Expression Profiles Of Transporters Research Articles

Expression Profile and Functional Activity of Peptide Transporters in Prostate Cancer Cells

Peptide transporters are expressed predominantly in intestinal and renal epithelial cells. The functional expression of peptide transporters is also identified in other types of tissues, such as glia cells, macrophages, and the epithelia of the bile duct, the lungs, and the mammary glands. However, their presence and role are poorly understood in carcinomas. We explored the expression profile and functional activity of peptide transporters in the prostate cancer cell lines LNCaP, PC-3, and DU145. Quantitative real time RT-PCR (qRT-PCR) and Western blot were used to evaluate the expression profile of peptide transporter 1 (PEPT1), peptide transporter 2 (PEPT2), peptide histidine transporter 1 (PHT1), and peptide histidine transporter 2 (PHT2) in these cells. LNCaP expresses high levels of PEPT2 and PHT1, while PC-3 demonstrates strong expression of PEPT1 and PHT1. DU145 shows only weak expression of PEPT1 and PHT1. Functional activities were studied in these cell lines using radiolabeled glycylsarcosine ([(3)H]Gly-Sar) and l-histidine ([(3)H]-l-histidine). The uptake of [(3)H]Gly-Sar and [(3)H]-l-histidine was time- and pH-dependent. A kinetic study showed that the uptake of Gly-Sar and l-histidine is saturable over the tested concentration range. The binding affinity (K(m)) and the maximal velocity (V(max)) exhibited in the three cell lines were consistent with the expression profiles we observed in qRT-PCR and Western blot analysis. A competitive inhibition study revealed that peptide transporters in prostate cancer cells exhibited broad substrate specificity with a preference for hydrophobic dipeptides, such as Leu-Leu. Fluorescence microscopy study revealed that the fluorescent dipeptide probe d-Ala-Lys-AMCA (a substrate of peptide transporters) specifically accumulated in the cytoplasm of LNCaP and PC-3, but not DU145 cells. Inhibiting the peptide transporter activity by Gly-Sar suppressed the growth of LNCaP and PC-3 cells. Our study indicated that PC-3 cells can be established as a new cell culture model for PEPT1 study, and LNCaP can be used as a model for PEPT2 study. Moreover, our results suggested that peptide transporters are overexpressed in prostate cancer cells and can be adopted as a promising target for tumor-specific drug delivery.

Gene expression of nutrient transporters and digestive enzymes in the yolk sac membrane and small intestine of the developing embryonic chick

Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption of nutrients posthatch. The uptake of nutrients is mediated by a variety of membrane-bound transporter proteins. The objective of this study was to determine the expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and embryonic intestine of egg-laying (Leghorn) and meat-producing (Cobb) chickens derived from 22 to 30 wk (young) and 45 to 50 wk (old) breeder flocks. Transporters examined included the peptide transporter PepT1, the glutamate/aspartate (EAAT3), cationic (CAT-1) and neutral (B0AT) amino acid transporters, and the fructose (GLUT5) and glucose (SGLT1) transporters. Digestive enzymes included aminopeptidase N (APN) and sucrase-isomaltase (SI). Expression of these genes was assessed by real-time PCR using the absolute quantification method in YSM at embryonic day (E) 11, 13, 15, 17, 19, 20, and 21 and intestine at E15, 17, 19, 20, and 21. The PepT1 and APN gene expression in the YSM increased until E15 and then decreased until E21, whereas expression in the intestine increased from E15 to E21. The B0AT showed a similar pattern, with greatest expression in the YSM occurring at E17/E19. The CAT1 and GLUT5 genes showed decreased expression in the YSM and increased expression in the intestine until E17/E19 and then a decrease until E21. Expression of SGLT1 and EAAT3 showed increased gene expression over time in both the intestine and YSM. Expression of SI showed little to no gene expression in the YSM, whereas the intestine exhibited consistently high levels of gene expression. In YSM and intestine, SI expression was greater in Leghorn than Cobb, whereas CAT1 and GLUT5 expression was greater in Cobb than Leghorn. Expression of the APN, CAT1, and SI genes was greater in embryos from young flocks than old flocks in YSM and intestine. These results demonstrate that the YSM expresses many of the digestive enzymes and nutrient transporters typically associated with the intestine and that these genes show tissue- and development-specific patterns of expression.

