Stromal cells extracted from ovarian cancer express functional multi drugs resistance proteins: ATP bindig casset and Major vault protein

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2072-2072
Author(s):  
A. Rafii ◽  
P. Mirshahi ◽  
A. Simon ◽  
A. Faussat ◽  
E. Ducros ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ovarian Carcinoma ◽  
Stromal Cells ◽  
Major Vault Protein ◽  
Cancer Resistance ◽  
Rt Pcr ◽  
Resistance Proteins ◽  
Lung Resistance Protein ◽  
Resistance Protein ◽  
Related Proteins

2072 Background: Stromal cells play a central role for the growth of tumor cells. The functional contribution of these cells in cancer therapy is poorly understood. Here we studied the presenceofthe proteins ABC (ATP binding Cassette) and MVP (Major vault protein) implicated in the multi drugs resistance (MDR) phenomena in stromal cells isolated from ascitis of patients with ovarian carcinoma. Methods: Stromal cells were extracted from ascitis of patients with ovarian carcinoma. The expression of MDR proteins as p-gp (Permeability-glycoprotein), BCRP (breast cancer resistance protein) and MRPs (multidrug related proteins) as well as LRP (lung resistance protein that is a MVP) was studied by two different technique (immunocytochemistry and flow cytometry) using specific antibodies against these proteins. The functionality of the pumps or efflux, was studied by incorporation of fluorescent probes, Rhodamine 123 and JC1, substrate for p-gp, calcéine-AM substrate for p-gp and MRPs and at last Mitoxantrone substratum of BCRP, in the presence of specific inhibitors: as the cyclosporine HAS, the GG918 and the MK571 for the pumps Pgp, BCRP and MRPs respectively. Then the expression of the genes was assessed by RT-PCR. Results: 1) The expression of the proteins ABC is confirmed by immunocytochemistry and by flow cytometry. The Pgp and LRP proteins were strongly expressed and they are functional, the MRP-1, 2, 3 and BCRP proteins are weakly expressed and the MRP-5 protein is not detected. 2) The RNAm corresponding to all of these proteins is found by RT-PCR in the stromal cells. Conclusions: All of these results suggest that the MDR proteins are present on the cells surface of the tumour cell microenvironnement. The functionality of these proteins allows supposing their implication in the phenomena of multi drugs resistance to chemotherapy. The interaction between cancer cells and stromal cells should be targeted during specific chemotherapy. No significant financial relationships to disclose.

scholarly journals Multiple drug resistance protein (MDR-1), multidrug resistance-related protein (MRP) and lung resistance protein (LRP) gene expression in childhood acute lymphoblastic leukemia

2004 ◽  
Vol 122 (4) ◽  
pp. 166-171 ◽  
Author(s):  
Elvis Terci Valera ◽  
Carlos Alberto Scrideli ◽  
Rosane Gomes de Paula Queiroz ◽  
Bianca Maria Ortelli Mori ◽  
Luiz Gonzaga Tone
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Multiple Drug Resistance ◽  
Lymphoblastic Leukemia ◽  
Multiple Drug ◽  
Rt Pcr ◽  
Free Survival ◽  
Lung Resistance Protein ◽  
Expression Of Genes ◽  
Resistance Protein ◽  
Mdr 1

CONTEXT: Despite the advances in the cure rate for acute lymphoblastic leukemia, approximately 25% of affected children suffer relapses. Expression of genes for the multiple drug resistance protein (MDR-1), multidrug resistance-related protein (MRP), and lung resistance protein (LRP) may confer the phenotype of resistance to the treatment of neoplasias. OBJECTIVE: To analyze the expression of the MDR-1, MRP and LRP genes in children with a diagnosis of acute lymphoblastic leukemia via the semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and to determine the correlation between expression and event-free survival and clinical and laboratory variables. DESIGN: A retrospective clinical study. SETTING: Laboratory of Pediatric Oncology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. METHODS: Bone marrow aspirates from 30 children with a diagnosis of acute lymphoblastic leukemia were assessed for the expression of messenger RNA for the MDR-1, MRP and LRP genes by semi-quantitative RT-PCR. RESULTS: In the three groups studied, only the increased expression of LRP was related to worsened event-free survival (p = 0.005). The presence of the common acute lymphoblastic leukemia antigen (CALLA) was correlated with increased LRP expression (p = 0.009) and increased risk of relapse or death (p = 0.05). The relative risk of relapse or death was six times higher among children with high LRP expression upon diagnosis (p = 0.05), as confirmed by multivariate analysis of the three genes studied (p = 0.035). DISCUSSION: Cell resistance to drugs is a determinant of the response to chemotherapy and its detection via RT-PCR may be of clinical importance. CONCLUSIONS: Evaluation of the expression of genes for resistance to antineoplastic drugs in childhood acute lymphoblastic leukemia upon diagnosis, and particularly the expression of the LRP gene, may be of clinical relevance, and should be the object of prospective studies.

