Expression Of Membrane Transporters Research Articles

Chaperone Stress 70 Protein (STCH) Binds and Regulates Two Acid/Base Transporters NBCe1-B and NHE1 *

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96-440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na(+)-dependent pHi recovery from NH4(+) pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na(+)/H(+) exchanger (NHE)-dependent pHi recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca(2+) ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pHi through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pHi regulation at times of cellular stress by enhancing the recovery from intracellular acidification.

The Effects of Copper on Na+/K+-ATPase and Aquaporin Expression in Two Euryhaline Invertebrates

We used immunocytochemical and fluorometric techniques to show that gill cells of two marine invertebrates, the crab Neohelice granulata (osmoregulator) and the clam Mesodesma mactroides (osmoconformer), increase the expression of membrane transporters [Na(+)/K(+)-ATPase and aquaporin (AQP1)] after whole-animals exposure (96h) to sublethal concentrations of copper in water of salinity 30ppt, when both clams and crabs are isosmotic with respect to the environmental medium. A plausible interpretation of our findings is that this increased expression in membrane transporters may serve as an attempt to ameliorate the deleterious effects of copper on the mechanisms involved in ion and volume regulation in gill cells.

Potential role of transporters in ocular pharmacokinetics

AbstractTraditionally pharmacokinetics has been considered to be dominated by passive drug diffusion and metabolism. During the last decade pharmacokinetic research has shown that active drug transport by membrane transporter proteins is much more common than previously thought. The role of membrane transporters in ocular tissues is poorly known, but literature search on transporter interactions of ocular drugs reveals that there is high potential for such interactions, but expression of membrane transporters in the ocular tissues is not well understood. Drug concentrations relative to the affinity of the transporters is another factor that affects the importance of these interactions. Pharmacokinetic simulations reveal that the active transporters probably have important role in the access of systemic drugs to the eye (across blood retina barrier) and in the case of controlled release formulations. For drug absorption from the regular eye drops transporters seem to have relatively minor influence.

Enhanced expression of membrane transporter and drug resistance in keloid fibroblasts

The mechanisms of keloid resistance to therapy and high recurrence rate remain undefined. This study explored the difference in drug resistance between keloid and normal fibroblasts. Fibroblasts derived from 9 patients with keloid and 9 skin donors were randomly combined into 3 keloid cell pools (3 cases per pool) and 3 normal cell pools (3 cases per pool) and were compared for their resistance to vincristine and mitoxantrone and related molecule expression. The results revealed stronger resistance to both vincristine and mitoxantrone with a higher survival rate in keloid cells than in normal cells (P<.05). The resistance of keloid fibroblasts could be largely abrogated by verapamil treatment. In addition, messenger RNA expression levels of multiple-drug resistance-1, ABCB5 (P-glycoprotein family member), and cytochrome P450 3A4 but not ABCG2 (ATP-binding cassette transporter) were significantly higher in passage 1 keloid fibroblasts than in passage 1 normal fibroblasts; no significant difference was found in latter passages. Immunohistochemistry staining revealed significantly more multiple-drug resistance-1-positive cells (round) in keloid tissue than in normal skin (mostly spindle shaped) (P<.05) but no significant difference in the percentage of positive cells in the 2 groups. Enhanced expression of membrane transporters and increased resistance to chemotherapy agents may contribute to the keloid's resistance to therapy and the high posttherapy recurrence rate.

