Downregulation of intestinal multidrug resistance transporter 1 in obese mice: Effect on its barrier function and role of TNF-α receptor 1 signaling.

OBJECTIVES: Multidrug resistance transporter 1 (Mdr-1) is a relevant component of the intestinal transcellular barrier that decreases absorption of oral drugs, thus modulating their bioavailability. Obese patients with metabolic disorders take medications that are subjected to intestinal metabolism and the Mdr-1-dependent barrier. This study evaluated the effect of a high-fat diet (HFD; 40% fat for 16 wk) on Mdr-1 expression and transport activity in C57BL/6 (C57) male mice. Comparable studies were performed in tumor necrosis factor α (TNF-α) receptor 1 knockout mice (R1KO) to delineate a possible role of TNF-α signaling. METHODS: mRNA expression was evaluated by real-time polymerase chain reaction and protein levels by western blotting and immunohistochemistry. Mdr-1 activity was assessed using the everted intestinal sac model, with rhodamine 123 as the substrate. Statistical comparisons were made using the Student t test or one-way analysis of variance followed by the post hoc Tukey test. RESULTS: Mdr-1 protein, as well as its corresponding Mdr1a and Mdr1b mRNA, was decreased in C57-HFD mice compared with controls. Immunohistochemical studies confirmed downregulation of Mdr-1 in situ. These results correlated with a 48% decrease in the basolateral to apical transport of rhodamine 123. In contrast, R1KO-HFD modified neither intestinal Mdr-1 mRNA nor its protein expression or activity. In addition, C57-HFD showed elevated intestinal TNF-α mRNA and protein (enzyme-linked immunosorbent assay) levels, whereas R1KO-HFD was undetectable or had a lower increase, respectively. CONCLUSIONS: This study demonstrated an impairment of the Mdr-1 intestinal barrier function induced by HFD as a consequence of downregulation of both Mdr-1 gene homologues, resulting in impaired Mdr-1 protein expression. Inflammatory response mediated by TNF-α receptor 1 signaling was likely involved.

Multidrug resistance-associated protein 2 is negatively regulated by oxidative stress in rat intestine via a posttranslational mechanism. Impact on its membrane barrier function.

Oxidative stress (OS) is a key factor in the development of gastrointestinal disorders, in which the intestinal barrier is altered. However, the Multidrug resistance-associated protein 2 (Mrp2) status, an essential component of the intestinal transcellular barrier exhibiting pharmaco-toxicological relevance by limiting the orally ingested toxicants and drugs absorption, has not been investigated. We here evaluated the short-term effect of OS on Mrp2 by treatment of isolated rat intestinal sacs with tert-butyl hydroperoxide (TBH) for 30 min. OS induction by TBH (250 and 500 µM) was confirmed by increased lipid peroxidation end products, decreased reduced glutathione (GSH) content and altered antioxidant enzyme activities. Under this condition, assessment of Mrp2 distribution between brush border (BBM) and intracellular (IM) membrane fractions, showed that Mrp2 protein decreased in BBM and increased in IM, consistent with an internalization process. This was associated with decreased efflux activity and, consequently, impaired barrier function. Subsequent incubation with N-Acetyl-L-Cysteine (NAC, 1 mM) reestablished GSH content and reverted concomitantly the alteration in Mrp2 localization and function induced by TBH. Cotreatment with a specific inhibitor of classic calcium-dependent Protein Kinase C (cPKC) implicated this kinase in TBH-effects. In conclusion, we demonstrated a negative posttranslational regulation of rat intestinal Mrp2 after short-term exposition to OS, a process likely mediated by cPKC and dependent on intracellular GSH content. The concomitant impairment of the Mrp2 barrier function may have implications in xenobiotic absorption and toxicity in a variety of human diseases linked to OS, with notable consequences on the toxicity/safety of therapeutic agents.

Role of interleukin 1 beta in the regulation of rat intestinal multidrug resistance-associated protein 2 under conditions of experimental endotoxemia.

