Role of the RNA binding protein IGF2BP1 in cancer multidrug resistance.

The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1), a member of a conserved family of single-stranded RNA-binding proteins (IGF2BP1-3), is expressed in a broad range of fetal tissues, placenta and more than sixteen cancer types but only in a limited number of normal adult tissues. IGF2BP1is required for the transport from nucleus to cytoplasm of certain mRNAs that play essential roles in embryogenesis, carcinogenesis, and multidrug resistance (MDR), by affecting their stability, translation, or localization. The purpose of this review is to gather and present information on MDR mechanisms in cancer and the significance of IGF2BP1 in this context. Within this review, we will provide an overview of IGF2BP1, including its tissue distribution, expression, molecular targets in the context of tumorigenesis and its inhibitors. Our main focus will be on elucidating the interplay between IGF2BP1 and MDR, particularly with regard to chemoresistance mediated by ABC transporters.

Extracellular Vesicles as Surrogates for the Regulation of the Drug Transporters ABCC2 (MRP2) and ABCG2 (BCRP).

Drug efflux transporters of the ATP-binding-cassette superfamily play a major role in the availability and concentration of drugs at their site of action. ABCC2 (MRP2) and ABCG2 (BCRP) are among the most important drug transporters that determine the pharmacokinetics of many drugs and whose overexpression is associated with cancer chemoresistance. ABCC2 and ABCG2 expression is frequently altered during treatment, thus influencing efficacy and toxicity. Currently, there are no routine approaches available to closely monitor transporter expression. Here, we developed and validated a UPLC-MS/MS method to quantify ABCC2 and ABCG2 in extracellular vesicles (EVs) from cell culture and plasma. In this way, an association between ABCC2 protein levels and transporter activity in HepG2 cells treated with rifampicin and hypericin and their derived EVs was observed. Although ABCG2 was detected in MCF7 cell-derived EVs, the transporter levels in the vesicles did not reflect the expression in the cells. An analysis of plasma EVs from healthy volunteers confirmed, for the first time at the protein level, the presence of both transporters in more than half of the samples. Our findings support the potential of analyzing ABC transporters, and especially ABCC2, in EVs to estimate the transporter expression in HepG2 cells.

Extracellular Vesicles and Cancer Multidrug Resistance: Undesirable Intercellular Messengers?

Cancer multidrug resistance (MDR) is one of the main mechanisms contributing to therapy failure and mortality. Overexpression of drug transporters of the ABC family (ATP-binding cassette) is a major cause of MDR. Extracellular vesicles (EVs) are nanoparticles released by most cells of the organism involved in cell-cell communication. Their cargo mainly comprises, proteins, nucleic acids, and lipids, which are transferred from a donor cell to a target cell and lead to phenotypical changes. In this article, we review the scientific evidence addressing the regulation of ABC transporters by EV-mediated cell-cell communication. MDR transfer from drug-resistant to drug-sensitive cells has been identified in several tumor entities. This was attributed, in some cases, to the direct shuttle of transporter molecules or its coding mRNA between cells. Also, EV-mediated transport of regulatory proteins (e.g., transcription factors) and noncoding RNAs have been indicated to induce MDR. Conversely, the transfer of a drug-sensitive phenotype via EVs has also been reported. Additionally, interactions between non-tumor cells and the tumor cells with an impact on MDR are presented. Finally, we highlight uninvestigated aspects and possible approaches to exploiting this knowledge toward the identification of druggable processes and molecules and, ultimately, the development of novel therapeutic strategies.

Extracellular Vesicles as Surrogates for Drug Metabolism and Clearance: Promise vs. Reality.

Drug-metabolizing enzymes (DMEs) and transporters play a major role in drug efficacy and safety. They are regulated at multiple levels and by multiple factors. Estimating their expression and activity could contribute to predicting drug pharmacokinetics and their regulation by drugs or pathophysiological situations. Determining the expression of these proteins in the liver, intestine, and kidney requires the collection of biopsy specimens. Instead, the isolation of extracellular vesicles (EVs), which are nanovesicles released by most cells and present in biological fluids, could deliver this information in a less invasive way. In this article, we review the use of EVs as surrogates for the expression and activity of DMEs, uptake, and efflux transporters. Preliminary evidence has been provided for a correlation between the expression of some enzymes and transporters in EVs and the tissue of origin. In some cases, data obtained in EVs reflect the induction of phase I-DMEs in the tissues. Further studies are required to elucidate to what extent the regulation of other DMEs and transporters in the tissues reflects in the EV cargo. If an association between tissues and their EVs is firmly established, EVs may represent a significant advancement toward precision therapy based on the biotransformation and excretion capacity of each individual.