Blood–brain barrier

Aβ= : amyloid β; ABC= : ATP-binding cassette; AD= : Alzheimer disease; AQP4= : aquaporin 4; BBB= : blood–brain barrier; CAM= : cell adhesion molecule; cAMP= : cyclic adenosine monophosphate; EAAT= : excitatory amino acid transporters; GTP= : guanosine triphosphate; HIF-1= : hypoxia-inducible factor–1; JAM= : junction-associated molecules; LRP= : low-density lipoprotein receptor-related protein; MMP= : matrix metalloproteinase; MS= : multiple sclerosis; NMO= : neuromyelitis optica; RAGE= : receptor for advanced glycosylation end-product; SLC= : soluble carrier; TGF= : transforming growth factor; TNF= : tumor necrosis factor; VE= : vascular endothelial; VEGF= : vascular endothelial growth factor; ZO= : zonula occludens Neural survival and signaling within the CNS require a highly controlled microenvironment that depends on the normal function of a neurovascular unit, which includes several interacting structures, including capillary endothelial cells, pericytes, astrocytes, and neuronal processes (figure 1). The blood–brain barrier (BBB) is a dynamic component of this unit and is a key interface for molecular exchange between the blood and the brain interstitial fluid. The endothelial cells that form the BBB are characterized by the presence of tight junctions, the polarized expression of membrane transporters and receptors responsible for the active transport of nutrients and efflux of potentially toxic substances, and the presence of enzymes that target neurotransmitters or their precursors. All these cells participate in reciprocal interactions that are critical for local regulation of blood flow, BBB permeability, homeostasis of the neuronal microenvironment, synaptic remodeling, neurogenesis, and angiogenesis. Impaired BBB function contributes to the pathophysiology of a wide range of neurologic disorders, including traumatic, vascular, inflammatory, neoplastic, toxic-metabolic, and degenerative disorders. The pathophysiologic mechanisms include disruption of tight junctions, altered transport of molecules, inflammatory responses, impaired astrocyte function, and brain hypoperfusion. However, the normal BBB may reduce the CNS bioavailability of potentially useful therapeutic agents. Thus, the BBB constitutes an important therapeutic target. The structure and multiple functions of the BBB and their involvement in neurologic disorders have been extensively reviewed.1,–,12 Figure 1 Neurovascular unit and molecular components of the physical blood–brain barrier (BBB) The BBB is an integral part of the neurovascular unit, which includes brain endothelial cells, pericytes, and smooth muscle cells; astrocytes; neuronal terminals; and circulating white blood cells interacting with the vessel wall. All these different cell types express a wide variety of receptors, ion channels, and transporters and their close proximity with each other allows for effective paracrine interactions. The BBB acts as a physical barrier because of the presence of …

Quantitative Proteomics of Transporter Expression in Brain Capillary Endothelial Cells Isolated from P-Glycoprotein (P-gp), Breast Cancer Resistance Protein (Bcrp), and P-gp/Bcrp Knockout Mice

The objective of this study was to quantitatively examine the protein expression of relevant transporters and other proteins in the brain capillary endothelial cells isolated from wild-type mice and P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and P-gp/Bcrp knockout mice. After the isolation of brain capillary endothelial cells, a highly sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring was used to determine the quantitative expression of membrane transporters at the blood-brain barrier (BBB) of the various mouse genotypes. Quantitative expression of 29 protein molecules, including 12 ATP-binding cassette transporters, 10 solute carrier transporters, five receptors, and two housekeeping proteins, was examined by quantitative proteomics in the four mouse genotypes. There was no significant difference in the expression of P-gp between the wild-type and Bcrp1(-/-) mice. Likewise, Bcrp expression was not significantly different between the wild-type and Mdr1a/b(-/-) mice. There was no significant difference in the expression of any of the measured proteins in the brain capillary endothelial cells across the genotypes, except for the lack of expression of the corresponding protein in the mice that had a genetic deletion of P-gp or Bcrp. In conclusion, using a quantitative proteomic approach, we have shown that there are no changes in the expression of several relevant transporters in brain capillary endothelial cells isolated from single and combination knockout mice. These data suggest that the mechanism behind the functional compensation between P-gp and Bcrp at the BBB is not related to compensatory changes in transporter expression.

Toward an Integrative Computational Model of the Guinea Pig Cardiac Myocyte

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca2+) release occurs at the nanodomain level, where openings of single L-type Ca2+ channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue level electromechanical function.

