Modulation of prostaglandin transport activity of SLCO2A1 by annexin A2 and S100A10.

Solute carrier organic anion transporter family member 2A1 (SLCO2A1) is a prostaglandin (PG) transporter and serves as the osmosensitive ATP-permeable maxi-anion channel (Maxi-Cl). Since a heterotetrameric complex of annexin A2 (ANXA2) and S100A10 is obligatory for the channel activity, the present study aimed to determine if they regulate SLCO2A1-mediated PG transport. This study examined PGE2 uptake and ATP release in Anxa2 and/or S100a10 knockout (KO) murine breast C127 cells. Deletion of Slco2a1 decreased PGE2-d4 uptake by wild-type (WT) cells in an isotonic medium (290 mosmol/kgH2O). Decreased osmolarity (135 mosmol/kgH2O) stimulated ATP release but did not affect PGE2 uptake kinetics, showing Km (1,280 nM) and Vmax (10.38 pmol/15 s/mg protein) similar to those in isotonic medium (1,227 nM and 10.65 pmol/15 s/mg protein), respectively, in WT cells. Deletion of Anxa2 associated with loss of S100a10 diminished SLCO2A1-mediated ATP release and uncompetitively inhibited PGE2 uptake with lowered Km (376 nM) and Vmax (2.59 pmol/15 s/mg protein). Moreover, the immunoprecipitation assay confirmed the physical interaction of ANXA2 with SLCO2A1 in WT cells. Enforcement of ANXA2 expression to Anxa2 KO cells partially restored PGE2 uptake and increased Km (744.3 nM) and Vmax (9.07 pmol/15 s/mg protein), whereas the uptake clearance (Vmax/Km) did not change much regardless of ANXA2 expression. These results suggest that an ANXA2/S100A10 complex modulates PG transport activity but osmolality has little effect on it; therefore, the bound form of SLCO2A1, which functions as a PG transporter and Maxi-Cl, may exist regardless of changes in the cell volume.NEW & NOTEWORTHY A previous study indicated that the ANXA2/S100A10 complex represents the regulatory component of SLCO2A1-mediated Maxi-Cl channel activity. The present study showed that apparent PGE2 uptake by C127 cells was osmoinsensitive and uncompetitively inhibited by loss of ANXA2 expression, demonstrating that ANXA2 is a regulatory factor of SLCO2A1-mediated PG transport activity.

An implication of the mitochondrial carrier SLC25A3 as an oxidative stress modulator in NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a type of steatosis not associated with excessive alcohol intake and includes nonalcoholic steatohepatitis (NASH), which can progress to advanced fibrosis and hepatocellular carcinoma. Mitochondrial dysfunction causes oxidative stress, triggering hepatocyte death and inflammation; therefore, the present study aimed to explore relationship between mitochondrial carriers and oxidative stress. Firstly, we established a high fat diet (HFD)-fed ICR mouse NAFLD model characterized by obesity with insulin resistance and found transcriptional upregulation of Slc25a17 and downregulation of Slc25a3 (isoform B) and Slc25a13 in their fatty liver. A mitochondrial phosphate and Cu carrier, SLC25A3, was further studied in wild-type (wt) and SLC25A3-defective HepG2 cells (C1 and C3). SLC25A3 deficiency had insignificant effect on mitochondrial membrane potential (MtMP) and oxygen consumption rate (OCR) in untreated cells but suppressed them when cells were exposed to oleic acid. C1 and C3 cells were prone to produce reactive oxygen species (ROS), and increased ROS was associated with reduced mRNA expression of glutathione peroxidase (GPX) 1 and glutathione disulfide reductase (GSX) in these cell lines. Interestingly, cytoplasmic and mitochondrial Cu accumulation significantly reduced in C1 cells, demonstrating a predominant contribution of SLC25A3 to Cu transport into mitochondrial matrix. Cytotoxicity of free fatty acids was unchanged between wt and SLC25A3-deficient cells. These results indicate that reduced expression of SLC25A3 in fatty liver contributes to electron leak from mitochondria by limiting Cu availability, rendering hepatocytes more susceptible to oxidative stress. This study provides evidence that SLC25A3 is a novel risk factor for developing NASH.