Multidrug resistance-associated protein 2 (Mrp2), expressed at the brush border membrane (BBM) of the enterocyte, is an ABC transporter with relevant intestinal barrier function. Its toxicological relevance lies in preventing absorption and tissue accumulation of dietary contaminants, drugs, and potentially harmful endogenous metabolites. Expression and activity of intestinal Mrp2 is downregulated in LPS-induced endotoxemia. In addition, confocal microscopy studies demonstrated internalization of the transporter to endocytic vesicles. Since IL-1ß plays an important role as early mediator of LPS-inflammatory responses, we evaluated whether IL-1ß mediates LPS-induced impairment of Mrp2 function. Two protocols were used: I) In vivo administration of LPS (5 mg/kg b.wt., i.p., single dose) to rats in simultaneous with administration of anti-IL-1ß (25 µg/kg b.wt., i.p., 4 doses), followed by studies of Mrp2 expression, localization and activity, 24 h after LPS administration; II) In vitro incubation of isolated intestinal sacs with IL-1ß (10 ng/mL) for 30 min, followed by analysis of Mrp2 activity and localization. We found that in vivo immunoneutralization of IL-1ß partially prevented the decrease of Mrp2 protein expression and activity as well as its internalization to intracellular domains induced by LPS. Involvement of IL-1ß in the alteration of Mrp2 localization and activity was more directly demonstrated in isolated intestinal sacs, as incubation with IL-1ß resulted in detection of Mrp2 in intracellular regions of the enterocyte in simultaneous with alteration of transport activity. In conclusion, IL-1ß induces early internalization of intestinal Mrp2, which could partially explain loss of expression at the BBM under conditions of experimental endotoxemia. Concomitant impairment of Mrp2-dependent barrier function may have pathophysiological relevance since IL-1ß mediates the effect of many local and systemic inflammatory processes.

Reversion of down-regulation of intestinal multidrug resistance-associated protein 2 in fructose-fed rats by geraniol and vitamin C: Potential role of inflammatory response and oxidative stress.

Intestinal multidrug resistance-associated protein 2 is an ABC transporter that limits the absorption of xenobiotics ingested orally, thus acting as essential component of the intestinal biochemical barrier. Metabolic Syndrome (MetS) is a pathological condition characterized by dyslipidemia, hyperinsulinemia, insulin resistance, chronic inflammation, and oxidative stress (OS). In a previous study we demonstrated that MetS-like conditions induced by fructose in drinking water (10% v/v, during 21 days), significantly reduced the expression and activity of intestinal Mrp2 in rats. We here evaluated the potential beneficial effect of geraniol or vitamin C supplementation, natural compounds with anti-inflammatory and anti-oxidant properties, in reverse fructose-induced Mrp2 alterations. After MetS-like conditions were induced (21 days), animals were cotreated with geraniol or vitamin C or vehicle for another 14 days. Decreased expression of Mrp2 protein and mRNA due to fructose administration was reversed by geraniol and by vitamin C, consistent with restoration of Mrp2 activity evaluated in everted intestinal sacs. Concomitantly, increased intestinal IL-1ß and IL-6 levels induced by fructose were totally and partially counterbalanced, respectively, by geraniol administration. The intestinal redox unbalance generated by fructose was improved by geraniol and vitamin C, as evidenced by decreasing lipid peroxidation products and activity of Superoxide Dismutase and by normalizing glutathione reduced/oxidized glutathione ratio. The restoration effects exhibited by geraniol and vitamin C suggest that local inflammatory response and OS generated under MetS-like conditions represent important mediators of the intestinal Mrp2 down-regulation. Additionally, both agents could be considered of potential therapeutic value to preserve Mrp2 function under MetS conditions.

