Pgp Functional Activity Research Articles

Prevention of MDR development in leukemia cells by micelle-forming polymeric surfactant

Doxorubicin (Dox) incorporated in nanosized polymeric micelles, SP1049C, has shown promise as monotherapy in patients with advanced esophageal carcinoma. The formulation contains amphiphilic block copolymers, Pluronics, that exhibit the unique ability to chemosensitize multidrug resistant (MDR) tumors by inhibiting P-glycoprotein (Pgp) drug efflux system and enhancing pro-apoptotic signaling in cancer cells. This work evaluates whether a representative block copolymer, Pluronic P85 (P85) can also prevent development of Dox-induced MDR in leukemia cells. For in vitro studies murine lymphocytic leukemia cells (P388) were exposed to increasing concentrations of Dox with/without P85. For in vivo studies, BDF1 mice bearing P388 ascite were treated with Dox or Dox/P85. The selected P388 cell sublines and ascitic tumor-derived cells were characterized for Pgp expression and functional activity (RT-PCR, Western Blot, rhodamine 123 accumulation) as well as Dox resistance (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). The global gene expression was determined by oligonucleotide gene microarrays. We demonstrated that P85 prevented development of MDR1 phenotype in leukemia cells in vitro and in vivo as determined by Pgp expression and functional assays of the selected cells. Cells selected with Dox in the presence of P85 in vitro and in vivo exhibited some increases in IC 50 values compared to parental cells, but these values were much less than IC 50 in respective cells selected with the drug alone. In addition to mdr1, P85 abolished alterations of genes implicated in apoptosis, drug metabolism, stress response, molecular transport and tumorigenesis. In conclusion, Pluronic formulation can prevent development of MDR in leukemia cells in vitro and in vivo.

Influence of RARα gene on MDR1 expression and P-glycoprotein function in human leukemic cells

BackgroundMultidrug resistance (MDR) phenotype of malignant cells is the major problem in the chemotherapy of neoplasia. The treatment of leukemia with retinoids is aimed on the induction of leukemic cells differentiation. However the interconnections between retinoid regulated differentiation of leukemic cells and regulation of MDR remains unclear.MethodsFour lines of cultured leukemic cells of diverse types of differentiation were infected with RARα gene and stable transfectants were isolated. We investigated the differentiation of these cells as well as the expression of RARα and MDR1 genes and P-glycoprotein (Pgp, MDR protein) functional activity in these cells.ResultsAll RARα transfected sublines demonstrated the increase in the quantity of RARα mRNA. All these sublines became more differentiated. Intrinsic activity of MDR1 gene (but not Pgp functional activity) was increased in one of the transfectants. All-trans-retinoic acid (ATRA) induced Pgp activity in two of three infectants to a larger extent than in parental cells.ConclusionThe data show that RARα regulates MDR1/ Pgp activity in human leukemic cells, in the first place, Pgp activity induced by ATRA. These results show that RARα overexpression in leukemic cells could result in MDR.

Resistance to spontaneous apoptosis in acute myeloid leukaemia blasts is associated with p-glycoprotein expression and function, but not with the presence of FLT3 internal tandem duplications.

The ability of acute myeloid leukaemia (AML) blasts to survive in culture has been associated with poor patient response to chemotherapy. Other biological factors predicting an adverse outcome include p-glycoprotein (pgp) expression, which is associated with a reduced remission rate, and the presence of fms-like tyrosine kinase 3 gene (FLT3) internal tandem duplications (ITDs), predictive of a high rate of leukaemic relapse. Our previous work has indicated a drug efflux-independent role for pgp in apoptosis resistance. We measured spontaneous in vitro apoptosis in 58 primary AML samples to establish its relationship with functional and phenotypic pgp and with FLT3 ITDs. Cells were incubated for 48 h in a suspension culture, and the remaining viable cells were counted by flow cytometry. Median survival was 38% of baseline values. Resistance to spontaneous apoptosis was strongly associated with pgp (MRK-16 antibody) expression (P = 0.001) and with pgp functional activity (P < 0.001). FLT3 ITDs, found in 20 cases, were inversely associated with functional pgp activity: thus, the median pgp modulation ratio was 2.0 in FLT3 wild-type cases and 1.38 in ITD cases (P = 0.018). Also, the presence of FLT3 ITDs was not associated with in vitro apoptosis resistance. In conclusion, we have found that the presence of FLT3 ITDs is not related to AML blast survival in vitro, and is inversely associated with pgp activity, whereas pgp expression and activity are associated with resistance to spontaneous apoptosis. These results may help to explain the differing adverse effects of pgp (on remission induction) and FLT3 ITDs (on relapse) in AML.

Cytokine Regulation of P-Glycoprotein

P-glycoprotein (Pgp) is a membrane bound transporter involved in the disposition of many endogenous compounds and xenobiotics. Alterations in Pgp expression and activity can significantly affect the disposition of Pgp substrates. Infection and inflammatory stimuli have also been shown to alter drug disposition. However, the specific effects of inflammation on Pgp expression and activity are not well understood. This paper evaluates and summarizes the current literature on the effects of cytokines and inflammation on mRNA and protein expression as well as functional activity of Pgp in whole animal models, primary rodent hepatocytes and human carcinoma cell lines.

Expression of Pgp functional activity can be an additional prognostic factor determining tamoxifen efficacy in breast cancer patients

Expression of Pgp functional activity can be an additional prognostic factor determining tamoxifen efficacy in breast cancer patients

P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress.