N-glycosylation modifies prostaglandin E2 uptake by reducing cell surface expression of SLCO2A1.

SLCO2A1 functions as a prostaglandin (PG) influx transporter to facilitate intracellular oxidation of PGs and its defect causes dysregulation of PG signaling and metabolism. This study aimed to clarify effects of N-glycosylation on functional SLCO2A1 expression. Putative N-glycosylation site(s) (N134, N478, and/or N491) of human SLCO2A1 were mutated to Q and wild-type (WT) and mutant forms were expressed in HEK293 and human epithelial cells. Molecular weight of WT decreased to nearly 55 kDa by PNGase F treatment and was identical to that of triple mutant (TM, i.e., N134Q/N478Q/N491Q). Transport affinity of TM for PGE2 (Km of 392.7 nM) was comparable to that of WT (Km of 328.5 nM); however, immunoassays showed that TM cell surface expression remained at 24% of WT in HEK293 cells, resulting in a reduced cellular PGE2 uptake. These results suggest N-glycosylation modifies cellular PGE2 uptake by decreasing SLCO2A1 localization to the plasma membrane.

Assessment of hepatic prostaglandin E2 level in carbamazepine induced liver injury.

Objective. Carbamazepine (CBZ), a widely used antiepileptic drug, is one major cause of the idiosyncratic liver injury along with immune reactions. Conversely, prostaglandin E2 (PGE2) demonstrates a hepatoprotective effect by regulating immune reactions and promoting liver repair in various types of liver injury. However, the amount of hepatic PGE2 during CBZ-induced liver injury remains elusive. In this study, we aimed to evaluate the hepatic PGE2 levels during CBZ-induced liver injury using a mouse model. Methods. Mice were orally administered with CBZ at a dose of 400 mg/kg for 4 days, and 800 mg/kg on the 5th day. Results. Plasma alanine transaminase (ALT) level increased in some of mice 24 h after the last CBZ administration. Although median value of hepatic PGE2 amount in the CBZ-treated mice showed same extent as vehicle-treated control mice, it exhibited significant elevated level in mice with severe liver injury presented by a plasma ALT level >1000 IU/L. According to these results, mice had a plasma ALT level >1000 IU/L were defined as responders and the others as non-responders in this study. Even though, the hepatic PGE2 levels increased in responders, the hepatic expression and enzyme activity related to PGE2 production were not upregulated when compared with vehicle-treated control mice. However, the hepatic 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression and activity decreased significantly in responders when compared with control mice. Conclusions. These results indicate that elevated hepatic PGE2 levels can be attributed to the downregulation of 15-PGDH expression under CBZ-induced liver injury.

MicroRNAs in Apple-Derived Nanoparticles Modulate Intestinal Expression of Organic Anion-Transporting Peptide 2B1/SLCO2B1 in Caco-2 Cells.

Plant-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo to intestinal tissues. We previously reported that apple-derived nanoparticles (APNPs) downregulate the mRNA of the human intestinal transporter organic anion-transporting peptide 2B1 (OATP2B1)/SLCO2B1 and that the 3'-untranslated region (3'UTR) is required for the response to APNPs. Here, we investigated the involvement of microRNAs (miRNAs) in APNPs in suppressing OATP2B1 expression to demonstrate that APNP macromolecules directly interact with intestinal tissues. Using in silico analysis, seven apple miRNAs were predicted as candidate miRNAs that interact with the SLCO2B1-3'UTR. The APNP-mediated decrease in luciferase activity of pGL3/SLCO2B1-3'UTR was abrogated by inhibitors of mdm-miR-160a-e, -7121a-c, or -7121d-h. Each miRNA mimic reduced the endogenous expression of SLCO2B1 mRNA in Caco-2 cells. The luciferase activity of the truncated pGL3/SLCO2B1-3'UTR, which contains approximately 200 bp around each miRNA recognition element (MRE), was decreased by the miR-7121d-h mimic but decreased little by the other mimics. APNP also reduced the luciferase activity of truncated pGL3/SLCO2B1-3'UTR containing an MRE for miR-7121d-h. Thus, we demonstrated that mdm-miR-7121d-h contributes to the APNP-mediated downregulation of intestinal OATP2B1. Accordingly, plant macromolecules, such as miRNAs, may directly interact with intestinal tissues via nanoparticles. SIGNIFICANCE STATEMENT: This study demonstrates that mdm-miR7121d-h contained in apple-derived nanoparticles downregulated the mRNA expression of SLCO2B1 by interacting with SLCO2B1-3'-untranslated region directly and that SLCO2B1 mRNA might also be decreased by mdm-miR160a-e and -7121a-c indirectly. This finding that the specific apple-derived microRNAs influence human intestinal transporters provides a novel concept that macromolecules in foods directly interact with and affect the intestinal function of the host.

