ABSTRACT
The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation.
Subject(s)
Marine Toxins , Organic Anion Transporters, Sodium-Independent , Proto-Oncogene Proteins c-akt , Humans , Organic Anion Transporters, Sodium-Independent/metabolism , Organic Anion Transporters, Sodium-Independent/pharmacology , Solute Carrier Organic Anion Transporter Family Member 1B3/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Tumor Suppressor Protein p53/metabolism , HEK293 Cells , Microcystins/metabolism , Okadaic Acid/toxicity , Epithelial-Mesenchymal Transition , Phosphoprotein PhosphatasesABSTRACT
Cadmium (Cd) is a typical pollutant and carcinogen in environment. Exposure assessment of contaminants is an important component of occupational and environmental epidemiological studies. Early studies of Cd have focused on aquatic animals, chickens and rats. However, toxicological evaluation of Cd in pigs has not been reported. Therefore, twelve pigs were randomly divided into two groups (n = 6): the control group and the Cd group (Cd content: 15 ± 0.242 mg/kg feed) in this study, the experimental period was 30 d, and the toxic effects of Cd on the liver of weanling piglets were examined by antioxidant function, liver function, Cd content, histological examination and transcriptomics. The results showed that the changes of antioxidant function, liver function and Cd content were significant in the liver. Transcriptional profiling results showed that 399 differentially expressed genes (DEGs) were significantly up-regulated while 369 DEGs were remarkably down-regulated in Cd group, and which were concentrated in three ontologies: molecular function, cellular component and biological processes. Interestingly, significant changes in some genes of the cytochrome P450 enzyme (CYP450) and solute carrier (SLC) families have been observed and were consistent with qRT-PCR results. In conclusion, Cd could cause liver injury in weanling piglets and change the transcriptomic characteristics of liver. CYP450 and SLC families play an indispensable role in Cd-mediated hepatotoxicity. Importantly, changes in mRNA levels of CYP2B22, CYP7A1, CYP8B1, SLC26A8, SLC11A1, SLC27A2 and SLC22A7 induced by Cd have been reported for the first time. Our findings will provide a new insight for better assessing the mechanism of Cd toxicity to the liver.
Subject(s)
Cadmium/toxicity , Chemical and Drug Induced Liver Injury, Chronic/enzymology , Cytochrome P-450 Enzyme System/metabolism , Environmental Pollutants/toxicity , Animals , Antioxidants/pharmacology , Chemical and Drug Induced Liver Injury, Chronic/pathology , Liver/drug effects , Organic Anion Transporters, Sodium-Independent/genetics , Organic Anion Transporters, Sodium-Independent/pharmacology , RNA, Messenger/genetics , Swine , Transcriptome/drug effectsABSTRACT
Salvianolic acid B (SAB) has a high concentration in the liver, but the mechanism of its distribution in the liver is unclear. The aim of this study was to investigate the mechanisms of hepatic uptake of SAB. In this study, we first explored the uptake features of SAB in HepG2 cells and the effect of rifampicin on uptake. Then, we explored the effects of SAB on the uptake of pitavastatin in HepG2 cells. Finally, we established an HEK239T-OATP1B1 cell model to confirm whether OATP1B1 mediated the transport of SAB. Results showed that the uptake kinetic parameters Vmax and Km for SAB in HepG2 cells were 21.28 ± 2.06 pmol mg-1 per protein min-1 and 28.47 ± 7.36 µM, respectively. Rifampicin inhibited the uptake of SAB in HepG2 cells (IC50 was 5.85 ± 1.70 µM), and SAB affected the uptake of pitavastatin in HepG2 cells (IC50 was 27.67 ± 1.90 µM). The uptake kinetic parameters Vmax and Km for SAB in HEK239T-OATP1B1 were 60.03 ± 6.16 pmol mg-1 per protein min-1 and 87.24 ± 15.28 µM, respectively, whereas in EGFP-HEK293 cells were 14.04 ± 2.53 pmol mg-1 per protein min-1 and 56.53 ± 15.50 µM. The SAB had no effect of on the expression of OATP1B1 in HEK239T-OATP1B1 cells. In conclusion, this study demonstrated that OATP1B1 contributes to the transport and accumulation of SAB in the liver.