Regulation of PXR Function by Coactivator and Corepressor Proteins: Ligand Binding Is Just the Beginning.

The pregnane X receptor (PXR, NR1I2) is a nuclear receptor which exerts its regulatory function by heterodimerization with the retinoid-X-receptor α (RXRα, NR2B1) and binding to the promoter and enhancer regions of diverse target genes. PXR is involved in the regulation of drug metabolism and excretion, metabolic and immunological functions and cancer pathogenesis. PXR activity is strongly regulated by the association with coactivator and corepressor proteins. Coactivator proteins exhibit histone acetyltransferase or histone methyltransferase activity or associate with proteins having one of these activities, thus promoting chromatin decondensation and activation of the gene expression. On the contrary, corepressor proteins promote histone deacetylation and therefore favor chromatin condensation and repression of the gene expression. Several studies pointed to clear cell- and ligand-specific differences in the activation of PXR. In this article, we will review the critical role of coactivator and corepressor proteins as molecular determinants of the specificity of PXR-mediated effects. As already known for other nuclear receptors, understanding the complex mechanism of PXR activation in each cell type and under particular physiological and pathophysiological conditions may lead to the development of selective modulators with therapeutic potential.

Protective effect of genistein pre-treatment on paraquat hepatotoxicity in rats.

Paraquat (PQ), an herbicide widely used in agriculture, is considered a highly toxic compound. In hepatocytes, P-glycoprotein (P-gp/Abcb1) is a canalicular transporter involved in PQ extrusion from the cell. Previously, we demonstrated that genistein (GNT) induces P-gp in rat liver. In this study, the protective role of GNT pretreatment towards hepatic damage in a model of acute intoxication with PQ in rats, was investigated. Wistar rats were randomized in 4 groups: Control, GNT (5 mg/kg/day sc, 4 days), PQ (50 mg/kg/day ip, last day) and GNT+ PQ. Hepatic lipoperoxidation (LPO) was evaluated by the thiobarbituric acid reactive substances method. Hepatic levels of 4-hydroxynonenal protein adducts (4-HNEp-add) and glutathione-S-transferase alpha (GSTα) protein expression were evaluated by Western blotting. Hepatic glutathione levels and plasma levels of alanine transaminase (ALT) and aspartate transaminase (AST) were also measured. Biliary excretion of PQ was studied in vivo and in isolated perfused liver. PQ was quantified by HPLC. PQ significantly increased AST and ALT activities, malondialdehyde and 4-HNEp-add levels, whereby pretreatment with GNT ameliorated this effect. PQ biliary excretion remained unchanged after treatments in both experimental models. Hepatic GSTα expression was augmented in GNT group. GNT pretreatment increased hepatic glutathione levels in PQ + GNT group. These results agree with the lower content of 4-HNEp-adds in GNT + PQ group respect to PQ group. Unexpectedly, increased activity of P-gp did not enhance PQ biliary excretion. Thus, GNT protective mechanism is likely through the induction of GSTα which results in increased 4-HNE metabolism before formation of protein adducts.

Acute regulation of apical ABC transporters in the gut. Potential influence on drug bioavailability.

The extensive intestinal surface offers an advantage regarding nutrient, ion and water absorptive capacity but also brings along a high exposition to xenobiotics, including drugs of therapeutic use and food contaminants. After absorption of these compounds by the enterocytes, apical ABC transporters play a key role in secreting them back to the intestinal lumen, hence acting as a transcellular barrier. Rapid and reversible modulation of their activity is a subject of increasing interest for pharmacologists. On the one hand, a decrease in transporter activity may result in increased absorption of therapeutic agents given orally. On the other hand, an increase in transporter activity would decrease their absorption and therapeutic efficacy. Although of less relevance, apical ABC transporters also contribute to disposition of drugs systemically administered. This review article summarizes the present knowledge on the mechanisms aimed to rapidly regulate the activity of the main apical ABC transporters of the gut: multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). Regulation of these mechanisms by drugs, drug delivery systems, drug excipients and nutritional components are particularly considered. This information could provide the basis for controlled regulation of bioavailability of therapeutic agents and at the same time would help to prevent potential drug-drug interactions.