Toxicological implication of prostaglandin transporter SLCO2A1 inhibition by cigarette smoke in exacerbation of lung inflammation.

We reported that bleomycin (BLM)-induced pulmonary fibrosis was exacerbated in the prostaglandin transporter gene (Slco2a1)-deficient mice (Slco2a1(-/-)). Because cigarette smoke (CS) contributes to creating a profibrotic milieu in the respiratory region, the present study aimed to investigate the impact of CS on SLCO2A1-associated pathogenesis in the lungs of BLM-instilled mice. Bronchoalveolar lavage (BAL) fluid cell analysis indicated more severe inflammation in Slco2a1(-/-) on day 5 after BLM intratracheal instillation, and Slco2a1 deletion increased mRNA expression of pro-inflammatory cytokines (Tnf-α and Il-1ß) and chemokine (Ccl5) in BAL cells. Male Slco2a1(-/-) exhibited significantly higher amounts of released Il-1ß in BAL fluid, compared with female Slco2a1(-/-), male or female Slco2a1(+/+) group. The amount of PGE2 collected in BAL fluid tended to increase in Slco2a1(-/-) compared with Slco2a1(+/+) group, whereas the PGE2 concentrations in lung tissues were comparable between both groups. Besides, PGE2 accumulated more in BAL fluid of male than that of female mice. Therefore, Slco2a1-deficient male mice were found to be more susceptible to BLM-treatment. Moreover, CS extracts (CSE) significantly reduced initial PGE2 uptake by rat type1 alveolar epithelial cell-like (AT1-L) cells and human SLCO2A1-transfected cells. Exposure of AT1-L cells to CSE resulted in decreased mRNA expression of Slco2a1, suggesting that CS modulates SLCO2A1 function. These results indicate that exacerbated lung inflammation is attributed to an increase in Il-1ß peptide and PGE2 accumulation in the alveolar space, which exhibits a male predominance. SLCO2A1 inhibition by CSE is considered to be a new rationale for the lung toxicity of CS.

Identification of the Uptake Transporter Responsible for Distribution of Acotiamide into Stomach Tissue.

The acetylcholinesterase inhibitor, acotiamide, improves gastric motility and is clinically used to treat functional dyspepsia. The present study aimed to identify the transporters involved in the distribution of acotiamide in stomach tissue. Acotiamide uptake by the gastric cancer-derived model cell line, Hs746 T, was Na+- and pH-independent. The initial uptake velocity of acotiamide was saturable with increasing concentrations of acotiamide and was inhibited by selective serotonin reuptake inhibitors, which are potent inhibitors of the plasma membrane monoamine transporter (PMAT). The uptake of acotiamide by PMAT gene-transfected HEK293 cells was saturable, with similar Km (197.9 µM) values to those of uptake by Hs 746T cells (106 µM). Moreover, immunoreactivity of PMAT was found in the gastric smooth muscle and vascular endothelial cells. These results suggest that PMAT contributes to the distribution of acotiamide in the stomach, where it exerts its pharmacological effects.

Prostaglandin Transporter OATP2A1/SLCO2A1 Is Essential for Body Temperature Regulation during Fever.