Distribución de espacios verdes y actividades públicas gratuitas para realizar actividad física en la ciudad de Buenos Aires / Distribution of green spaces and public activities free for physical activity in the city of Buenos Aires

El sedentarismo está aumentando en la Ciudad Autónoma de Buenos Aires (CABA). Es importante estudiar el ambiente construido que promueve la realización de actividad física (AF). Nuestro objetivo fue analizar la distribución del espacio verde (EV) apto para realizar AF, así como la oferta estatal de actividades deportivas gratuitas en la ciudad. Se realizó un estudio ecológico analizando y mapeando datos según fuentes e informes oficiales de CABA para 2015. Hicimos un análisis por número absoluto, superficie, densidad poblacional y valor promedio del metro cuadrado construido por comuna. Además, realizamos un análisis cualitativo según imágenes satelitales de la ciudad. La mediana de espacio verde apto para AF era de 2,6 m2/habitante (rango intercuartílico de 1,0 a 4,6). La menor cantidad estuvo en las comunas céntricas, más densamente pobladas, así como en la mayoría de las de menor valor del terreno (una de estas tenía buena cantidad de EV, pero con un ambiente construido que podría limitar la realización de AF). En cambio, en cuanto a las actividades deportivas gratuitas, a menor valor del terreno había mayor oferta. Estos resultados deben analizarse junto con condiciones ambientales y de seguridad para la planificación integral de la ciudad. (AU)

Sedentary lifestyle is increasing in Buenos Aires City (CABA). It is important to study the built environment that promotes physical activity (PA). Our objective was to analyze the distribution of the green spaces or urban open spaces (GS) suitable for PA, as well as the state offer of free sports activities in the city. We did an ecological study, analyzing and mapping data according to sources and official reports of CABA for 2015. We performed analyses by absolute number, area, population density and average value of the built squared meter for each district (comuna). In addition, we did a qualitative analysis according to satellite images of the city. The median of green space suitable for PA was 2.6 m2 / inhabitant (interquartile range 1.0 to 4.6). The smallest amount was in the central districts, more densely populated, as well as in most of the lower landvalue ones (one of these had a good amount of GS, but with a built environment that could limit the practice of PA). In contrast, regarding free sports activities, the lower the value of the land, the greater the state offer. These results must be analyzed along with environmental and safety conditions for an integral planning of the city. (AU)

Inhibition of multidrug resistance-associated protein 2 (MRP2) activity by the contraceptive nomegestrol acetate in HepG2 and Caco-2 cells.

Multidrug resistance-associated protein 2 (MRP2) plays a key role in hepatic and intestinal disposition of endo- and xenobiotics. Several therapeutic agents modulate MRP2 activity resulting in pharmacological interactions. Nomegestrol acetate (NMGA) is a progestogen increasingly used in contraceptive formulations. The aim of this work was to evaluate the effect of NMGA on MRP2 activity in HepG2 and Caco-2 cells as models of human hepatocytes and enterocytes, respectively. NMGA (5, 50 and 500 nM; 48 h) decreased MRP2-mediated transport of 2,4-dinitrophenyl-S-glutathione in HepG2 cells, with no effect on MRP2 protein expression. Acute exposure (1 h) to the same concentrations of NMGA failed to affect MRP2 activity, ruling out an inhibitory action directly induced by the drug. In contrast, acute incubation with a lysate of HepG2 cells pre-treated with NMGA, containing potential metabolites, reproduced MRP2 inhibition. Preincubation of lysates with sulfatase but not with ß-glucuronidase abolished the inhibitory action, strongly suggesting participation of NMGA sulfated derivatives. Western blot studies in plasma vs. intracellular membrane fractions ruled out internalization of MRP2 to be responsible for the impairment of transport activity. MRP2-mediated transport of 5(6)-carboxy-2',7'-dichlorofluorescein was not affected in Caco-2 cells incubated for 48 h with either 5, 50 or 500 nM NMGA. Conversely, acute exposure (1 h) of Caco-2 cells to NMGA-treated HepG2 lysates decreased MRP2 activity, being this effect also prevented by pre-treatment of the lysates with sulfatase. Taken together, these findings demonstrate an inhibitory effect of NMGA sulfated metabolites on hepatic and intestinal MRP2 function. Extrapolated to the in vivo situation, they suggest the possibility of pharmacological interactions with coadministered drugs.

Biphasic modulation of cAMP levels by the contraceptive nomegestrol acetate. Impact on P-glycoprotein expression and activity in hepatic cells.