Novel Aspects of Extracellular Vesicles in the Regulation of Renal Physiological and Pathophysiological Processes.

Extracellular vesicles (EV) are nanosized particles released by a large variety of cells. They carry molecules such as proteins, RNA and lipids. While urinary EVs have been longer studied as a source of biomarkers for renal and non-renal disorders, research on EVs as regulatory players of renal physiological and pathological processes has experienced an outbreak recently in the past decade. In general, the microenvironment and (patho)physiological state of the donor cells affect the cargo of the EVs released, which then determines the effect of these EVs once they reach a target cell. For instance, EVs released by renal epithelial cells modulate the expression and function of water and solute transporting proteins in other cells. Also, EVs have been demonstrated to regulate renal organogenesis and blood flow. Furthermore, a dual role of EVs promoting, but also counteracting, disease has also been reported. EVs released by renal tubular cells can reach fibroblasts, monocytes, macrophages, T cells and natural killer cells, thus influencing the pathogenesis and progression of renal disorders like acute kidney injury and fibrosis, nephrolithiasis, renal transplant rejection and renal cancer, among others. On the contrary, EVs may also exert a cytoprotective role upon renal damage and promote recovery of renal function. In the current review, a systematic summary of the key studies from the past 5 years addressing the role of EVs in the modulation of renal physiological and pathophysiological processes is provided, highlighting open questions and discussing the potential of future research.

Functional role of miR-148a in oropharyngeal cancer: influence on pregnane X receptor and P-glycoprotein expression.

MicroRNAs are short noncoding RNAs of about 19-25 nucleotides that usually target the 3' untranslated regions of mRNAs thus mediating post-transcriptional regulation of gene expression. Previous data indicate a role for miR-148a in the regulation of the pregnane X receptor (PXR/NR1I2), a nuclear receptor that regulates the expression of drug transporters like P-glycoprotein (P-gp/ABCB1). Our study investigated the effect of miR-148a on the post-transcriptional regulation of PXR and its target gene ABCB1 in oropharyngeal cancer cell lines (OPSCC). miR-148a was over-expressed and knocked-down in three OPSCC cell lines (HNO41, HNO206, and HNO413) by transfection with miR-148a mimic and miR-148a antagomir, respectively. Expression of miR-148a, NR1I2, and ABCB1 mRNA was quantified via real-time qPCR, protein expression of PXR was assessed by immunoblotting. Transfection of miR-148a mimic led to increased miR-148a levels in all cell lines and transfection of miR-148a antagomir reduced miR-148a expression in HNO206 and HNO413. Whereas these changes had no significant effect on PXR mRNA expression, protein expression was reduced in HNO41 by transfection with miR-148a and increased in HNO413 by transfection with miR-148a antagomir. Transfection of miR-148a downregulated ABCB1 mRNA in all cell lines, whereas antagonizing miR-148a had no significant effect. Our data demonstrate a modulation of PXR/NR1I2 and ABCB1 expression in OPSCC by miR-148a, however the effect was not uniform in all cell lines and depended on the range of expression of miR-148 and the genotype of rs1054190 SNP in NR1I2 3'UTR. Thus, our findings argue against an unequivocal association between miR-148a and PXR levels in OPSCC.

The phytoestrogens daidzein and equol inhibit the drug transporter BCRP/ABCG2 in breast cancer cells: potential chemosensitizing effect.

PURPOSE: The soy isoflavone genistein has been described to up-regulate breast cancer resistance protein (BCRP) and, thus, enhance chemoresistance in breast cancer cells. The aim of this work was to assess the effect of long- and short-term incubation with daidzein, the second most abundant soy isoflavone and its metabolite equol on the expression and activity of P-glycoprotein, multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and BCRP in breast cancer cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with phytoestrogen concentrations within the range achieved in individuals with a high isoflavone intake. Transporter expression was evaluated at protein and mRNA level through western blot and qRT-PCR, respectively. Transporter activity was determined using doxorubicin, mitoxantrone and carboxy-dichlorofluorescein as substrates. RESULTS: Daidzein (5 µM) up-regulated MRP2- and down-regulated MRP1 protein expressions in MCF-7 and MDA-MB-231 cells, respectively. Both effects were ER-dependent, as determined using the antagonist ICI 182,780. The decrease in MRP1 mRNA in MDA-MB-231 cells indicates a transcriptional mechanism. On the contrary, MRP2 induction in MCF-7 cells takes place post-transcriptionally. Whereas changes in the transporter expression had a minor effect on the transporter activity, acute incubation with daidzein, R-equol and S-equol led to a strong inhibition of BCRP activity and an increase in the IC50 of BCRP substrates. CONCLUSIONS: In contrast to previous reports for genistein, daidzein and equol do not provoke a major up-regulation of the transporter expression but instead an inhibition of BCRP activity and sensitization to BCRP substrates.