Prostaglandin E2 (PGE2) in the hypothalamus is a principal mediator of the febrile response. However, the role of organic anion transporting polypeptide 2A1 (OATP2A1/SLCO2A1), a prostaglandin transporter, in facilitating this response is unknown. Here, we investigated the effect of Slco2a1 deficiency on the body core temperature (Tc) and on the PGE2 concentration in hypothalamus interstitial fluid (Cisf) and CSF (Ccsf) of lipopolysaccharide (LPS; 100 µg/kg, i.p.)-treated mice of both sexes. Slco2a1-/- mice did not develop a febrile response. Ccsf was increased in Slco2a1+/+ and Slco2a1-/- mice, and Ccsf of Slco2a1-/- mice was well maintained at 5 h after LPS injection (1160 pg/ml) compared with Slco2a1+/+ mice (316 pg/ml). A microdialysis study revealed that Cisf peaked at 2 h after LPS injection in Slco2a1+/+ mice (841 pg/ml), whereas the increase in Cisf was negligible in Slco2a1-/- mice. The PGE2 plasma concentration in Slco2a1-/- mice (201 pg/ml) was significantly higher than that in Slco2a1+/+ mice (54 pg/ml) at 1 h after LPS injection, whereas the two groups showed similar PGE2 concentrations in the hypothalamus. Strong Oatp2a1 immunoreactivity was observed in F4/80-positive microglia and perivascular cells and in brain capillary endothelial cells. The changes in Tc and Cisf seen in LPS-injected Slco2a1+/+ mice were partially attenuated in monocyte-/macrophage-specific Slco2a1-/- (Slco2a1Fl/Fl/LysMCre/+) mice. Thus, OATP2A1 facilitates the LPS-induced febrile response by maintaining a high level of Cisf, possibly by regulating PGE2 secretion from F4/80-positive glial cells and/or facilitating PGE2 transport across the blood-brain barrier. These findings suggest that OATP2A1 is a useful therapeutic target for neuroinflammation.SIGNIFICANCE STATEMENT Fever is a physiological response caused by pyrogen-induced release of prostaglandin E2 (PGE2) in the hypothalamus, which plays a central role in regulating the set-point of body temperature. However, it is unclear whether the prostaglandin transporter OATP2A1/SLCO2A1 is involved in this response. We show here that LPS-induced fever is associated with increased PGE2 concentration in hypothalamus interstitial fluid (Cisf), but not in CSF (Ccsf), by means of a microdialysis study in global Slco2a1-knock-out mice and monocyte-/macrophage-specific Slco2a1-knock-out mice. The results suggest that OATP2A1 serves as a regulator of Cisf in F4/80-positive glial cells. OATP2A1 was detected immunohistochemically in brain capillary endothelial cells and, therefore, may also play a role in PGE2 transport across the blood-brain barrier.

Experimental Evidence for Resecretion of PGE2 across Rat Alveolar Epithelium by OATP2A1/SLCO2A1-Mediated Transcellular Transport.

Prostaglandin transporter Oatp2a1/Slco2a1 is expressed at the apical (AP) membranes of type-1 alveolar epithelial (AT1) cells. To investigate the role of OATP2A1 in prostaglandin E2 (PGE2) handling by alveolar epithelium, we studied PGE2 transport across and secretion from monolayers of rat AT1-like (AT1-L) cells obtained by trans-differentiation of type-2 alveolar epithelial cells isolated from male Wistar rats. Rat AT1-L cells expressed Oatp2a1/Slco2a1, together with smaller amounts of Mrp4/Abcc4 and Oct1/Slc22a1 PGE2 uptake was saturable with Km 43.9 ± 21.9 nM. Transcellular transport of PGE2 across AT1-L cells grown on permeable filters in the AP-to-basolateral (BL) direction was 5-fold greater than that in the reverse direction and was saturable with Km 118 ± 26.8 nM; it was significantly inhibited by OATP inhibitors bromosulfophthalein (BSP) and suramin, and an MRP4 inhibitor, Ceefourin 1. We simultaneously monitored the effects of BSP on the distribution of PGE2 produced by bradykinin-treated AT1-L cells and PGE2-d4 externally added on the AP side of the cells. In the presence of BSP, PGE2 increased more rapidly on the AP side, whereas PGE2-d4 decreased more slowly on the AP side. The decrease in PGE2-d4 from the AP side corresponded well to the increase on the BL side, indicating that intracellular metabolism did not occur. These results suggest that Oatp2a1 and Mrp4 mediate transepithelial transport of PGE2 in the AP-to-BL direction. Therefore, OATP2A1 may be an important regulator of PGE2 in alveolar epithelium by reducing secretion of PGE2 and facilitating "resecretion" of PGE2 present in the alveolar lumen to the interstitial space or blood.