Chemosensitization of Diffuse Large B Cell Lymphoma by Demethylating Nucleoside Analogues

Abstract 1617Diffuse Large B cell Lymphoma (DLBCL) is a heterogeneous disease in which 40 to 50% of the patients will either no respond or relapse within the first five years after the administration of combinatorial chemotherapy. Taking together the observed occurrence of aberrant DNA methylation patterning in DLBCL and the possibility that hypermethylation might suppress genes that could play a role in chemo-responsiveness, we hypothesized that DNMT inhibitors might induce re-expression of genes in a least a subset of resistant DLBCLs in order to induce chemosensitization.To characterize the responsiveness of a genetically diverse set of DLBCL cells to DNMT inhibitors, a panel of 30 DLBCL cell lines was exposed to increasing concentrations of 5-aza-2'-deoxycytidine (decitabine), 5-azacytidine and 6 DLBCL-active chemotherapy agents. We found that resistance to demethylating nucleoside analogues is mostly non-overlapping with resistance to other chemotherapy drugs used in DLBCL. Features such as cellular uptake and retention on H3-decitabine at 24 h and 48 h, TP53 mutation, expression of membrane transporters and nucleoside metabolic enzymes that have been suggested to influence sensitivity of other tumor types were not associated with the response to decitabine suggesting that the difference in sensitivity among the panel of DLBCL may be biological in nature. To guide combinatorial therapy of drugs we performed a supervised analysis of gene expression profiles of 6 decitabine sensitive and 8 resistant cell lines. We next used C-mapping to screen for drugs that induce the differential expression of this signature, based on the hypothesis that such drugs would synergize with decitabine. Six drugs signatures from C-map were positively associated with our decitabine signature, including doxorubicin and mitoxantrone, which are highly active drugs in DLBCL. Accordingly, in chemo-sensitive cells, short-course low-dose administration of decitabine synergizes the anti-lymphoma effect of doxorubicin in vitro and in mice DLBCL xenograft experiments. The synergistic effect on cell viability was independent of whether the drugs were administered sequentially or concurrently. Therefore, for DLBCL cells individually responsive to 5-aza-2'-deoxycytidine and doxorubicin, combination therapy resulted in enhanced therapeutic efficacy. We wondered whether more decitabine and doxorubicin-resistant DLBCL cells may benefit from longer exposure to decitabine. Accordingly, low-dose (no significant DNA damage induction) longer administration of decitabine induced DNA demethylation, growth retardation and, most remarkably, doxorubicin sensitization in chemotherapy-resistant DLBCL cells in vitro and in mice xenografts. Tumor chemosensitization was achieved without toxicity to normal tissues. To identify candidate epigenetically silenced genes that might contribute to doxorubicin resistance, we compared the gene expression and DNA methylation profiles of the panel of five doxorubicin-resistant vs. six doxorubicin-sensitive cell lines. We found 15 unique genes specifically hypermethylated and repressed in resistant cell lines. Among these, we found 2 that were significally reactivated in doxorubicin-resistant cells after prolonged low-dose administration of decitabine, arguing that cell re-programming occurs upon treatment with a demethylating agent. One of these two genes was SMAD1. We found that SMAD1 is epigenetically silenced in resistant DLBCL cells. Accordingly, DNA demethylating agents reactivated SMAD1 and rescue experiments showed that it contributed to the chemosensitization effect in DLBCL cells and in 5 primary patients samples treated ex vivo. In two different cohorts of patients, both SMAD1 expression and methylation were correlated with outcome and overall survival in DLBCL patients, after correction for IPI and COO subtypes. Pre and post 5'azacytidine treatment biopsies in a high risk DLBCL patient confirmed SMAD1 demethylation and chemo-sensitization in the clinical setting, delineating a personalized strategy for the clinical use of DNMT inhibitors.In summary, our work helped to found potential meaningful combinations for treatment of DLBCL, exposed a tumor-suppressive role for SMAD1 in DLBCL biology and defined an epigenetically mechanism used by lymphoma cells to escape TGFB growth inhibitory effects. Disclosures:Off Label Use: Use of DNA demethylating agents prior to classical chemotherapy regimen in DLBCL. Martin:Millennium Pharmaceuticals Inc.: Employment, Research Funding. Leonard:Millenium: Consultancy; Hospira: Consultancy; Biogen IDEC: Consultancy; Cephalon: Consultancy; Calistoga: Consultancy; Johnson and Johnson: Consultancy; Celgene: Consultancy; Cell Therapeutics: Consultancy; Immunomedics: Honoraria; Pfizer: Consultancy; Sanofi Aventis: Consultancy; EMD Serono: Consultancy; glaxosmithkline: Consultancy; Novartis: Consultancy; Abbott: Consultancy; Seattle Genetics: Consultancy.