ABC Transporters: Regulation and Association with Multidrug Resistance in Hepatocellular Carcinoma and Colorectal Carcinoma.

For most cancers, the treatment of choice is still chemotherapy despite its severe adverse effects, systemic toxicity and limited efficacy due to the development of multidrug resistance (MDR). MDR leads to chemotherapy failure generally associated with a decrease in drug concentration inside cancer cells, frequently due to the overexpression of ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1), multidrug resistance-associated proteins (MRPs/ABCCs), and breast cancer resistance protein (BCRP/ABCG2), which limits the efficacy of chemotherapeutic drugs. The aim of this review is to compile information about transcriptional and post-transcriptional regulation of ABC transporters and discuss their role in mediating MDR in cancer cells. This review also focuses on drug resistance by ABC efflux transporters in cancer cells, particularly hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) cells. Some aspects of the chemotherapy failure and future directions to overcome this problem are also discussed.

Modulation of ABC Transporters by Nuclear Receptors: Physiological, Pathological and Pharmacological Aspects.

ABC transporters are membrane proteins mediating the efflux of endo- and xenobiotics. Transporter expression is not static but instead is subject to a dynamic modulation aiming at responding to changes in the internal environment and thus at maintaining homeostatic conditions. Nuclear receptors are ligand modulated transcription factors that get activated upon changes in the intracellular concentrations of the respective agonists and bind to response elements within the promoter of ABC transporters, thus modulating their expression and, consequently, their activity. This review compiles information about transporter regulation by nuclear receptors classified according to the perpetrator compounds and the biological effects resulting from the regulation. Modulation by hormone receptors is involved in maintaining endocrine homeostasis and may also lead to an altered efflux of other substrates in cases of altered hormonal levels. Xenobiotic receptors play a key role in limiting the accumulation of potentially harmful compounds. In addition, their frequent activation by therapeutic agents makes them common molecular elements mediating drug-drug interactions and cancer multidrug resistance. Finally, lipid and retinoid receptors are usually activated by endogenous molecules, thus sensing metabolic changes and inducing ABC transporters to counteract potential alterations. Furthermore, the axis nuclear receptor-ABC transporter constitutes a promising therapeutic target for the treatment of several disease states like cancer, atherosclerosis and dyslipidemia. In the current work, we summarize the information available on the pharmacological potential of nuclear receptor modulators and discuss their applicability in the clinical practice.

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.

Human papilloma virus (HPV) 18 proteins E6 and E7 up-regulate ABC transporters in oropharyngeal carcinoma. Involvement of the nonsense-mediated decay (NMD) pathway.

Oropharyngeal cancer incidence increased dramatically in the last decades, being infection with human papillomaviruses (HPV) a determinant of this trend. Concerning etiology, treatment response and prognosis, HPV+ and HPV- oropharyngeal cancers constitute different disease entities. The underlying molecular background is not completely understood. ATP-binding cassette (ABC) transporters mediate the efflux of anticancer drugs and are regulated by changes in the intracellular milieu. Furthermore, a role in cancer pathogenesis besides drug transport was reported. We evaluated the effect of transfection with E6 and E7 oncogenes from HPV16 and HPV18 on ABC transporters in oropharyngeal cancer cells. HPV18E6/E7 up-regulated P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and MRP2 expression in HNO206 cells and breast cancer resistance protein (BCRP) in HNO206 and HNO413 cells. While P-gp was regulated translationally, MRP1, MRP2 and BCRP up-regulation resulted from mRNA stabilization. For MRP1 and MRP2, the nonsense-mediated decay pathway was involved. In general, resistance to substrates of up-regulated transporters was increased. Transfection with oncogenes individually indicated a major role of HPV18E7. Our findings suggest ABC transporters as molecular players leading to differences in the pathogenesis of HPV+ and HPV- oropharyngeal cancer.

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.