Effect of endogenous multidrug resistance 1 and P-glycoprotein expression on anticancer drug resistance in colon cancer cell lines.

P-glycoprotein (P-gp, multidrug resistance 1 (MDR1)) overexpression confers multidrug resistance to cancer cells, and P-gp in cell lines transfected with MDR1 or selected with chemotherapeutics significantly affect the anticancer drug efficacy. Although human cancer cell line panels consisting of defined tumor cell lines expressing endogenous P-gp have been used to screen drugs in pharmaceutical industries, endogenous P-gp affecting in vitro anticancer drug efficacy is unclear. The impact of P-gp expression on anticancer drug efficacy was assessed by using five colon cancer cell lines expressing varying endogenous P-gp levels and by selecting from the Cancer Cell Line Encyclopedia (CCLE). mRNA expression of MDR1 was considered as a surrogate of the protein expression of its gene product, P-gp, in CL-11, C2BBe1 and RKO cells, whereas P-gp protein expression in plasma membranes or crude membrane fractions was lower than expected from mRNA expression in CW-2 and CL-40 cells. The EC50 of paclitaxel and vinorelbine decreased in the presence of a P-gp inhibitor in CW-2 and CL-11 cells that highly express P-gp. No significant alterations in EC50 were observed in the CL-40, C2BBe1 and RKO cells, which show lower P-gp expression. Accordingly, the apparent in vitro efficacy of anticancer drugs could be underestimated if the endogenous P-gp expression is higher than in CL-11 cells. The effect of P-gp needs to be carefully evaluated in cell lines that highly express P-gp, which account for 1.5% of cancer cell lines, including all cancer types, and 14.5% of colon cancer cell lines in CCLE, considering the protein expression levels in plasma membranes.

Impact of Breast Cancer Resistance Protein Expression on the In Vitro Efficacy of Anticancer Drugs in Pancreatic Cancer Cell Lines.

Breast cancer resistance protein (BCRP) overexpression confers multidrug resistance to cancer cells, and the efficacy of anticancer drugs has been reported to be significantly affected by BCRP in cell lines transfected with BCRP or selected with drugs. It is unclear whether the in vitro efficacy of anticancer drugs is affected by endogenous BCRP, although cancer cell line panels consisting of defined tumor cell lines with endogenous BCRP have been used to screen for anticancer drugs in the pharmaceutical industry. We assessed the impact of BCRP expression on efficacy of anticancer drugs using pancreatic cancer cell lines expressing varying levels of endogenous BCRP. Pancreatic cancer cell lines were selected from the Cancer Cell Line Encyclopedia (CCLE). The EC50 of 7-ethyl-10-hydroxycamptothecin (SN-38), topotecan, and mitoxantrone decreased in the presence of a BCRP inhibitor in PANC-1 and AsPC-1 cells, which exhibit high BCRP expression. However, no significant alterations in EC50 were observed in HPAF-II, SW 1990, and MIA PaCa-2, which show moderate or low BCRP expression. The shift of EC50 of anticancer drugs with and without a BCRP inhibitor increased with an increase of BCRP mRNA expression levels; however, the shift was obvious only in cells highly expressing BCRP. Thus, the in vitro efficacy of anticancer drugs on cell proliferation may be minimally affected by BCRP in most pancreatic cancer cell lines, considering that 72% of pancreatic cancer cell lines in CCLE show moderate or low BCRP expression. The effect of BCRP should be carefully evaluated in pancreatic cell lines that highly express BCRP.

Apple-Derived Nanoparticles Modulate Expression of Organic-Anion-Transporting Polypeptide (OATP) 2B1 in Caco-2 Cells.