ABC transporters are key players in drug excretion with alterations in their expression and activity by therapeutic agents potentially leading to drug-drug interactions. The interaction potential of nomegestrol acetate (NMGA), a synthetic progestogen increasingly used as oral contraceptive, had never been explored. In this work we evaluated (1) the effect of NMGA on ABC transporters in the human hepatic cell line HepG2 and (2) the underlying molecular mechanism. NMGA (5, 50 and 500 nM) increased P-glycoprotein (P-gp) expression at both protein and mRNA levels and reduced intracellular calcein accumulation, indicating an increase also in transporter activity. This up-regulation of P-gp was corroborated in Huh7 cells and was independent of the classical progesterone receptor. Instead, using a siRNA-mediated silencing approach, we demonstrated the involvement of membrane progesterone receptor α. Moreover, we found that the activation of this receptor by NMGA led to a falling-rising profile in intracellular cAMP levels and protein kinase A activity over time, ultimately leading to transcriptional P-gp up-regulation. Finally, we identified inhibitory G protein and phosphodiesterases as mediators of this novel biphasic modulation. These results demonstrate the ability of NMGA to selectively up-regulate hepatic P-gp expression and activity and constitute the first report of ABC transporter modulation by membrane progesterone receptor α. If a similar regulation took place in vivo, decreased bioavailability and therapeutic efficacy of NMGA-coadministered P-gp substrates could be expected. This holds special importance considering long-term administration of NMGA and broad substrate specificity of P-gp.

Acute regulation of multidrug resistance-associated protein 2 localization and activity by cAMP and estradiol-17ß-D-glucuronide in rat intestine and Caco-2 cells.

Multidrug resistance-associated protein 2 (MRP2) is an ATP-dependent transporter expressed at the brush border membrane of the enterocyte that confers protection against absorption of toxicants from foods or bile. Acute, short-term regulation of intestinal MRP2 activity involving changes in its apical membrane localization was poorly explored. We evaluated the effects of dibutyryl-cAMP (db-cAMP), a permeable analog of cAMP, and estradiol-17ß-D-glucuronide (E217G), an endogenous derivative of estradiol, on MRP2 localization and activity using isolated rat intestinal sacs and Caco-2 cells, a model of human intestinal epithelium. Changes in MRP2 localization were studied by Western blotting of plasma membrane (PM) vs. intracellular membrane (IM) fractions in both experimental models, and additionally, by confocal microscopy in Caco-2 cells. After 30 min of exposure, db-cAMP-stimulated sorting of MRP2 from IM to PM both in rat jejunum and Caco-2 cells at 10 and 100 µM concentrations, respectively, with increased excretion of the model substrate 2,4-dinitrophenyl-S-glutathione. In contrast, E217G (400 µM) induced internalization of MRP2 together with impairment of transport activity. Confocal microscopy analysis performed in Caco-2 cells confirmed Western blot results. In the particular case of E217G, MRP2 exhibited an unusual pattern of staining compatible with endocytic vesiculation. Use of selective inhibitors demonstrated the participation of cAMP-dependent protein kinase and classic calcium-dependent protein kinase C in db-cAMP and E217G effects, respectively. We conclude that localization of MRP2 in intestine may be subjected to a dynamic equilibrium between plasma membrane and intracellular domains, thus allowing for rapid regulation of MRP2 function.

Glucagon-like peptide 2 prevents down-regulation of intestinal multidrug resistance-associated protein 2 and P-glycoprotein in endotoxemic rats.