ABCB5 expression and cancer stem cell hypothesis in oral squamous cell carcinoma

IntroductionThe vast majority of oral cancers are squamous cell carcinomas (OSCC). The effectiveness of adjuvant cytostatic chemotherapy for OSCC is frequently restricted due to an inducible cellular mechanism called multidrug resistance (MDR) and a putative cancer stem cell (CSC) compartment in human carcinogenesis expressing multidrug efflux pumps. The novel human ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] acts as an energy-dependent drug efflux transporter and marks tumour cells of a putative CSC compartment. However, to date, there is no link between ABCB5 expression and OSCC. Materials and methodsExpression of ABCB5 was analysed in OSCC specimen (n=191) and cancer cell lines (BICR3, BICR56) by immunohistochemistry, real-time polymerase chain reaction (RT-PCR) analysis and western blotting. Scanned images were digitally analysed using ImageJ and the immunomembrane plug-in. ABCB5 expression on protein level was correlated with clinical characteristics and impact on survival. ABCB5 was co-labelled with CD44 in immunohistochemical and immunofluorescence double labelling experiments. Expression subgroups were identified by receiver operating characteristics (ROC) analysis. ResultsHigh ABCB5 expression was significantly associated with tumour progression and recurrence of the tumour. Multivariate analysis demonstrated high ABCB5 expression as an independent prognostic factor (p=0.0004). Immunohistochemical and immunofluorescence double labelling experiments revealed ABCB5 expression by CD44+ cancer cells. ABCB5 specificity was confirmed by western blot and RT-PCR analysis. ConclusionsFor the first time, this study provides evidence that ABCB5 expression in OSCC might be associated with tumour formation, metastasis and a putative CSC compartment. One of the principal mechanisms for protecting putative cancer stem cells is through the expression of multifunctional efflux transporters from the ABC gene family, like ABCB5. This provides one mechanism in which putative cancer stem cells could survive and may lead to tumour relapse. Knowledge of expression profiles of ABC transporters and other genes involved in MDR will likely help therapeutic optimisation for cancer patients in clinic. However, this hypothesis requires further in vitro and in vivo studies.

Defining new criteria for selection of cell-based intestinal models using publicly available databases

BackgroundThe criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies.ResultsWe made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics.ConclusionsThis study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

Expression Profiles of Metabolic Enzymes and Drug Transporters in the Liver and along the Intestine of Beagle Dogs

Beagle dogs are widely used in preclinical pharmacokinetic, safety, and formulation studies. However, little is known about intestinal and hepatic distribution of major enzymes and transporters involved in oral absorption and presystemic drug metabolism. We characterized mRNA levels of CYP3A12, CYP3A26, CYP2D15, UGT1A6, ABCB1 (MDR1), ABCC1 (MRP1), ABCG2 (BCRP), SLC15A1 (PEPT1), and SLC22A1 (OCT1) in dog liver and along the intestine by real-time quantitative reverse transcription-polymerase chain reaction. Tissue protein levels of CYP2D15, MDR1, and PEPT1 were obtained by Western blot. Gene distribution and expression variability was statistically described by a generalized additive mixed model smoothing function and correspondence analysis. Results were compared with the expression pattern known for the human orthologs. Hepatic mRNA levels for metabolic enzymes were generally higher than those for membrane transporters, whereas in the intestine the opposite was observed. Hepatic mRNA levels followed the order CYP2D15 > UGT1A6 ≈ CYP3A26 > ABCB1 ≈ SLC15A1 ≈ SLC22A1 > ABCG2 > ABCC1 ≈ CYP3A12. Along the gut, the genes were differentially distributed with greatest expression in duodenum/upper jejunum (ABCG2), middle jejunum (ABCB1 and SLC15A1), or in cecum/colon (ABCC1 and CYP2D15). CYP3A12, CYP3A26, SLC22A1, and UGT1A6 had a rather uniform expression. Intestinal mRNA profiles of CYP2D15, ABCB1, and SLC15A1 correlated with the respective protein levels. Canine CYP3A12/26, CYP2D15, and ABCB1 colonic distributions differed from those of human orthologs, whereas UGT1A6, ABCC1, ABCG2, SLC15A1, and SLC22A1 were comparable to those of humans in both small and large intestine. We aim to apply these data to better interpret pharmacokinetic studies in dogs with respect to their human relevance.