Subject(s)
Benzofurans/therapeutic use , Biological Transport/physiology , Liver/metabolism , Organic Anion Transporters, Sodium-Independent/therapeutic use , Solute Carrier Organic Anion Transporter Family Member 1B3/therapeutic use , Benzofurans/pharmacology , Humans , Organic Anion Transporters, Sodium-Independent/pharmacology , Solute Carrier Organic Anion Transporter Family Member 1B3/pharmacologyABSTRACT
PURPOSE: Organic anion-transporting polypeptides (OATP) mediate the cellular uptake of a broad range of drugs. The hydrophobic anticancer drug, paclitaxel (PTX), was recently identified as a substrate for OATP1B3 in vitro. We investigated the role of Oatp1a/1b transporters in the pharmacokinetics of PTX in vivo, as well as their impact at different dose levels of PTX and methotrexate (MTX). EXPERIMENTAL DESIGN: Recently generated Slco1a/1b(-/-) (lacking all Oatp1a/1b transporters) and wild-type mice were intravenously dosed with 2, 10, or 50 mg/kg of PTX, or with 10, 50, or 500 mg/kg of MTX, and plasma and tissue drug concentrations were measured. RESULTS: In spite of its hydrophobicity, PTX systemic exposure (at 10 mg/kg) was increased by greater than 2-fold in Slco1a/1b(-/-) mice compared with wild-type, whereas PTX liver uptake was reduced by about 2-fold. Oatp1a/1b transporters displayed a high impact on PTX and MTX pharmacokinetics over a broad dose range. For MTX, even at 500 mg/kg, saturation of Oatp1a/1b was not observed, with a 3.4-fold increase in plasma and 30-fold decrease in liver levels in Slco1a/1b(-/-) mice compared with wild-type. Although beginning saturation of Oatp1a/1b was observed at the highest dose of PTX, plasma levels in Slco1a/1b(-/-) mice were still 1.7-fold increased and liver levels 1.5-fold decreased compared with wild-type. CONCLUSION: Oatp1a/1b transporters play a pronounced role in determining plasma levels and tissue distribution of MTX and PTX, thus affecting even highly hydrophobic drugs. Variation in OATP1A/1B transporter activity, due to genetic variation, inhibition, and/or tumor expression might affect toxicity and therapeutic efficacy of these anticancer drugs.
Subject(s)
Organic Anion Transporters, Sodium-Independent/pharmacology , Organic Anion Transporters/pharmacology , Organic Anion Transporters/physiology , Organic Cation Transport Proteins/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Dose-Response Relationship, Drug , Drug Interactions , Hydrophobic and Hydrophilic Interactions , Liver/metabolism , Liver-Specific Organic Anion Transporter 1 , Methotrexate/pharmacokinetics , Mice , Mice, Knockout , Paclitaxel/pharmacokineticsABSTRACT
The purpose of this study was to invent an extracellular inhibitor selective for the plasma membrane Ca(2+) pump(s) (PMCA) isoform 1. PMCA extrude Ca(2+) from cells during signalling and homeostasis. PMCA isoforms are encoded by 4 genes (PMCA1-4). Pig coronary artery endothelium and smooth muscle express the genes PMCA1 and 4. We showed that the endothelial cells contained mostly PMCA1 protein while smooth muscle cells had mostly PMCA4. A random peptide phage display library was screened for binding to synthetic extracellular domain 1 of PMCA1. The selected phage population was screened further by affinity chromatography using PMCA from rabbit duodenal mucosa which expressed mostly PMCA1. The peptide displayed by the selected phage was termed caloxin 1b3. Caloxin 1b3 inhibited PMCA Ca(2+)-Mg(2+)-ATPase in the rabbit duodenal mucosa (PMCA1) with a greater affinity (inhibition constant=17±2 µM) than the PMCA in the human erythrocyte ghosts (PMCA4, inhibition constant=45±4 µM). The affinity of caloxin 1b3 was also higher for PMCA1 than for PMCA2 and 3 indicating its selectivity for PMCA1. Consistent with an inhibition of PMCA1, caloxin 1b3 addition to the medium increased cytosolic Ca(2+) concentration in endothelial cells. Caloxin 1b3 is the first known PMCA1 selective inhibitor. We anticipate caloxin 1b3 to aid in understanding PMCA physiology in endothelium and other tissues.