Therapeutic resistance resulting from mutations in Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways

Chemotherapy remains a commonly used therapeutic approach for many cancers. Indeed chemotherapy is relatively effective for treatment of certain cancers and it may be the only therapy (besides radiotherapy) that is appropriate for certain cancers. However, a common problem with chemotherapy is the development of drug resistance. Many studies on the mechanisms of drug resistance concentrated on the expression of membrane transporters and how they could be aberrantly regulated in drug resistant cells. Attempts were made to isolate specific inhibitors which could be used to treat drug resistant patients. Unfortunately most of these drug transporter inhibitors have not proven effective for therapy. Recently the possibilities of more specific, targeted therapies have sparked the interest of clinical and basic researchers as approaches to kill cancer cells. However, there are also problems associated with these targeted therapies. Two key signaling pathways involved in the regulation of cell growth are the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. Dysregulated signaling through these pathways is often the result of genetic alterations in critical components in these pathways as well as mutations in upstream growth factor receptors. Furthermore, these pathways may be activated by chemotherapeutic drugs and ionizing radiation. This review documents how their abnormal expression can contribute to drug resistance as well as resistance to targeted therapy. This review will discuss in detail PTEN regulation as this is a critical tumor suppressor gene frequently dysregulated in human cancer which contributes to therapy resistance. Controlling the expression of these pathways could improve cancer therapy and ameliorate human health.

The role of paclitaxel in the development and treatment of multidrug resistant cancer cell lines

Paclitaxel (PTX) is used for treatment of wide range of solid tumors, but its efficacy is often limited by appearance of multidrug resistance (MDR). We explored the MDR induced by PTX in human colon cancer DLD1 and glioblastoma U87 cell lines. After confirmation of the cross-resistance to other anticancer agents in newly established DLD1-TxR and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased. Flow cytometry analyzes showed that the accumulation of P-glycoprotein (P-gp) substrates (rhodamine 123 and doxorubicin) was significantly lower in DLD1-TxR and U87-TxR compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression and glutathione (GSH) concentration was observed in U87-TxR. The analysis of cell cycle kinetics revealed extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX treatment, while glioblastoma cells died during interphase (G1, S or G2). The secretion of vascular endothelial growth factor (VEGF) was inhibited by single PTX treatment of colon cancer and in continuous treatment of glioblastoma cell lines. In conclusion, continuous PTX treatment caused the over-expression of P-gp and acquisition of MDR in colon cancer and glioblastoma cell lines, while some mechanisms of MDR and tumor progression such as GSH detoxification system and VEGF secretion were suppressed. Hence, the present results implicate that PTX is an important clinical tool for colon cancer and glioblastoma treatment even in the presence of MDR.

Point mutations in human guanylate kinase account for acquired resistance to anticancer nucleotide analogue PMEG

Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S 35N V 168F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs.

Enrichment for Breast Cancer Cells with Stem/Progenitor Properties by Differential Adhesion

Cancer stem cells are commonly isolated by cell sorting for surface antigens that typify stem cells. This technique is very expensive, requiring advanced, high-speed sorters and high-quality antibodies, and yields are often low. Some stem cells can be isolated based on ability to exclude dyes, conferred by expression of membrane transporters, but this property is not universal. Mammary stem cells are known to down-regulate cell-cell junctions and exhibit mesenchymal behaviors in vitro. We predicted that such cells should be readily detachable from tissue-culture plastic and that this might serve as a basis for their isolation from differentiated cells. We found that immortalized or transformed mammary epithelial cells can indeed be resolved into trypsin-sensitive and trypsin-resistant populations. The former are mesenchymal in morphology and expression profile and are enriched in stem cell properties such as mammosphere-forming ability, drug resistance, and CD44 stem cell antigen relative to the trypsin-resistant population. The latter, in contrast, are cobblestone in morphology, epithelial in expression profile, and deficient in mammosphere formation. After several rounds of differential trypsinization, the trypsin-sensitive pool had 80-fold higher mammosphere-forming ability than the trypsin-resistant population and 20-fold higher than the starting population. This resolution compares favorably with other enrichment methods. Thus, for relatively differentiated epithelial cell types, differential adhesion may serve as an enrichment strategy to increase the stem cell pool for subsequent manipulations.