Abstract LB-345: Differences in metformin transporter expression between breast tumor and breast cancer cell lines: selecting a relevant breast cancer cell model for metformin therapy

Abstract Purpose: Recent studies show that the anti-diabetic drug metformin induces significant anticancer effects in several cancers including breast cancer via activation of intracellular AMPK. Because of its hydrophilic nature and net positive charge at physiologic pHs, metformin requires cation-selective transporters such as OCT1-3, PMAT and MATE1-2 to enter cells and activate AMPK. Expression of these transporters in cancer cells would determine the intracellular concentrations of metformin that can be achieved for anticancer effects. Therefore, elucidating metformin transport in breast cancer is central to understanding optimum dosing and transporter-associated inter-subject variability in its anticancer efficacy. However, there is limited information on metformin transport in breast cancer. Studies in our laboratory suggest that cation-selective transporters are expressed at negligible levels in MCF-7 cells, a widely used in vitro breast cancer model, which is not consistent with the robust anticancer effect of metformin in xenograft mouse models and clinical studies. Therefore, this study aims at determining a) metformin transporter expression in human breast tumor, b) if metformin transporter expression in breast cancer cell lines is relevant to that in breast tumor tissue and c) there is differential metformin transporter expression in tumor and non-malignant breast tissue. Methods: Total RNA isolated from breast cancer cell lines MCF-7, SK-BR-3, MDA-MB-231, ZR-75-1, BT-474, MDA-MB-435S, MDA-MB-468, BT-20 and BT-549 and from 14 pairs of human breast tumor and adjacent non-malignant breast tissues was subjected to.real-time PCR to determine relative expression levels of OCT1-3, PMAT, MATE1 and 2. Results: Most cell lines expressed low levels of candidate cation-selective transporters. MDA-MB-468 and BT-549 showed high expression of MATE1 and MDA-MB-231 expressed high levels of OCT3 and MATE1. In tumor and non-malignant breast tissue, the predominant transporters were OCT3 and PMAT. Nine of the total 14 tumor samples showed downregulation of OCT3 by 85% and PMAT by 66% compared to non-malignant tissue. Conclusions: The expression profile of cation-selective transporters is different between tumor and normal breast tissue and between breast tumor tissue and cell lines. Therefore, one needs to use caution in choosing a relevant in vitro cell model for investigating anticancer efficacy of metformin whose uptake is transporter-mediated. Expression data suggest that OCT3 and PMAT may play a major role in metformin intracellular uptake in breast cancer. MDA-MB-231 may be a useful model to assess the metformin anticancer effects in breast cancer. Clearly, genetic variability in metformin transporters may lead to differential responses to metformin anticancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-345. doi:1538-7445.AM2012-LB-345

Expression profile and thyroid hormone responsiveness of transporters and deiodinases in early embryonic chicken brain development

We used the chick embryo to study the mechanisms regulating intracellular TH availability in developing brain. TH-transporters OATP1C1 and MCT8, and deiodinases D1, D2, and D3 were expressed in a region-specific way, well before the onset of endogenous TH secretion. Between day 4 and 10 of development MCT8 and D2 mRNA levels increased, while OATP1C1 and D3 mRNA levels decreased. D2 and D3 mRNAs were translated into active protein, while no D1 activity was detectable. Injection of THs into the yolk 24h before sampling increased TH levels in the brain and resulted in decreased OATP1C1 and increased MCT8 expression in 4-day-old embryos. A compensatory response in deiodinase activity was only observed at day 8. We conclude that THs are active in the early embryonic brain and TH-transporters and deiodinases can regulate their availability. However, the absence of clear compensatory mechanisms at day 4 makes the brain more vulnerable for changes in maternal TH supply.

Statins modulate the expression profile of ATP-binding cassette transporters in human neuroblastoma cells

Statins modulate the expression profile of ATP-binding cassette transporters in human neuroblastoma cells

Signal profile on Gd-EOB-DTPA-enhanced MR imaging in non-alcoholic steatohepatitis and liver cirrhosis induced in rats: correlation with transporter expression