During ischaemia/reperfusion, cells of the blood-brain barrier are subjected to oxidative stress. This study uses primary cultured rat brain endothelial cells to examine the effect of such stresses on expression of multidrug transporters. H(2)O(2) up to 500 microm applied to cell monolayers caused a concentration-dependent increase in expression of P-glycoprotein (Pgp) but not of multidrug resistance-associated protein (Mrp1). Concentrations > 250 microm H(2)O(2) decreased cell viability. Application of 100 microm H(2)O(2) caused a significant increase after 48 h in Pgp functional activity, as assessed from [(3)H]vincristine accumulation experiments. At this concentration, H(2)O(2) produced a transient increase within 10 min followed by a sustained decrease in levels of intracellular reactive oxygen species (iROS), detectable by flow cytometry. Reoxygenation of cell monolayers after 6 h hypoxia gave rise to a similar transient increase in iROS and this also led to increased Pgp expression by 24 h. Increases were also observed within 4 h after both H(2)O(2) and hypoxia/reoxygenation treatments in mdr1a and mdr1b mRNA. Evidence suggests this was due to enhanced transcription rather than mRNA stabilization. Therefore, oxidative stress, by changing Pgp expression, may affect movement of Pgp substrates in and out of the brain.

P-glycoprotein in the catfish intestine: inducibility by xenobiotics and functional properties

The p-glycoprotein (pgp)-mediated multixenobiotic resistance (MXR) mechanism of aquatic animals has been associated with protection against pollution. Recent studies in mammals suggest that intestinal pgp may modulate intestinal bioavailability of dietary xenobiotics. In order to further delineate this mechanism in the catfish, these studies: (1) examined the pgp-related distribution in the intestine and liver of catfish, (2) evaluated the MXR response following exposure to various dietary xenobiotics and a prototypic pgp inducer and (3) evaluated pgp functional activity in membrane vesicles, using prototypic substrates and inhibitors. For this purpose, catfish were exposed in vivo to the pgp inducer vincristine (VIN), and the xenobiotics β-naphthoflavone (BNF), benzo[ a]pyrene (BaP), and 3,4,3′,4′-tetrachlorobiphenyl (TCB). Membrane vesicles, prepared from liver and intestine (proximal and distal sections) of control and exposed catfish, were subjected to SDS PAGE, Western Blot, and detection with the pgp C219 monoclonal antibody. Transport activity was evaluated in vitro using the pgp substrate [ 3H]vinblastine (VBL), and the pgp inhibitor verapamil (VP). Immunoblot studies demonstrated a pgp-related protein of approximately 170 kDa in the intestine and liver of catfish. This protein appears to be very susceptible to degradation, and was present in higher levels in the liver, in comparison to the intestine, where regional differences were not observed. Dietary exposure to the pgp substrate VIN, or the xenobiotics BNF, BaP, and TCB, did not appear to affect pgp-related reactivity. Transport studies with VBL indicate that the pgp-related protein of the catfish intestine displays classic pgp-mediated multidrug resistance (MDR) characteristics, such as energy-dependency, and sensitivity to VP. These studies suggest that the pgp-related protein in the catfish intestine and liver is not only immunochemically, but also functionally related to the mammalian MDR. Moreover, the results presented indicate that pgp-related reactivity and transport in intestinal vesicles of catfish may be influenced by factors including method sensitivity, sample collection, sample preparation, and immunoblot conditions.

#405 Evaluation of the expression and functional activity of PGP, MRP and LRP in pediatric acute lymphoblastic leukemia

#405 Evaluation of the expression and functional activity of PGP, MRP and LRP in pediatric acute lymphoblastic leukemia

Studies of P‐glycoprotein in chronic myelogenous leukaemia patients: expression, activity and correlations with CD34 antigen

Over-expression of the P-glycoprotein (Pgp), transmembrane drug efflux pump, has been shown to cause multidrug resistance of tumour cells (MDR). To investigate the clinical significance of Pgp expression for chronic myeloid leukaemia (CML) diagnosis and monitoring we have studied 38 CML patients in various phases of the disease (chronic phase, CP; accelerated phase, AP; blast crisis, BC). Anti-Pgp monoclonal antibody UIC2 and FACScan analysis were used. Pgp functional activity was investigated by evaluation of verapamil influence upon rhodamine 123 efflux from the cells. Correlations between Pgp and CD34 expression were investigated. In CP, Pgp-expressing cells were found in 2/14 patients; in one of them Pgp proved to be non-functional. There were few Pgp-expressing cells in AP cases. The group of BC patients consisted of cases resistant to chemotherapy. This gave us the opportunity to consider whether drug resistance of BC CML patients is preferentially connected with Pgp-mediated MDR. 11/22 BC patients had 20% or more of Pgp-expressing blasts in the peripheral blood. In all four Pgp+ BC cases studied for Pgp activity this protein was functional. Only 4/22 BC patients demonstrated large (40% or more) fractions of Pgp+ blasts. Moreover, sequential studies of 11 BC CML patients during treatment revealed an increase in the number of Pgp-expressing cells in only two cases. This suggests that Pgp+ cells did not often accumulate in BC CML patients due to chemotherapy and are the cause of drug resistance in only a few cases. A positive correlation between Pgp and CD34 expression was found (r = 0.69; P = 0.0004). 3/22 BC CML patients had large fractions of both Pgp+ and CD34+ blasts in their peripheral blood. The BC CML patients with this immunophenotype of blast cells may represent a subtype of BC CML resistant to treatment due to Pgp overexpression.