scholarly journals Isolation and Characterization of Human Synovial Fluid-Derived Mesenchymal Stromal Cells from Popliteal Cyst

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Fang Li ◽  
Jianglin Chen ◽  
Mengjia Gong ◽  
Yang Bi ◽  
Chengchen Hu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Synovial Fluid ◽  
Stromal Cells ◽  
Pediatric Patients ◽  
Cyst Fluid ◽  
Popliteal Cyst ◽  
Cell Marker ◽  
Related Proteins

Mesenchymal stem cells (MSCs) are multipotent progenitor cells in adult tissues. The aim of this study is to isolate and identify synovial fluid-derived mesenchymal stromal cells (SF-MSCs) from the popliteal cyst fluid of pediatric patients. SF-MSCs were collected from the popliteal cyst fluid of pediatric patients during cystectomy surgery. After cyst fluid extraction and adherent culturing, in vitro morphology, growth curve, and cell cycle were observed. The expression of stem cell surface markers was analyzed by flow cytometry, and expression of cell marker protein was detected by immunofluorescence. SF-MSCs were cultured in osteogenic, adipogenic, and chondrogenic differentiation medium. The differentiation potential of SF-MSCs was analyzed by alkaline phosphatase (Alizarin Red), Oil Red O, and Alcian blue. Antibody detection of human angiogenesis-related proteins was performed compared with bone marrow mesenchymal stem cells (BM-MSCs). The results show that SF-MSCs from the popliteal cyst fluid of pediatric patients showed a shuttle appearance and logarithmic growth. Flow cytometry analysis revealed that SF-MSCs were negative for hematopoietic lineage markers (CD34, CD45) and positive for MSC markers (CD44, CD73, CD90, and CD105). Interstitial cell marker (vimentin) and myofibroblast-like cell marker alpha-smooth muscle actin (α-SMA) were positive. These cells could differentiate into osteogenic, adipogenic, and chondrogenic lineages, respectively. Several types of human angiogenesis-related proteins were detected in the cell secretory fluid. These results show that we successfully obtained SF-MSCs from the popliteal cyst fluid of pediatric patients, which have the potential to be a valuable source of MSCs.

MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3536-3536
Author(s):  
Neeta Shirvaikar ◽  
Leah A. Marquez-Curtis ◽  
Andrew Shaw ◽  
A. Robert Turner ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Flow Cytometry ◽  
Steady State ◽  
Western Blot ◽  
Lipid Rafts ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Membrane Lipid ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3536 Poster Board III-473 Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34+ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34+ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34+ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34+ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34+ cells is necessary for proMMP-2 activation as media conditioned by CD34+ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation. Disclosures: No relevant conflicts of interest to declare.

Treatment Failure Is Strongly Predicted by P-Glycoprotein (Pgp) Function but Not by Multidrug Resistance Protein (MRP-1), Breast Cancer Resistance Protein (BCRP) or Lung Resistance Protein (LRP) in Acute Myeloid Leukemia (AML) Patients 60 Years and Older Receiving Intensive Chemotherapy (CALGB 9720/9760).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2349-2349 ◽  
Author(s):  
Maria R. Baer ◽  
Nicholas W. Cuviello ◽  
Jennifer S. Shoemaker ◽  
Robert C. Barrier ◽  
Michael A. Caligiuri ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Odds Ratio ◽  
De Novo ◽  
Efflux Pump ◽  
Risk Groups ◽  
Drug Efflux ◽  
Cancer Resistance ◽  
Lung Resistance Protein ◽  
Psc 833 ◽  
Resistance Protein