To compare the transporter expression and signal profile on Gd-EOB-DTPA-enhanced MRI between non-alcoholic steatohepatitis (NASH) and cirrhotic liver induced in rats, and investigate the correlation of the transporter expression and fibrosis rate in both diseases. Forty-eight rats were divided into four groups of 12: TAA (cirrhosis), NASH 7- and 10-week, and control groups. Each group was divided into two subgroups: Group 1 for MRI and Group 2 for transporter examinations. The relative enhancement of the TAA group was significantly lower than those of other groups (p < 0.01). The T(max) and T(1/2) of the NASH 10-week group was significantly prolonged in comparison with the TAA group (p < 0.01). There was no significant difference in the oatp1 expression, whereas the mrp2 expression of the TAA group was significantly higher than those of other groups (p < 0.01). There was no significant correlation between the fibrosis rate and oatp1 expression, whereas a paradoxical correlation was found between the fibrosis rate and mrp2 expression (NASH: negative correlation, r = 0.91, p < 0.01; TAA: positive correlation, r = 0.85, p < 0.01). Our findings showed that the mrp2 expression in cirrhosis increases in comparison with NASH, and there was a paradoxical correlation between the fibrosis rate and mrp2 expression.

Comparison of leukemia inhibitory factor-induced intracellular signalling in different trophoblastic cell lines

Understanding the mechanisms of drug resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) is essential to develop novel chemotherapies for non-small cell lung cancer (NSCLC). Therefore, we analyzed the expression and function of ATP-binding cassette (ABC) transporters in EGFR TKI-resistant NSCLC.In three newly established AG1478-resistant NSCLC cell lines, we evaluated the expression profile of ABC transporters and genotyping of ABCG2 by real-time polymerase chain reaction and elucidated their function by Hoechst dye efflux analyses. The growth-inhibitory effect of the topoisomerase I inhibitor Hoechst 33342, which is extruded by ABCG2, was also investigated in these cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay.In AG1478-resistant cells, significantly less ABCG2 was expressed, and the ratios of the cells with a strong ability to extrude Hoechst dye were remarkably smaller than in the parent cells. Because of the ABCG2 downregulation and loss of function due to C421A/C421A homozygosity, PC-14AG50R was thus considered to be more sensitive to Hoechst 33342 than the parental cells. All AG1478-resistant cells were more sensitive to the combination of Hoechst 33342 and AG1478 than to single agent.Resistance to EGFR TKI in NSCLC is associated with the downregulation of ABCG2 expression. A topoisomerase I inhibitor alone or in combination with EGFR TKI might offer a promising strategy for treating NSCLC that is resistant to EGFR TKI.

The effect of sphingomyelin synthase 2 (SMS2) deficiency on the expression of drug transporters in mouse brain

Sphingomyelin synthase (SMS), the last enzyme involved in the biosynthesis of sphingomyelin (SM), plays a critical role in the constitution of cell membrane and has impact on the expression of membrane proteins. SMS2, one of two SMS enzymes, is predominantly located in the plasma membrane, and is mainly expressed in the brain. Therefore, it is conceivable that SMS2 deficiency may have impact on expression of some membrane proteins, such as membrane-bound drug transporters. Using SMS2 gene deficient mouse brain tissues, we studied the gene and protein expression profiles of drug transporters, ERM proteins (ezrin/radixin/moesin) and the cytoskeleton protein, β-actin, in mouse brain by RT-PCR, western blot and immunohistochemistry analysis. We found that the mRNA expression of Mdr1 rather than the other drug transporters was significantly decreased in the SMS2 deficient brain. Accordingly, the expression and the function of Pgp (Mdr1/P-glycoprotein) were significantly downregulated in brain. In addition, the substantially downregulated expression of ezrin and β-actin was also observed in the SMS2 deficient brain. The immunohistochemistry analysis further revealed the suppressed expression of Pgp, ezrin and β-actin in both cortex and paraventricular areas of SMS2 knockout mice. Furthermore, both Pgp and β-actin were found to be co-immunoprecipitated with ezrin from the total brain lysate, suggesting the association between Pgp, ezrin and β-actin in the brain. These results indicate that SMS2 participates in the expression regulation of drug transporters, particularly Pgp, and suggest that SMS2 may be a potential target for enhancing drug access to the brain.