Multidrug resistance-associated protein 2 (Mrp2, ABCC2) and P-glycoprotein (P-gp, ABCB1) constitute essential components of the intestinal biochemical barrier that prevent incorporation of food contaminants, drugs or toxic metabolites into the blood stream. Endotoxemia induced in rats by administration of bacterial lipopolysaccharide (LPS) results in elevated intestinal permeability and toxicity of xenobiotics in part associated with down-regulation of expression and activity of Mrp2 and P-gp. We evaluated the protective effect of glucagon-like peptide 2 (GLP-2), a peptide hormone with enterotrophic properties, on Mrp2 and P-gp alterations induced by single i.p. injection of LPS (5mg/kg b.wt.) to rats. Two different protocols of GLP-2 administration, namely prevention and reversion, were examined. The prevention protocol consisted of 7s.c. injections of GLP-2 (125µg/kg b.wt.) administered every 12h, starting 60h before LPS administration. The reversion protocol consisted of 2 doses of GLP-2, starting 3h after LPS injection. Intestinal samples were collected 24h after LPS administration and expression (protein and mRNA) and activity of Mrp2 were evaluated in proximal jejunum whereas those of P-gp were studied in ileum. GLP-2 completely neutralized down-regulation of expression of Mrp2 and P-gp and loss of their respective activities induced by LPS under prevention protocol. GLP-2 was also able to prevent internalization of both transporters from the apical membrane of the enterocyte to intracellular compartments, as detected by confocal microscopy. LPS induced an increase in IL-1ß and oxidized glutathione tissue levels, which were also counterbalanced by GLP-2 administration. In contrast, the reversion protocol failed to attenuate Mrp2 and P-gp down-regulation induced by LPS. We conclude that GLP-2 can prevent down-regulation of intestinal expression and activity of Mrp2 and P-gp in endotoxemic rats and that IL-1ß and oxidative stress constitute potential targets of GLP-2 protective effects.

Intestinal multidrug resistance-associated protein 2 is down-regulated in fructose-fed rats.

Expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2) and glutathione-S-transferase (GST) were examined in fructose fed Wistar rats, an experimental model of metabolic syndrome. Animals were fed on (a) control diet or (b) control diet plus 10% w/vol fructose in the drinking water. Mrp2 and the α class of GST proteins as well as their corresponding mRNAs were decreased, suggesting a transcriptional regulation by fructose. Confocal microscopy studies reaffirmed down-regulation of Mrp2. Everted intestinal sacs were incubated with 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl- S-glutathione (DNP-SG; model Mrp2 substrate), was measured in the same compartment to estimate Mrp2 activity. Excretion of DNP-SG was substantially decreased by fructose treatment, consistent with simultaneous down-regulation of Mrp2 and GST. In addition, the effect of fructose on intestinal barrier function exerted by Mrp2 was evaluated in vivo using valsartan, a recognized Mrp2 substrate of therapeutic use. After intraduodenal administration as a bolus, intestinal absorption of valsartan was increased in fructose-drinking animals. Fructose administration also induced oxidative stress in intestinal tissue as demonstrated by significant increases of intestinal lipid peroxidation end products and activity of the antioxidant enzyme superoxide dismutase, by a decreased GSH/GSSG ratio. Moreover, fructose treatment conduced to increased intestinal levels of the proinflammatory cytokines IL-ß1 and IL-6. Collectively, our results demonstrate that metabolic syndrome-like conditions, induced by a fructose-rich diet, result in down-regulation of intestinal Mrp2 expression and activity and consequently in an impairment of its barrier function.

Modulation of expression and activity of intestinal multidrug resistance-associated protein 2 by xenobiotics.

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is a transporter that belongs to the ATP-binding cassette (ABC) superfamily. In the intestine, it is localized to the apical membrane of the enterocyte and plays a key role in limiting the absorption of xenobiotics incorporated orally. MRP2 may also play a role in systemic clearance of xenobiotics available from the serosal side of the intestine. MRP2 transports a wide range of substrates, mainly organic anions conjugated with glucuronic acid, glutathione and sulfate and its expression can be modulated by xenobiotics at transcriptional- and post-transcriptional levels. Transcriptional regulation is usually mediated by a group of nuclear receptors. The pregnane X receptor (PXR) is a major member of this group. Relevant drugs described to up-regulate intestinal MRP2 via PXR are rifampicin, spironolactone and carbamazepine, among others. The constitutive androstane receptor (CAR, NR1I3) was also reported to modulate MRP2 expression, phenobarbital being a typical activator. Dietary compounds, including micronutrients and other natural products, are also capable of regulating intestinal MRP2 expression transcriptionally. We have given them particular attention since the composition of the food ingested daily is not necessarily supervised and may result in interactions with therapeutic drugs. Post-transcriptional regulation of MRP2 activity by xenobiotics, e.g. as a consequence of inhibitory actions, is also described in this review. Unfortunately, only few studies report on drug-drug or nutrient-drug interactions as a consequence of modulation of intestinal MRP2 activity by xenobiotics. Future clinical studies are expected to identify additional interactions resulting in changes in efficacy or safety of therapeutic drugs.