Subject(s)
Calcium/metabolism , Endothelial Cells/drug effects , Organic Anion Transporters, Sodium-Independent/pharmacology , Plasma Membrane Calcium-Transporting ATPases/antagonists & inhibitors , Animals , Ca(2+) Mg(2+)-ATPase/antagonists & inhibitors , Ca(2+) Mg(2+)-ATPase/metabolism , Cell Membrane/drug effects , Cell Membrane/enzymology , Coronary Vessels/cytology , Coronary Vessels/enzymology , Endothelial Cells/enzymology , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Humans , In Vitro Techniques , Intestinal Mucosa/drug effects , Intestinal Mucosa/enzymology , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/enzymology , Organ Specificity , Plasma Membrane Calcium-Transporting ATPases/metabolism , Rabbits , Solute Carrier Organic Anion Transporter Family Member 1B3 , SwineABSTRACT
5-Hydroxymethylfurfural (HMF) is formed when sugars are acidified or heated. It is present at high levels in numerous foods. HMF is inactive in standard genotoxicity tests, but can be metabolized to a chemically reactive intermediate, 5-sulfooxymethylfurfural (SMF), which is mutagenic and carcinogenic. We recently found that direct parental administration of SMF to mice leads to abundant acute necrosis and proteinaceous casts in the proximal tubules as the dominating toxicological effect. Since proximal tubule cells actively mediate the excretion of many organic anions, we hypothesized that transporter-mediated uptake of SMF into the cells could be the reason for this selective organotoxicity. To test this hypothesis, we used human embryonic kidney (HEK293) cells stably expressing human (h) OAT1 or OAT3. SMF was a competitive inhibitor of p-aminohippurate uptake by hOAT1 and estrone sulfate uptake by hOAT3 with K(i) values of 225 microM and 1.5mM, respectively. Moreover, the initial rates of SMF uptake were 5.2- and 3.1-fold higher in cells expressing hOAT1 and hOAT3, respectively, than in control HEK293 cells. Likewise, the sensitivity of hOAT1- and hOAT3-expressing cells to SMF cytotoxicity was significantly higher than that of control cells, and was reduced by addition of probenecid, an inhibitor of OATs. Taken together, these results indicate that OAT1 and OAT3 mediate the uptake of SMF into proximal tubule cells and thereby may be involved in SMF-induced nephrotoxicity.