Progesterone and Estrogen Modulate the Expression of Xenobiotic Transporters in Cultured Mammary Epithelial Cells

Milk is an important source of nutrition, but it can become contaminated when mothers (or cows) ingest drugs or are exposed to toxins in the environment. Some toxins are reportedly concentrated in milk although the underlying mechanisms are not well‐defined, especially across species. Importantly, in vitro models to assess potential exposure through milk are lacking. The goal for these studies is to determine whether a bovine mammary epithelial cell line, BME‐UV, expresses putative drug transporters and to test for hormone‐induced changes in transporter expression profile or activity. Real time PCR was employed to evaluate the expression of xenobiotic transporters in polarized BME‐UV cell monolayers cultured in the presence or absence of progesterone and/or estradiol. P‐glycoprotein (P‐gp) mRNA expression was reduced following exposure to β‐estradiol or progesterone alone, but significantly decreased when the two hormones were used in combination. In contrast, expression of organic anion transporter 3 increased with hormone exposure. Since expression of P‐gp is reduced in lactating mammary epithelia, our results showing a similar decrease in P‐gp expression with hormone exposure in BME‐UV cells suggests that this cell line recapitulates observations in native tissues and thus will provide a powerful tool to quantify transport of distinct drugs or environmental contaminants. [NIH P20‐RR017686 Core C]

Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors

We have obtained, for the first time, a quantitative protein expression profile of membrane transporters and receptors in human brain microvessels, that is, the blood-brain barrier (BBB). Brain microvessels were isolated from brain cortexes of seven males (16-77 years old) and protein expression of 114 membrane proteins was determined by means of a liquid chromatography-tandem mass spectrometric quantification method using recently established in-silico peptide selection criteria. Among drug transporters, breast cancer resistance protein showed the most abundant protein expression (8.14 fmol/μg protein), and its expression level was 1.85-fold greater in humans than in mice. By contrast, the expression level of P-glycoprotein in humans (6.06 fmol/μg protein) was 2.33-fold smaller than that of mdr1a in mice. The organic anion transporters reported in rodent BBB, that is, multidrug resistance-associated protein, organic anion transporter and organic anion-transporting polypeptide family members, were under limit of quantification in humans, except multidrug resistance-associated protein 4 (0.195 fmol/μg protein). Among detected transporters and receptors for endogenous substances, the glucose transporter 1 level was similar to that of mouse, while the L-type amino acid transporter 1 level was fivefold smaller than that of mouse. These findings should be useful for understanding human BBB function and its differences from that in mouse.

Intensified expressions of a monocarboxylate transporter in consistently renewing tissues of the mouse

Monocarboxylates-lactate and ketone bodies-can compensate for glucose as energy sources under certain physical conditions. To identify the main energy source used in self-renewing tissues, expression profiles of monocarboxylate transporters (MCTs) were mainly investigated immunohistochemically in the gastrointestinal tract, skin, and bone marrow of mice, with reference to glucose transporters. In the small intestine, MCT1-immunoreactive epithelial cells accumulated in crypts with a selective immunolabeling along the basolateral membrane of cells. BrdU-labeled dividing cells were included in the cryptal MCT1-immunoreactive foci. The skin displayed an intense and extensive immunoreactivity for MCT1 in the hair bulge, which gives rise to the epidermis, hair, and sebaceous gland. The stratified squamous epithelium in the esophagus contained MCT1-immunoreactive cells in the basal layer but frequently lacked GLUT1-immunoreactive cells. The bone marrow was largely immunoreactive for MCT1 but not for GLUT1, suggesting the active production and utilization of monocarboxylates for hematopoiesis under hypoxic conditions. These findings support the idea that monocarboxylates are favorite energy sources in self-renewing tissues.

Membrane Drug Transporters and Chemoresistance in Human Pancreatic Carcinoma

Pancreatic cancer ranks among the tumors most resistant to chemotherapy. Such chemoresistance of tumors can be mediated by various cellular mechanisms including dysregulated apoptosis or ineffective drug concentration at the intracellular target sites. In this review, we highlight recent advances in experimental chemotherapy underlining the role of cellular transporters in drug resistance. Such contribution to the chemoresistant phenotype of tumor cells or tissues can be conferred both by uptake and export transporters, as demonstrated by in vivo and in vitro data. Our studies used human pancreatic carcinoma cells, cells stably transfected with human transporter cDNAs, or cells in which a specific transporter was knocked down by RNA interference. We have previously shown that 5-fluorouracil treatment affects the expression profile of relevant cellular transporters including multidrug resistance proteins (MRPs), and that MRP5 (ABCC5) influences chemoresistance of these tumor cells. Similarly, cell treatment with the nucleoside drug gemcitabine or a combination of chemotherapeutic drugs can variably influence the expression pattern and relative amount of uptake and export transporters in pancreatic carcinoma cells or select for pre-existing subpopulations. In addition, cytotoxicity studies with MRP5-overexpressing or MRP5-silenced cells demonstrate a contribution of MRP5 also to gemcitabine resistance. These data may lead to improved strategies of future chemotherapy regimens using gemcitabine and/or 5-fluorouracil.