Interaction of foods with intestinal transporters has generally been ascribed to small molecules, but recently, edible-plant-derived nanoparticles (NPs) have been suggested to affect intestinal function. Here, we examined the effects of NPs contained in edible fruits on intestinal transporters. Apple-derived NPs (APNPs) were isolated by ultracentrifugation and characterized by measurement of particle size distribution and electron microscopy. Human epithelial colorectal adenocarcinoma (Caco-2) cells internalized fluorescently labeled APNPs, suggesting that fruit-derived NPs would be internalized into intestinal epithelial cells in vivo. We found that the mRNA expression levels of several transporters, including organic-anion-transporting polypeptide (OATP) 2B1, were changed in APNP-treated Caco-2 cells. The protein expression and activity of OATP2B1 were also decreased by APNP exposure, as determined by Western blotting and measurements of [3H]estrone-3-sulfate uptake by Caco-2 cells, respectively. These actions required intact APNPs, because sonication or boiling abrogated the effects. Since the content of apple-derived small molecules in APNPs was negligible, the observed decrease of OATP2B1 expression appears to be mediated by large molecules in the APNPs. We further found that the 3'-untranslated region of the OATP2B1 gene was required for the response to APNPs, suggesting that microRNA in the APNPs might be involved. These results propose a novel mechanism, in which large molecules such as microRNA in food could affect intestinal transporters through food-derived NPs, which also demonstrates that food-derived NPs should be useful for delivery of biologically active large molecules to intestinal tissues.

Different Efflux Transporter Affinity and Metabolism of 99mTc-2-Methoxyisobutylisonitrile and 99mTc-Tetrofosmin for Multidrug Resistance Monitoring in Cancer.

BACKGROUND: Little is known about the affinity and stability of 99mTc-labeled 2-methoxyisobutylisonitrile (99mTc-MIBI) and tetrofosmin (99mTc-TF) for imaging of multiple drug resistance transporters in cancer. We examined the affinity of 99mTc-labeled compounds for these transporters and their stability. METHODS: 99mTc-MIBI and 99mTc-TF were incubated in vesicles expressing P-glycoprotein (MDR1), multidrug resistance-associated protein (MRP)1-4, or breast cancer resistance protein with and without verapamil (MDR1 inhibitor) or MK-571 (MRP inhibitor). Time activity curves of 99mTc-labeled compounds were established using SK-N-SH neuroblastoma, SK-MEL-28 melanoma, and PC-3 prostate adenocarcinoma cell lines, and transporter expression of multiple drug resistance was measured in these cells. The stability was evaluated. RESULTS: In vesicles, 99mTc-labeled compounds had affinity for MDR1 and MRP1. 99mTc-TF had additional affinity for MRP2 and MRP3. In SK-N-SH cells expressing MDR1 and MRP1, MK-571 produced the highest uptake of both 99mTc-labeled compounds. 99mTc-MIBI uptake with inhibitors was higher than 99mTc-TF uptake with inhibitors. 99mTc-TF was taken up more in SK-MEL-28 cells expressing MRP1 and MRP2 than PC-3 cells expressing MRP1 and MRP3. 99mTc-MIBI was metabolized, whereas 99mTc-TF had high stability. CONCLUSION: 99mTc-MIBI is exported via MDR1 and MRP1 (MRP1 > MDR1) at greater levels and more quickly compared to 99mTc-TF, which is exported via MDR1 and MRP1-3 (MRP1 > MDR1; MRP1, 2 > MRP3). Because 99mTc-MIBI is metabolized, clinical imaging for monitoring MDR and shorter examination times may be possible with an earlier scanning time on late phase imaging. 99mTc-TF has high stability and accurately reflects the function of MDR1 and MRP1-3.

Membrane Transporters Contributing to PGE2 Distribution in Central Nervous System.

Prostaglandin (PG) E2 is a well-established lipid mediator that plays a role in diverse functions and diseases of the brain. Cyclooxygenase and PGE synthase have been extensively studied as molecular determinants of extracellular concentration of PGE2 near prostanoid E receptors since the brain has limited capacity of PG metabolism. There is accumulating evidence that several members of the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies regulate PGE2 distribution in brain capillary endothelial cells, choroid plexus (CP) and arachnoid epithelium, and different parenchyma cells such as neuronal and glial cells. These transporters may mediate entry and exit of PGE2 at blood-brain and blood-cerebrospinal fluid boundaries, resulting in brain distribution of PGE2. However, their roles in neuroinflammation and disease progression remain unclear. In this review, current knowledge on transporters involved in brain distribution of PGE2 is summarized, and especially, potentials of organic anion transporting polypeptide (OATP) and organic anion transporter (OAT) family members are discussed as molecular determinants of PGE2 concentration in the brain.