Subject(s)
Epithelial Cells/drug effects , Furaldehyde/analogs & derivatives , Kidney Tubules, Proximal/pathology , Kidney/drug effects , Organic Anion Transport Protein 1/antagonists & inhibitors , Organic Anion Transporters, Sodium-Independent/antagonists & inhibitors , Cell Line , Cells, Cultured , Dose-Response Relationship, Drug , Epithelial Cells/physiology , Furaldehyde/pharmacology , Humans , Kidney/metabolism , Kidney/pathology , Male , Organic Anion Transport Protein 1/pharmacology , Organic Anion Transporters, Sodium-Independent/pharmacologyABSTRACT
BACKGROUND: Evidence is accumulating that bile acids are involved in colon cancer development, but their molecular mechanisms remain unexplored. Bile acid has been reported to be associated with induction of the cyclooxygenase-2 (COX-2) gene. Because the human liver-specific organic anion transporter-2 (LST-2/OATP8/OATP1B3) is expressed in gastrointestinal cancers and might transport bile acids to the intracellular space, we studied the molecular mechanisms by which bile acids induce the transcription of COX-2, and the role of LST-2 in colonic cell lines. METHODS: Transcriptional activity of COX-2 was measured using a human COX-2 promoter-luciferase assay under various concentrations of bile acids. Electrophoresis mobility shift assays (EMSAs) for peroxisome proliferators-activated receptor (PPAR) alpha and cyclic AMP responsive element (CRE) were performed. RESULTS: The COX-2 promoter was induced by lithocholic acid (LCA), deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA). Deletion and site-directed mutation analyses showed that CRE is the responsive element for LCA. An adenovirus expression system revealed that LST-2 is responsible for induction of COX-2. By EMSA using oligonucleotides of CRE, we observed formation of a specific protein-DNA complex, which was inhibited by a specific antibody against PPARalpha and CRE. A PPARalpha-specific agonist induced transcription of COX-2. CONCLUSION: These results indicate that COX-2 is transcriptionally activated by the addition of LCA, CDCA, and DCA and that LST-2 plays an important role by transporting bile acid to the intracellular space. Moreover, LCA-dependent COX-2 gene activation consists of a transcriptional complex including PPARalpha and CRE-binding protein. Thus, this induction of COX-2 may participate in carcinogenesis and progression of colorectal cancer cells.
Subject(s)
Bile Acids and Salts/pharmacology , Colorectal Neoplasms/genetics , Cyclooxygenase 2/genetics , Gene Expression Regulation/drug effects , PPAR alpha/pharmacology , Transcription, Genetic/drug effects , Adenoviridae , Cell Line, Tumor , Colorectal Neoplasms/metabolism , Cyclic AMP Response Element-Binding Protein , Electrophoretic Mobility Shift Assay , Humans , Lithocholic Acid/pharmacology , Organic Anion Transporters, Sodium-Independent/metabolism , Organic Anion Transporters, Sodium-Independent/pharmacology , PPAR alpha/metabolism , Promoter Regions, Genetic , Solute Carrier Organic Anion Transporter Family Member 1B3 , Transcriptional Activation , TransfectionABSTRACT
In rats, the secretion of p-aminohippurate (PAH) by the kidney is higher in males (M) than in females (F). The role of the major renal PAH transporters, OAT1 and OAT3, in the generation of these gender differences, as well as the responsible hormones and mechanisms, has not been clarified. Here we used various immunocytochemical methods to study effects of gender, gonadectomy, and treatment with sex hormones on localization and abundance of OAT1 and OAT3 along the rat nephron. Both transporters were localized to the basolateral membrane: OAT1 was strong in proximal tubule S2 and weak in the S3 segments, whereas OAT3 was stained in proximal tubule S1 and S2 segments, thick ascending limb, distal tubule, and in principal cells along the collecting duct. Gender differences in the expression of both transporters in adult rats (M > F) were observed only in the cortical tubules. OAT1 in the cortex was strongly reduced by castration in adult M, whereas the treatment of castrated M with testosterone, estradiol, or progesterone resulted in its complete restitution, further depression, or partial restitution, respectively. In adult F, ovariectomy weakly increased, whereas estradiol treatment of ovariectomized F strongly decreased, the expression of OAT1. The expression of OAT3 in the M and F cortex largely followed a similar pattern, except that ovariectomy and progesterone treatment showed no effect, whereas in other tissue zones gender differences were not observed. In prepubertal rats, the expression of OAT1 and OAT3 in the kidney cortex was low and showed no gender differences. Our data indicate that gender differences in the rat renal cortical OAT1 and OAT3 (M > F) appear after puberty and are determined by both a stimulatory effect of androgens (and progesterone in the case of OAT1) and an inhibitory effect of estrogens.