Downregulated ABCG2 Enhances Sensitivity to Topoisomerase I Inhibitor in Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-Resistant Non-small Cell Lung Cancer

Understanding the mechanisms of drug resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) is essential to develop novel chemotherapies for non-small cell lung cancer (NSCLC). Therefore, we analyzed the expression and function of ATP-binding cassette (ABC) transporters in EGFR TKI-resistant NSCLC. In three newly established AG1478-resistant NSCLC cell lines, we evaluated the expression profile of ABC transporters and genotyping of ABCG2 by real-time polymerase chain reaction and elucidated their function by Hoechst dye efflux analyses. The growth-inhibitory effect of the topoisomerase I inhibitor Hoechst 33342, which is extruded by ABCG2, was also investigated in these cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. In AG1478-resistant cells, significantly less ABCG2 was expressed, and the ratios of the cells with a strong ability to extrude Hoechst dye were remarkably smaller than in the parent cells. Because of the ABCG2 downregulation and loss of function due to C421A/C421A homozygosity, PC-14AG50R was thus considered to be more sensitive to Hoechst 33342 than the parental cells. All AG1478-resistant cells were more sensitive to the combination of Hoechst 33342 and AG1478 than to single agent. Resistance to EGFR TKI in NSCLC is associated with the downregulation of ABCG2 expression. A topoisomerase I inhibitor alone or in combination with EGFR TKI might offer a promising strategy for treating NSCLC that is resistant to EGFR TKI.

The Impact of Resuscitated Fecal Peritonitis on the Expression of the Hepatic Bile Salt Transporters in a Porcine Model

Sepsis is often associated with cholestatic liver dysfunction caused by changes in the expression profile of hepatic bile salt transporters. However, in rodent endotoxin models, the role of ischemic hepatitis caused by liver hypoperfusion cannot be delineated. We hypothesized that hepatocytes change their expression pattern of bile salt transporters during early severe sepsis despite adequate resuscitation. Fifteen anesthetized and instrumented pigs were randomized to either fecal peritonitis (n = 8) or control (n = 7). Resuscitation was performed by hydroxyethyl starch and norepinephrine infusion. Hemodynamic parameters and markers of cholestatic and ischemic hepatic dysfunction were recorded. At baseline and after 21 h, messenger RNA (mRNA) and protein expression of bile salt transporters was determined by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively, on in vivo liver biopsies. All resuscitated septic pigs developed a normotensive hyperdynamic circulation with increased portal flow. After 21 h of peritonitis, no signs of biochemical or histological cholestasis were present. Na-taurocholate cotransporting polypeptide and bile salt export pump mRNA were downregulated by 83% (P = 0.001) and 67% (P = 0.001), respectively, in comparison with controls, whereas multidrug resistance-associated protein 4 (MRP4) mRNA was upregulated by 85% (P = 0.02). Bile salt export pump and MRP2 staining were downregulated in septic pigs. During early porcine fluid-resuscitated sepsis, hepatic basolateral influx (Na-taurocholate cotransporting polypeptide) and canalicular efflux (bile salt export pump) of bile salts were downregulated without hemodynamic signs of hepatic hypoperfusion or biochemical signs of cholestasis. In parallel, the basolateral escape transport (MRP4) was markedly upregulated, possibly as an early adaptive response to counteract hepatocellular accumulation of toxic bile acids.