Abstract Older AML patients have low complete remission (CR) rates and short disease-free (DFS) and overall (OS) survival; unfavorable karyotypes, prior myelodysplastic syndrome (MDS) and multidrug resistance (MDR) mediated by the cellular drug efflux pump Pgp have been implicated. The significance of other MDR proteins expressed in AML cells, including the cellular drug efflux pumps MRP-1 and BCRP and the major vault protein LRP, is unknown. We correlated MDR parameters with response in 170 previously untreated AML patients ≥60 years old receiving ADE (cytarabine 100 mg/m2, daunorubicin 60 mg/m2 and etoposide 100 mg/m2 induction for 7+3+3 days and consolidation for 5+2+2 days) (CALGB 9720). Pretreatment blasts were studied by flow cytometry for expression of Pgp, MRP-1, BCRP and LRP with the MRK-16, MRPm6, BXP-21 and LRP56 antibodies; for efflux of mitoxantrone, a substrate for Pgp, MRP-1 and BCRP; and for modulation of mitoxantrone efflux by the Pgp, MRP-1 and BCRP modulators PSC-833, MK-571 and fumitremorgin C (FTC) and by cyclosporine A (CsA), which modulates all three proteins (CALGB 9760). Karyotypes of 141 patients (83%) were centrally reviewed and classified as favorable, intermediate or adverse with respect to likelihood of CR, cumulative incidence of relapse (CIR) and OS (Blood2002;100:4325). Ages were 60 to 89 years (median=71). 135 patients (79%) had de novo AML, 35 (21%) prior MDS and 9 (5%) therapy-related AML. Karyotypes were favorable for CR in 2 (1%), intermediate in 105 (74%), adverse in 23 (16%) and unclassified in 11; favorable for CIR in 2 (1%), intermediate in 92 (65%), adverse in 31 (22%) and unclassified in 16; and favorable for OS in 2 (1%), intermediate in 88 (62%), adverse in 40 (28%) and unclassified in 11. CR rate was 46% and median DFS and OS 6.2 (95% CI:5.0–8.8) and 8.1 (95% CI:5.3–9.5) months. Karyotype risk groups (favorable/intermediate vs. adverse) were associated with CR (n=130; p=0.0105) but not with DFS or OS, and age and prior MDS were not associated with CR, DFS or OS. Pgp, MRP-1, BCRP and LRP were expressed in 31, 39, 50 and 52% of samples. Mitoxantrone efflux occurred in 68% and was inhibited by PSC-833, MK-571, FTC and CsA in 30, 5, 7 and 35%. No MDR parameter correlated with age, prior MDS or karyotype risk groups. CR rate, DFS and OS did not differ in patients with (n=115) vs without efflux, but patients with, vs without, PSC-833 modulation of efflux, indicating Pgp function, were less likely to achieve CR (24% vs. 55%; p=0.0022) and had shorter OS (p=0.0066). In multivariate analysis adjusting for karyotype risk, patients (n=88) with PSC-833 efflux modulation remained less likely to achieve CR (odds ratio=0.21; 95% CI:0.07–0.64). Patients with efflux with, vs without, CsA modulation were also less likely to achieve CR (25% vs. 57%; p=0.0015), even adjusting for karyotype risk (odds ratio=0.29; 95% CI:0.11–0.78), likely due to effect on Pgp. MK-571 and FTC modulation did not predict CR or OS, and no MDR parameter predicted DFS. Thus Pgp function is the only MDR parameter associated with outcome, and strongly predicts CR induction failure. These data support development of Pgp modulators and modulation regimens that can be safely administered to older patients with AML with Pgp function.

scholarly journals Enantioselective Drug Recognition by Drug Transporters

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3062 ◽  
Author(s):  
Yuichi Uwai
Keyword(s):  
Drug Transporters ◽  
Organic Anion ◽  
Cancer Resistance ◽  
Multidrug Resistance Proteins ◽  
Inhibitory Effects ◽  
Organic Anion Transporting Polypeptides ◽  
Resistance Proteins ◽  
Anion Transporters ◽  
Resistance Protein ◽  
The Relationship

Drug transporters mediate the absorption, tissue distribution, and excretion of drugs. The cDNAs of P-glycoprotein, multidrug resistance proteins (MRPs/ABCC), breast cancer resistance protein (BCRP/ABCG2), peptide transporters (PEPTs/SLC15), proton-coupled folate transporters (PCFT/SLC46A1), organic anion transporting polypeptides (OATPs/SLCO), organic anion transporters (OATs/SLC22), organic cation transporters (OCTs/SLC22), and multidrug and toxin extrusions (MATEs/SLC47) have been isolated, and their functions have been elucidated. Enantioselectivity has been demonstrated in the pharmacokinetics and efficacy of drugs, and is important for elucidating the relationship with recognition of drugs by drug transporters from a chiral aspect. Enantioselectivity in the transport of drugs by drug transporters and the inhibitory effects of drugs on drug transporters has been summarized in this review.