The Role of the Placenta in Fetal Exposure to Xenobiotics: Importance of Membrane Transporters and Human Models for Transfer Studies

The placenta is a key organ in fetal growth and development because it controls maternal-to-fetal exchanges of nutrients and hormones. It also interferes with drug delivery to the fetus by expressing active membrane transporters and xenobiotic metabolism enzymes. Developing strategies to understand the role of the placenta in drug delivery is a challenge in toxicology. Despite common physiological functions, the placentas of different species are heterogeneous in their morphology and in their expression of membrane transporters and metabolizing proteins. These characteristics raise the difficulty of obtaining a good representative model of human placental transfer. To date, different in vitro, in vivo, and ex vivo tools have been used to elucidate transport and metabolism processes in the human placenta. This study recapitulates the typical features of human placenta and then presents the placental enzymes of xenobiotic metabolism, ATP-binding cassette transporters, solute carrier transporters, and their role in fetal exposure to xenobiotics. The study also compares the characteristics of different models of human placenta, in terms of membrane localization of transporters, and the expression of xenobiotic metabolism enzymes. The use of these models for toxicological studies, in particular xenobiotic transfer, is described, and the advantages and limits of each model are summarized.

Phase II study of CP-4126, a gemcitabine-lipid conjugate, in patients with advanced pancreatic cancer.

e14674 Background: Uptake of hydrophilic nucleoside analogues into tumor cells is determined by membrane transporter expression. Clinical response to gemcitabine (gem) is correlated with expression...

Gene Expression Patterns and Tumor Uptake of18F-FDG,18F-FLT, and18F-FEC in PET/MRI of an Orthotopic Mouse Xenotransplantation Model of Pancreatic Cancer

Our aim was to use PET/MRI to evaluate and compare the uptake of 18F-FDG, 3-deoxy-3-18F-fluorothymidine (18F-FLT), and 18F-fluorethylcholine (18F-FEC) in human pancreatic tumor cell lines after xenotransplantation into SCID mice and to correlate tumor uptake with gene expression of membrane transporters and rate-limiting enzymes for tracer uptake and tracer retention. Four weeks after orthotopic inoculation of human pancreatic carcinoma cells (PancTuI, Colo357, and BxPC3) into SCID mice, combined imaging was performed with a small-animal PET scanner and a 3-T MRI scanner using a dedicated mouse coil. Tumor-to-liver uptake ratios (TLRs) of the tracers were compared with gene expression profiles of the tumor cell lines and both normal pancreatic tissue and pancreatic tumor tissue based on gene microarray analysis and quantitative polymerase chain reaction. 18F-FLT showed the highest tumor uptake, with a mean TLR of 2.3, allowing correct visualization of all 12 pancreatic tumors. 18F-FDG detected only 4 of 8 tumors and had low uptake in tumors, with a mean TLR of 1.1 in visible tumors. 18F-FEC did not show any tumor uptake. Gene array analysis revealed that both hexokinase 1 as the rate-limiting enzyme for 18F-FDG trapping and pancreas-specific glucose transporter 2 were significantly downregulated whereas thymidine kinase 1, responsible for 18F-FLT trapping, was significantly upregulated in the tumor cell lines, compared with normal pancreatic duct cells and pancreatic tumor tissue. Relevant genes involved in the uptake of 18F-FEC were predominantly unaffected or downregulated in the tumor cell lines. In comparison to 18F-FDG and 18F-FEC, 18F-FLT was the PET tracer with the highest and most consistent uptake in various human pancreatic tumor cell lines in SCID mice. The imaging results could be explained by gene expression patterns of membrane transporters and enzymes for tracer uptake and retention as measured by gene array analysis and quantitative polymerase chain reaction in the respective cell lines. Thus, standard molecular techniques provided the basis to help explain model-specific tracer uptake patterns in xenotransplanted human tumor cell lines in mice as observed by PET.