Nucleoside transporter expression profiles in human cardiac tissue show striking individual variability with overall predominance of hENT1

Nucleoside transporters (NTs) are integral membrane transport proteins that modulate the flux of nucleosides such as adenosine across cell membranes. Two families of NTs exist, the concentrative NTs (CNTs, SLC28) and the equilibrative NTs (ENTs, SLC29). CNTs and ENTs transport anti-cancer and anti-viral nucleoside analog drugs and ENTs are also targets of drugs used to treat cardiac pathologies. Levels of some NT profiles have been shown to relate to clinical outcomes in the use of nucleoside analog drugs. However, currently, patient NT profile is not assessed prior to pharmacological administration of analog drugs. Here we describe a reliable method to determine a complete individual NT expression profile from human tissue using quantitative real-time PCR. We developed this assay on tissue (right atrial appendage, left internal mammary, aorta) from individuals undergoing cardiac surgery and compared these findings to the NT expression profiles in pooled whole heart tissue (normal and diseased). Data show that hENT1 is the most abundantly expressed NT, with highest expression levels in the aorta. However, NT expression profiles are highly variable among individuals and changes in NT expression between normal and diseased tissues were observed. These data are the first to describe the RNA expression patterns of all seven NT isoforms in the human heart. The methodology described here may be useful for quantitatively characterizing complete NT expression profiles in any human target tissue.

Hematopoietic stem cells exhibit a specific ABC transporter gene expression profile clearly distinct from other stem cells

BackgroundATP-binding cassette (ABC) transporters protect cells against unrelated (toxic) substances by pumping them across cell membranes. Earlier we showed that many ABC transporters are highly expressed in hematopoietic stem cells (HSCs) compared to more committed progenitor cells. The ABC transporter expression signature may guarantee lifelong protection of HSCs but may also preserve stem cell integrity by extrusion of agents that trigger their differentiation. Here we have studied whether non-hematopoietic stem cells (non-HSCs) exhibit a similar ABC transporter expression signature as HSCs.ResultsABC transporter expression profiles were determined in non-hematopoietic stem cells (non-HSCs) from embryonic, neonatal and adult origin as well as in various mature blood cell types. Over 11,000 individual ABC transporter expression values were generated by Taqman Low Density Arrays (TLDA) to obtain a sensitivity comparable with quantitative real-time polymerase chain reactions. We found that the vast majority of transporters are significantly higher expressed in HSCs compared to non-HSCs. Furthermore, regardless their origin, non-HSCs exhibited strikingly similar ABC transporter expression profiles that were distinct from those in HSCs. Yet, sets of transporters characteristic for different stem cell types could be identified, suggesting restricted functions in stem cell physiology. Remarkably, in HSCs we could not pinpoint any single transporter expressed at an evidently elevated level when compared to all the mature blood cell types studied.ConclusionsThese findings challenge the concept that individual ABC transporters are implicated in maintaining stem cell integrity. Instead, a distinct ABC transporter expression signature may be essential for stem cell function. The high expression of specific transporters in non-HSCs and mature blood cells suggests a specialized, cell type dependent function and warrants further functional experiments to determine their exact roles in cellular (patho)physiology.

Pachymic acid stimulates glucose uptake through enhanced GLUT4 expression and translocation

In an effort to investigate the effect and mechanism of Poria cocos on glucose uptake, six lanostane-type triterpenoids were isolated and analyzed. Among them, pachymic acid displayed the most significant stimulating activity on glucose uptake in 3T3-L1 adipocytes. The effect of pachymic acid on the expression profile of glucose transporters in differentiated 3T3-L1 adipocytes was also analyzed. Our results demonstrated that pachymic acid induced an increase in GLUT4, but not GLUT1, expression at both the mRNA and protein levels. The role of GLUT4 was further confirmed using the lentiviral vector-derived GLUT4 short hairpin RNA (shRNA). The stimulating activity of pachymic acid on glucose uptake was abolished when the endogenous GLUT4 expression was suppressed in 3T3-L1 adipocytes. In addition to increased GLUT4 expression, pachymic acid stimulated GLUT4 redistribution from intracellular vesicles to the plasma membrane in adipocytes. Exposure of the differentiated adipocytes to pachymic acid increased the phosphorylation of insulin receptor substrate (IRS)-1, AKT and AMP-activated kinase (AMPK). The involvement of PI3K and AMPK in the action of pachymic acid was further confirmed as PI3K and AMPK inhibitors completely blocked the pachymic acid-mediated activities in adipocytes. In addition, pachymic acid was shown to induce triglyceride accumulation and inhibit lipolysis in differentiated adipocytes. Taken together, we demonstrated the insulin-like activities of this compound in stimulating glucose uptake, GLUT4 gene expression and translocation, and promoting triglyceride accumulation in adipocytes. Our study provides important insights into the underlying mechanism of hypoglycemic activity of P. cocos.