Differential Expression of P-Glycoprotein, but Not of MRP, LRP and BCRP in Leukemic Stem Cells Compared to More Differentiated CD34+ CD38+ Acute Myeloid Leukemia Blasts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2360-2360
Author(s):  
Lorena L. Figueiredo-Pontes ◽  
Maria C. Pintao ◽  
Luciana C. Oliveira de Oliveira ◽  
Leandro F. Dalmazzo ◽  
Rafael H. Jacomo ◽  
...  
Keyword(s):  
Stem Cell ◽  
De Novo ◽  
High Expression ◽  
Cancer Resistance ◽  
Differentiation Potential ◽  
Lung Resistance Protein ◽  
P Glycoprotein ◽  
Resistance Protein ◽  
The Mean ◽  
D Values

Abstract The identity and nature of the leukemic stem cell (LSC) is an area of intense research interest since it may lead to a better understanding of the leukemogenic process and development of specific therapies. By definition, the LSC retains the stem cell properties of self-renewal, high proliferative capacity, predominant quiescent cell cycle status and differentiation potential, and is biologically distinct from more differentiated blasts. Here we address the question whether the expression of membrane transporters associated with multidrug resistance (MDR) phenotype is differentially expressed in LSC (defined as CD34+,CD38−CD123+) and more mature blasts CD34+CD38+ and the bulk leukemic population. Twenty-one patients with AML de novo were selected based on CD34 positivity by the leukemic blasts. MDR protein expression was measured by flow cytometry with the monoclonal antibodies against P-glycoprotein (P-gp), MDR-related protein (MRP), Lung-resistance protein (LRP) and breast cancer resistance protein (BCRP). In all experiments, at least one hundred thousand events were acquired in a FACScalibur flow cytometer and analysis was performed using the Cell Quest software. The expression levels of MDR transporters were analyzed using: the Kolmogorov-Smirnov test, categorizing D value for descriptive purposes as high if D ≥ 0.30, low if 0.2 ≤ D < 0.3 and negative if D < 0.20; and the mean channel fluorescence index (MFI), defined by the difference between the mean channel of fluorescence (MCF) for each antigen and MCF of the respective isotypic control. LSCs represented 0.33% (0.01–3.13%) of the total population of leukemic blasts. The values of D and MFI for MRP, LRP and BCRP in LSC, CD34+CD38+ and bulk leukemic population were not statistically different. In contrast, D values for P-gp were higher in LSCs (the mean ± Standard Error: 0.71 ± 0.10) compared to bulk leukemic population (0.38 ± 0.10, P=0.004) and to the CD34+CD38+ (0.21 ± 0.04, P=0.05) subset. In agreement, MFI values for P-gp were 146.7 ± 66.1, 38.2 ± 11.0 and 62.78 ± 16.4 in LSCs, blasts, and CD34+CD38+ respectively. Based on the LSC analysis, 95%, 85% and 57% of AML cases were found to have high expression (D ≥ 0.3) of MRP, LRP and BCRP, respectively. Similarly, 95%, 86% and 62% of the patients were thus categorized when the bulk population and 100%, 95% and 62% when CD34+CD38+ subsets were analyzed. Our results demonstrate that P-gp is overexpressed in LSCs compared to more differentiated subsets. Considering that P-gp-mediated drug efflux is the best characterized cellular mechanism of MDR, its constitutively high expression in LSCs may protect them from genotoxins and provide survival advantage. In addition, its conceivable that previously described higher incidence of MDR phenotype at relapse may reflect the selection of an intrinsic MDR+ LSC clone.

Expression of Lung Resistance Protein and Correlation with Other Drug Resistance Proteins and Outcome in Myelodysplastic Syndromes

1998 ◽  
Vol 29 (5-6) ◽  
pp. 547-551 ◽  
Author(s):  
Pascale Lepelley ◽  
Stephanie Poulain ◽  
Nathalie Grardel ◽  
Claude Preudhomme ◽  
Alain Cosson ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Myelodysplastic Syndromes ◽  
Resistance Proteins ◽  
Lung Resistance Protein ◽  
Lung Resistance ◽  
Resistance Protein
Sign in / Sign up

Export Citation Format

Share Document