Progress of Drug Transport Study Based on Absolute Quantitative Method for Membrane Transporter Proteins

Knowing the amount of membrane transporter expression in human tissue is one of the key issues in the rational and reliable prediction of pharmacokinetic profiles in humans. Recently, we have developed a simultaneous and highly sensitive method for the absolute quantification of multiple membrane transporter proteins in mammalian tissues. To develop quantitative analysis of high molecular-weight membrane proteins, we have solved problems using proteomics technology as follows: 1) The target proteins are detected via tryptic peptides that can be dissolved and analyzed with LC-MS/MS, while membrane protein is difficult to dissolve. 2) LC-MS/MS in multiple reaction monitoring (MRM) mode produce a highly sensitive and selective response for transporter proteins with low expression by separation from highly abundant molecules. 3) Analyte specificity for each peptide was demonstrated in amino acid sequences using multiple MRM detection. Selection of the peptide probe was very important for highly sensitive analysis with LC-MS/MS. We set criteria for peptide selection using an informatics approach. Peptides without unstable residue, double basic residues, and integral membrane domain were selected as useful probes. The developed method will provide an inclusive assay platform for all transporter proteins expressed in both animal/human tissues and will contribute to progress in drug discovery and development.

Asbestos induces doxorubicin resistance in MM98 mesothelioma cells via HIF-1

Human malignant mesothelioma (HMM), which is strongly related to asbestos exposure, exhibits high resistance to many anticancer drugs. Asbestos fibre deposition in the lung may cause hypoxia and iron chelation at the fibre surface. Hypoxia-inducible factor (HIF)-1alpha, which is upregulated by a decreased availability of oxygen and iron, controls the expression of membrane transporters, such as P-glycoprotein (Pgp), which actively extrude the anticancer drugs. The present study aimed to assess whether asbestos may play a role in the induction of doxorubicin resistance in HMM cells through the activation of HIF-1alpha and an increased expression of Pgp. After 24-h incubation with crocidolite asbestos or with the iron chelator dexrazoxane, or under hypoxia, HMM cells were tested for HIF-1alpha activation, Pgp expression, accumulation of doxorubicin and sensitivity to its toxic effect. Crocidolite, dexrazoxane and hypoxia caused HIF-1alpha activation, Pgp overexpression and increased resistance to doxorubicin accumulation and toxicity. These effects were prevented by the co-incubation with the cell-permeating iron salt ferric nitrilotriacetate, which caused an increase of intracellular iron bioavailability, measured as increased activity of the iron regulatory protein-1. Crocidolite, dexrazoxane and hypoxia induce doxorubicin resistance in human malignant mesothelioma cells by increasing hypoxia-inducible factor-1alpha activity, through an iron-sensitive mechanism.

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.

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; andthe 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.

Active Drug Delivery by Heterologous Expression of Membrane Transporters

AbstractFor Abstract see ChemInform Abstract in Full Text.

Active drug delivery by heterologous expression of membrane transporters

The oligopeptide transporter PEPT1 is predominantly expressed in the brush-border membranes of small intestinal epithelial cells, where it plays pivotal roles in the efficient absorption of di-/tripeptides. PEPT1 has enormous potential as an oral drug delivery target, because it also mediates the intestinal absorption of peptide-mimetic and nonpeptide substrates. We demonstrated that the peptide derivation of amino acid-related drugs is applicable to improve their intestinal absorption. We have found that oligopeptide transport activity is also expressed in cancer cell lines. The tissue distribution of bestatin was significantly increased in solid tumors that overexpress PEPT1 in nude mice. Orally administered bestatin strongly suppressed tumor growth. These results provided the first demonstration of the tumor-selective delivery of a drug by specific transport activity. The absolute degree of PEPT1 mRNA expression in the small intestine was determined using real-time PCR in rats. Starvation of the animals increased the mRNA expression level profoundly in the upper small intestine. The longitudinal expression pattern was well correlated with the intestinal transport of cefadroxil in rats. We constructed a recombinant adenovirus vector encoding PEPT1 cDNA. Heterologous expression of PEPT1 in the liver was successfully achieved by simple intvavenous. Administration of the vector, resulting in increased liver distribution of [3H] carnosine. In situ perfusion of the brain with the vector doubled the brain distribution of cefadroxil. Heterologous expression of the drug transporter in vivo could be a useful approach for drug delivery.