication of intracellular rhodamine-123 distribution model in bladder cancer resistant cells differing from sensitive cells / 中华泌尿外科杂志

Objective To observe the change of intracellular rhodamine 123 distribution model in bladder cancer sensitive cells(T24) and resistant cells(TADM),and to explore the cause of the change and its relation with multidrug resistance(MDR). Methods T24 and TAMD cells were cultured together with rhodamine 123 for a certain period.The intracellular distribution and intensity of fluorescence were detected by interactive laser cytometer(ILC). Results In T24 cells,rhodamine 123 was located mainly in the perinuclear membrane area,and in TADM cells,rhodamine 123 was concentrated mainly at the two poles of nucleus.After the extracellular rhodamine 123 was washed,in T24 cells ,the intracellular fluorescent intensity decayed slowly.It took more than 40 minutes for T24 cells to eliminate intracellular rhodamine 123 completely.By contrast,in TADM cells, the intracellular fluorescent intensity decayed rapidly.It took only 15 minutes for TADM cells to eliminate intracellular rhodamine 123 completely. Conclusions (1)Intracellular rhodamine 123 was rapidly pumped out,which is the main cause of MDR in TADM;(2)The intracellular rhodamine 123 distribution model in TADM differ from that in T24,which may be another cause of its MDR phenotype.

Best protecting dosage of phycocyanin on PC12 cells after anoxia/re-oxygen in vitro / 中国海洋药物

Objective To study the best protecting dosage of phycocyanin on PC12 cells after anoxia/re-oxygen in vitro.Methods The anoxia/re-oxygen cell models were established with cultured PC12 cells and treated by phycocyanin.The absorbency of PC12 cells was determined by MTT method to observe the activity and number of PC12 cells after anoxia/re-oxygen.The fluorescent intensity in PC12 cells labeled by Rhodamine123 was measured with laser scanning confocal microscope to observe the mitochondrial membrane potential after anoxia/re-oxygen.Results After anoxia/re-oxygen,the absorbency and fluorescent intensity in PC12 cells were significantly lower than those in control group.After treatment with phycocyanin,the absorbency of PC12 cell in test groups was higher than that in model group,especially in 5?g?mL-1 subgroup,P

The prevention of acute graft versus host disease by Rhodamine 123-mediated photodynamic therapy / 中国病理生理杂志

AIM: To evaluate the effect and safety of Rhodamine 123 (Rh123)-mediated photodynamic treatment (PDT) on acute graft versus host disease. METHODS: An acute graft versus host disease (aGVHD) mice model was established using C57B/6 mice as donors and BALB/c mice as recipients. Mixed lymphocytic cells were cultured with Rh123 (50 nmol/L) and irradiated by argon laser 30 mW/cm2 for 3 min, then transplanted to BALB/c recipient mice mixed with donor bone marrow. Hepatopoietic recovery, aGVHD occurrence, survival time after transplantation and pathological changes were observed. In addition, CD3+CD69+ positive rates of MLC were examined by flowcytometry. RESULTS: Occurrence of aGVHD decreased, degree of pathological manifestation became milder, survival rates were higher than non PDT groups. CD3+CD69+ rates of MLC cells treated with photodynamic therapy (PDT) and cultured for 24 h significantly decreased. CONCLUSION: Rh123-mediated PDT can effectively prevent aGVHD of allogeneic bone marrow transplantation in mice.

PHOTODYNAMIC EFFECT OF HEMATOPORPHYRIN DERIVATIVE (HPD) ON MITOCHONDRIA AND SUCCINATE DEHYDROGENASE OF HUMAN GASTRIC CANCER CELLS IN DIFFERENT PHASES OF CELL CYCEL / 解剖学报

Synchronous cells in different phase (G_1, S, G_2, M) of cell cycle were obtained from human gastric low-differentiated mucous adenocarcinoma cells (MGC 80-3), which werec ultured with nitrous oxide under high pressure and blocked with overdosage of TdR. Dynamic changes of the mitochondria stained with Rhodamine-123 and succinate dehydrogenase demonstrated by cytochemical method were observed in various phase of the cells at 0, 24, 36, 60 hours after treatment with HPD plus red light. The results showed that mitochonodria of all four phases are of impairment immediately after photoradiation, SDH reactivity is decreased slightly at 24 hours, the activities of SDH is the weakest. As time goes on, we observed that mitochondria gradually recovered to its original structure and then SDH returned to its normal level. The recovery rate of mitochondria and SDH was in the following order, i. e. S, G_1, G_2, andM. The relationship between these changes and cell killing is briefly discussed.

SELECTIVE TOXICITY OF RHODAMINE 123 ON CARCINOMA CELL IN VITRO / 解剖学报

The selective toxicity of the mitochondria-specific cationic fluorescent dye rhodamine 123 (Rh-123) on HeLa cells in culture was studied. In this report, we demonstrate that with continuous exposure, Rh-123 markedly inhibited the growth of HeLa cells but had little effect on normal human kidney fibroblasts. With continuously exposed to Rh-123, the growth rate, colony forming ability, and mitotic index of HeLa cells were decreased. The mechanism of toxicity of Rh-123 on HeLa ceils was investigated by EM and enzyme cytochemistry stain. The mitochondria of carcinoma cells were the main targets for the inhibitory action of Rh-123, since they selectively accumulated the dye. At the dosage of Rh-123 which was toxic to HeLa cells, the structure and function of mitochondria were disrupted, as the mitochondriarelated enzymes, i. e., ATPase, LDH, and SDH were inhibited. The possible mechanism of the action of Rh-123 on HeLa cells is briefly discussed.