The pregnane X receptor (PXR) and the nuclear receptor corepressor 2 (NCoR2) modulate cell growth in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide. The pregnane X receptor (PXR) is a nuclear receptor regulating several target genes associated with cancer malignancy. We here demonstrated a significant effect of PXR on HNSCC cell growth, as evidenced in PXR knock-down experiments. PXR transcriptional activity is more importantly regulated by the presence of coactivators and corepressors than by PXR protein expression. To date, there is scarce information on the regulation of PXR in HNSCC and on its role in the pathogenesis of this disease. Coactivator and corepressor expression was screened through qRT-PCR in 8 HNSCC cell lines and correlated to PXR activity, determined by using a reporter gene assay. All cell lines considerably expressed all the cofactors assessed. PXR activity negatively correlated with nuclear receptor corepressor 2 (NCoR2) expression, indicating a major role of this corepressor in PXR modulation and suggesting its potential as a surrogate for PXR activity in HNSCC. To test the association of NCoR2 with the malignant phenotype, a subset of three cell lines was transfected with an over-expression plasmid for this corepressor. Subsequently, cell growth and chemoresistance assays were performed. To elucidate the mechanisms underlying NCoR2 effects on cell growth, caspase 3/7 activity and protein levels of cleaved caspase 3 and PARP were evaluated. In HNO97 cells, NCoR2 over-expression decreased cell growth, chemoresistance and increased cleaved caspase 3 levels, caspase activity and cleaved PARP levels. On the contrary, in HNO124 and HNO210 cells, NCoR2 over-expression increased cell growth, drug resistance and decreased cleaved caspase 3 levels, caspase activity and cleaved PARP levels. In conclusion, we demonstrated a role of PXR and NCoR2 in the modulation of cell growth in HNSCC. This may contribute to a better understanding of the highly variable HNSCC therapeutic response.

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.

The trypanocidal benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2).

Oxidative stress is a frequent cause underlying drug-induced hepatotoxicity. Benznidazole (BZL) is the only trypanocidal agent available for treatment of Chagas disease in endemic areas. Its use is associated with side effects, including increases in biomarkers of hepatotoxicity. However, BZL potential to cause oxidative stress has been poorly investigated. Here, we evaluated the effect of a pharmacologically relevant BZL concentration (200µM) at different time points on redox status and the counteracting mechanisms in the human hepatic cell line HepG2. BZL increased reactive oxygen species (ROS) after 1 and 3h of exposure, returning to normality at 24h. Additionally, BZL increased glutathione peroxidase activity at 12h and the oxidized glutathione/total glutathione (GSSG/GSSG+GSH) ratio that reached a peak at 24h. Thus, an enhanced detoxification of peroxide and GSSG formation could account for ROS normalization. GSSG/GSSG+GSH returned to control values at 48h. Expression of the multidrug resistance-associated protein 2 (MRP2) and GSSG efflux via MRP2 were induced by BZL at 24 and 48h, explaining normalization of GSSG/GSSG+GSH. BZL activated the nuclear erythroid 2-related factor 2 (Nrf2), already shown to modulate MRP2 expression in response to oxidative stress. Nrf2 participation was confirmed using Nrf2-knockout mice in which MRP2 mRNA expression was not affected by BZL. In summary, we demonstrated a ROS increase by BZL in HepG2 cells and a glutathione peroxidase- and MRP2 driven counteracting mechanism, being Nrf2 a key modulator of this response. Our results could explain hepatic alterations associated with BZL therapy.

Modulation of Expression and Activity of ABC Transporters by the Phytoestrogen Genistein. Impact on Drug Disposition.

ATP binding cassette (ABC) transporters are involved in drug absorption, distribution and elimination. They also mediate multidrug resistance in cancer cells. Isoflavones, such as genistein (GNT), belong to a class of naturally-occurring compounds found at high concentrations in commonly consumed soya based-foods and dietary supplements. GNT and its metabolites interact with ABC transporters as substrates, inhibitors and/or modulators of their expression. This review compiles information about regulation of ABC transporters by GNT with special emphasis on the three major groups of ABC transporters involved in excretion of endo- and xenobiotics as follows: Pglycoprotein (MDR1, ABCB1), a group of multidrug resistance associated proteins (MRPs, ABCC subfamily) and ABCG2 (BCRP), an ABC half-transporter. The impact of these regulations on potential GNT-drug interactions is further considered.