The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter.

Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance.

The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 µM) for 48 h exhibited a dose-response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics.

Regulation of multidrug resistance proteins by genistein in a hepatocarcinoma cell line: impact on sorafenib cytotoxicity.

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 µM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 µM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 µM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 µM) and MRP2 induction by GNT (only at 10 µM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.

Estrogen receptor-α mediates human multidrug resistance associated protein 3 induction by 17α-ethynylestradiol. Role of activator protein-1.

Previously, we have demonstrated that 17α-ethynylestradiol (EE) induces rat multidrug-resistance associated protein 3 (Mrp3, Abcc3) expression transcriptionally through estrogen receptor-α (ER-α) activation. We explored the effect of EE on MRP3 expression of human origin. HepG2 cells were transfected with ER-α and incubated with EE (1-10-50 µM) for 48 h. MRP3 protein and mRNA levels were measured by Western blotting and Real time PCR, respectively. EE up-regulated MRP3 protein and mRNA at 50 µM only in ER-α(+)-HepG2 cells. The in silico analysis of mrp3 promoter region demonstrated absence of estrogen response elements, but showed several Ap-1 binding sites. We further evaluated the potential involvement of the transcription factors c-JUN and c-FOS (members of Ap-1) in MRP3 up-regulation. ER-α(+) HepG2 cells were incubated with EE and c-FOS and c-JUN levels measured by Western blotting in nuclear extracts. EE up-regulated only c-JUN. Experiments of overexpression and knock-down of c-JUN by siRNA further demonstrated that this transcription factor is indeed implicated in MRP3 upregulation by EE. Co-immunoprecipitation assay demonstrated that EE induces c-JUN/ER-α interaction, and chromatin immunoprecipitation assay showed that this complex is recruited to the AP-1 binding consensus element present at the position (-1300/-1078 bp) of human mrp3 promoter. We conclude that EE induces MRP3 expression through ER-α, with recruitment of ER-α in complex with c-JUN to the human mrp3 promoter.

Pregnane X receptor mediates the induction of P-glycoprotein by spironolactone in HepG2 cells.

We evaluated the effect of spironolactone (SL), a well-known inducer of biotransformation and elimination pathways, on the expression and activity of P-glycoprotein (P-gp/ABCB1/MDR1), a major xenobiotic transporter, in HepG2 cells, as well as the potential mediation of pregnane X nuclear receptor (PXR). Cells were exposed to SL (1, 5, 10, 20 or 50 µM) for 48 h. Expression of P-gp and its mRNA levels were estimated by Western blotting and real time PCR, respectively. P-gp activity was inversely correlated with the ability of the cells to accumulate the model substrate rhodamine 123 (Rh123, 5 µM), in the presence or absence of verapamil (50 µM), a P-gp inhibitor. At the highest dose of SL tested, P-gp and MDR1 mRNA levels were significantly increased (73 and 108%) with respect to control cells. Rh123 accumulation was concomitantly reduced and verapamil was able to abolish this effect, confirming P-gp participation. Additionally, we tested the cytotoxicity of doxorubicin, a model substrate of P-gp, under inducing conditions. HepG2 cells treated with SL exhibited higher viability, i.e. less doxorubicin toxicity, than control cells, consistent with P-gp up-regulation. When HepG2 cells were treated with SL in the presence of ketoconazole (KTZ), a non-specific nuclear receptor inhibitor, the up-regulation of P-gp was suppressed. To further identify the nuclear receptor involved, cells were transfected with a siRNA directed against human PXR, leading to a 74% decrease in PXR protein levels, which totally abolished SL induction of P-gp. We conclude that SL up-regulates P-gp expression, likely at transcriptional level, and its efflux activity in HepG2 cells. This effect is mediated by PXR. Thus, ligands of PXR such as SL may alter the disposition and toxicity of other xenobiotics, including drugs of therapeutic use, that are P-gp substrates.