Contribution of equilibrative nucleoside transporter(s) to intestinal basolateral and apical transports of anticancer trifluridine.

Trifluridine (FTD) exhibits anticancer activities after its oral administration despite its hydrophilic nature. It was previously reported that concentrative nucleoside transporter (CNT) 1 mediates the apical uptake of FTD in human small intestinal epithelial cells (HIECs). In the present study, FTD was also identified as a substrate for equilibrative nucleoside transporter (ENT) 1 and ENT2 in transporter gene-transfected cells. An immunocytochemical analysis revealed that ENT1 was expressed at the basolateral and apical membranes of HIECs. Cellular accumulation increased in the presence of S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT selective inhibitor. Cytotoxicity in HIEC monolayers at low FTD concentrations was increased by NBMPR, and this may have been due to inhibition of the ENT-mediated basolateral transport of FTD by NBMPR. These results suggest that ENTs reduce the intestinal cytotoxicity of FTD by facilitating its basolateral efflux. On the other hand, the intracellular accumulation and cytotoxicity of FTD in HIECs were decreased at higher concentrations of FTD by NBMPR, and this may have been due to the NBMPR inhibition of the apical uptake of FTD, which has been suggested to be mediated by CNTs and ENTs. In conclusion, ENTs were responsible for intestinal transepithelial permeation by mediating the basolateral efflux of FTD after its uptake by CNT1 from the apical side, resulting in decreases in its intracellular accumulation and intestinal toxicity in humans. Equilibrative nucleoside transporters may also partially contribute to the low-affinity uptake of FTD across the apical membrane along with high-affinity CNT1.

Contribution of equilibrative nucleoside transporters 1 and 2 to gemcitabine uptake in pancreatic cancer cells.

Hepatic arterial infusion (HAI) chemotherapy is expected to be a more effective and safer method to treat the hepatic metastasis of pancreatic cancer than intravenous (iv) administration because of higher tumor exposure and lower systemic exposure. To clarify the uptake mechanism of nucleoside anticancer drugs, including gemcitabine (GEM), in pancreatic cancer, we investigated the uptakes of radiolabeled uridine (a general substrate of nucleoside transporters) and GEM in pancreatic cancer cell lines MIA-PaCa2 and As-PC1. Uridine uptake was inhibited by non-labeled GEM and also by S-(4-nitrobenzyl)-6-thioinosine (NBMPR; an inhibitor of equilibrative nucleoside transporters, ENTs) in a concentration-dependent manner, suggesting that ENTs contribute to uridine uptake in pancreatic cancer cells. As for GEM, saturable uptake was mediated by high- and low-affinity components with Km values of micromolar and millimolar orders, respectively. Uptake was inhibited in a concentration-dependent manner by NBMPR and was sodium ion-independent. Moreover, the concentration dependence of uptake in the presence of 0.1 µM NBMPR showed a single low-affinity site. These results indicated that the high- and low-affinity sites correspond to hENT1 and hENT2, respectively. The results indicated that at clinically relevant hepatic concentrations of GEM in GEM-HAI therapy, the metastatic tumor exposure of GEM is predominantly determined by hENT2 under unsaturated conditions, suggesting that hENT2 expression in metastatic tumor would be a candidate biomarker for indicating anticancer therapy with GEM-HAI.

Comparative Evaluation of Dehydroepiandrosterone Sulfate Potential to Predict Hepatic Organic Anion Transporting Polypeptide Transporter-Based Drug-Drug Interactions.

Pharmacokinetic drug-drug interactions (DDIs) on hepatic organic anion transporting polypeptides (OATPs) are important clinical issues. Previously, we reported that plasma dehydroepiandrosterone sulfate (DHEAS) could serve as an endogenous probe to predict OATP-based DDIs in monkeys using rifampicin as an OATP inhibitor. Since the contribution of hepatic OATPs to the changes in plasma DHEAS by rifampicin remains unclear, however, we performed an in vivo pharmacokinetic study to explore this issue. Since plasma DHEAS concentrations were low in our rat model, the disposition of externally administered DHEAS was evaluated. Intravenously administered DHEAS was recovered mainly in bile (29.1%) and less in urine (2.95%). The liver tissue-to-plasma concentration ratio (Kpliver) decreased from 41.8 to 5.07 by rifampicin, and this decrement was consistent with the decrease in distribution volume from 247 to 59 ml/rat. Comparison of the in vitro IC50 of rifampicin for DHEAS uptake by isolated rat hepatocytes and in vivo plasma rifampicin concentration suggested that the effect of rifampicin on the plasma DHEAS concentration was explained mostly by the inhibition of hepatic OATPs, demonstrating that DHEAS could be a biomarker of hepatic OATP activity. Next, previously reported rifampicin-induced changes in plasma concentrations evaluated as an AUC ratio (AUCR) of possible probe compounds were compared on the basis of rifampicin dose/body surface area. The AUCR values of endogenous compounds and i.v. administered statins, for which possible DDIs in the intestinal absorption process can be excluded, increased proportionally to the rifampicin dose. Simultaneous measurement of these endogenous compounds could be effective biomarkers for the prediction of OATP-based DDIs.

Role of OATP2A1 in PGE(2) secretion from human colorectal cancer cells via exocytosis in response to oxidative stress.

Chronic inflammation induced by reactive oxygen species is associated with increased risk of developing colorectal cancer (CRC), and prostaglandin E2 (PGE2), which serves as a key mediator of inflammatory responses, plays an important role in CRC initiation and progression. Therefore, in the present study, we aimed to investigate the role of prostaglandin transporter OATP2A1/SLCO2A1 in the changes of PGE2 disposition in CRC cells in response to oxidative stress. H2O2 induced translocation of cytoplasmic OATP2A1 to plasma membranes in LoVo and COLO 320DM cells, but not in Caco-2 cells. The shift of subcellular OATP2A1 was abolished in the presence of anti-oxidant N-acetyl-L-cysteine or an inhibitor of protein kinase C, which evokes exocytosis. Exposure of LoVo cells to H2O2 caused an increase in the amount of extracellular PGE2 without changing the sum of intra- and extracellular PGE2. OATP2A1 knockdown decreased extracellular PGE2 in LoVo cells. In addition, extracellular PGE2 was significantly reduced by exocytosis inhibitor cytochalasin D, suggesting that H2O2-induced PGE2 release occurs in an exocytotic manner. Furthermore, mRNA expression of vascular endothelial growth factor (VEGF) was significantly reduced in LoVo cells by knockdown of OATP2A1. These results suggest that cytoplasmic OATP2A1 likely facilitates PGE2 loading into suitable intracellular compartment(s) for efficient exocytotic PGE2 release from CRC cells exposed to oxidative stress.

Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells.

Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways.

Local Drug-Drug Interaction of Donepezil with Cilostazol at Breast Cancer Resistance Protein (ABCG2) Increases Drug Accumulation in Heart.

Clinical reports indicate that cardiotoxicity due to donepezil can occur after coadministration with cilostazol. We speculated that the concentration of donepezil in heart tissue might be increased as a result of interaction with cilostazol at efflux transporters such as P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2), which are expressed in many tissues including the heart, and our study tested this hypothesis. First, donepezil was confirmed to be a substrate of both BCRP and P-glycoprotein in transporter-transfected cells in vitro. Cilostazol inhibited BCRP and P-glycoprotein with half-inhibitory concentrations of 130 nM and 12.7 µM, respectively. Considering the clinically achievable unbound plasma concentration of cilostazol (about 200 nM), it is plausible that BCRP-mediated transport of donepezil would be affected by cilostazol in vivo. Indeed, in an in vivo rat study, we found that coadministration of cilostazol significantly increased the concentrations of donepezil in the heart and brain, where BCRP functions as a part of the blood-tissue barrier, whereas the plasma concentration of donepezil was unaffected. In addition, in vitro accumulation of donepezil in heart tissue slices of rats was significantly increased in the presence of cilostazol. These results indicate that donepezil-cilostazol interaction at BCRP may be clinically relevant in heart and brain tissues. In other words, the tissue distribution of drugs can be influenced by drug-drug interaction (DDI) at efflux transporters in certain tissues (local DDI) without any apparent change in plasma concentration (systemic DDI).