Significance of the Signal Intensity of Gadoxetic Acid-enhanced MR Imaging for Predicting the Efficacy of Hepatic Arterial Infusion Chemotherapy in Hepatocellular Carcinoma.

PURPOSE: We attempted to clarify the relationship between the signal intensity (SI) in the hepatobiliary phase of gadoxetic acid-enhanced magnetic resonance (MR) imaging and the efficacy of hepatic arterial infusion chemotherapy (HAIC) in hepatocellular carcinomas (HCCs). METHODS: We enrolled 14 patients with HCCs who underwent gadoxetic acid-enhanced MR imaging prior to HAIC using cisplatin and 5-fluorouracil. In the hepatobiliary phase, we calculated the SI of the HCCs and the background liver. In cases with multiple HCCs, we calculated the SI of the largest lesion. Patients were classified into high (n = 7) and low intensity (n = 7) groups based on the median value of the SI ratio (SI of the tumor/SI of the background liver). We analyzed progression-free survival using the Kaplan-Meier method and the log-rank test. In the 5 patients with a history of HCC surgery, we compared the expression of immunohistochemical organic anion-transporting polypeptide (OATP) 8 between the high and low intensity groups by chi-square test. RESULTS: The SI ratios were 0.568 ± 0.093 (mean ± standard deviation) in the high intensity group and 0.251 ± 0.086 in the low intensity group. Compared to the group with low signal intensity, the group with high signal intensity demonstrated significantly lower serum levels of alpha fetoprotein (AFP) (P = 0.0350), significantly higher progression-free survival (P = 0.0108), better differentiation of tumor grade at histologic examination (P = 0.0253), and significantly higher OATP8 expression (P = 0.0253). CONCLUSION: Patients with HCCs of high SI ratio in the hepatobiliary phase of gadoxetic acid-enhanced MR imaging can respond better to HAIC.

Novel No-Wash Luminogenic Probes for the Detection of Transporter Uptake Activity.

Luminogenic probes were designed and synthesized for the detection of uptake transporter activity in a lytic cell-based assay. These probes rely on a self-cleavable trimethyl lock quinone-cyanobenzothiazole (TMQ-CNBT) or trimethyl lock quinone-luciferin (TMQ-Luc) linked to the anion transporter substrate fluorescein. Upon cellular transport, the TMQ is reduced by viable cells, resulting in the facile intramolecular lactonization and rapid release of the bioluminescent reporter molecule. The uptake transporter activity can then be detected without removing and washing off the extracellular substrates. Six probes were tested with OATP1B1*1a and OATP1B3 overexpressing HEK293 cells, and all compounds showed up to 10.2-fold enhancement in uptake when compared to control cells. Uptake of TMQ-luciferin compounds 2, 4, and 6 increased linearly over time up to 30 min at a concentration ranging from 40 nM to 20 µM. The apparent Km values obtained at different time intervals up to 30 min were nearly identical for a given compound, which validates the 30 min window as appropriate for uptake transporter assays. The average apparent Km values ranged from 0.3 to 0.8 µM and 0.2 to 1.3 µM for OATP1B1*1a and OATP1B3, respectively, indicating good affinities to these anion transporters. Furthermore, uptake of compound 2 was inhibited by two inhibitors of OATP1B1*1a and OATP1B3: rifampicin and ritonavir. The preliminary results obtained from compound 2 exhibited a time-dependent, saturatable, and inhibitable nature of uptake, indicating the feasibility of using the probe for the detection of a transporter-mediated process. This add-and-read homogeneous assay may provide a convenient, rapid, and facile way to detect changes in transporter activity in a high-throughput format, and this assay design strategy could create a new platform for a general cell uptake assay for biomaterials in the future.

Insights into the diagnosis of hepatocellular carcinomas with hepatobiliary MRI.

The incidence of hepatocellular carcinomas (HCCs) has increased worldwide in line with an improved screening by high-resolution imaging of cirrhotic livers. Besides abdominal ultrasonography and computerised tomography, magnetic resonance imaging (MRI) is an important tool to detect HCCs. With commercialisation of MR hepatobiliary contrast agents that cross membrane transporters in hepatocytes or tumour cells, MRI adds new information to detect and characterise HCCs. When tumour cells lose organic anion transporting polypeptides (OATP1B1/B3) in cell membranes facing sinusoidal blood, tumours appear hypointense (decreased contrast agent concentrations) in comparison to surrounding normal or cirrhotic liver that retains OATP1B1/B3 expression. However, expression, regulation, and prognostic significance of transporter evolution along carcinogenesis are not completely known. Moreover, understanding signal intensities in focal lesions also relies on transport functions of cellular efflux transporters. This manuscript reviews all the publications that associate liver imaging with hepatobiliary contrast agents and expression of transporters. The regulation of transporters along carcinogenesis to anticipate the prognosis of focal lesions is also included.

Reduced organic anion transporter expression is a risk factor for hepatocellular carcinoma in chronic hepatitis C patients: a propensity score matching study.

OBJECTIVES: Recent reports indicated that reduced SLC22A7 (a gene-encoding organic anion transporter 2) expression in noncancerous liver tissue predicts hepatocellular carcinoma (HCC) recurrence after curative resection. Our study aimed to elucidate the association between SLC22A7 expression and HCC development in chronic hepatitis C patients. METHODS: HCC recurrence after local ablation therapy and SLC22A7 expression in noncancerous liver tissue were analyzed in 20 patients. Subsequently, the association between de novo HCC development and SLC22A7 expression was examined at baseline in 38 hepatitis C patients without HCC who subsequently developed HCC as well as in 76 hepatitis C patients who did not develop HCC and were matched for age, gender and stage of fibrosis. RESULTS: In the patients whose HCC had been cured, reduced SLC22A7 expression in noncancerous liver tissue was significantly associated with a high incidence of multifocal HCC recurrence. In patients without HCC at baseline, cumulative incidence of de novo HCC development was significantly higher with a reduced SLC22A7 expression than with a normal expression (p = 0.01). This difference remained significant among patients without known risk factors for HCC like age and advanced fibrosis. CONCLUSION: Reduced SLC22A7 expression in the liver indicates a significant risk for HCC development in chronic hepatitis C, independently of other risk factors.

Organic anion transporting polypeptides expressed in pancreatic cancer may serve as potential diagnostic markers and therapeutic targets for early stage adenocarcinomas.

PURPOSE: Organic Anion Transporting Polypeptides (OATPs) are expressed in various epithelial tissues in the body. Because they can be expressed in cancers and because they can transport anticancer drugs, OATPs could be potential targets for cancer therapy. Therefore we examined their expression in human pancreatic ductal adenocarcinomas. METHODS: Expression of all 11 human OATPs was measured at the mRNA level and OATPs with highest expression were characterized at the protein level. RESULTS: Transcripts of SLCO1B3, SLCO2A1, SLCO3A1 and SLCO4A1 were detected in all the tested pancreatic tissues. OATP1B3, OATP2A1, OATP3A1 and OATP4A1 protein expression was confirmed in these tissues and expression of all four transporters increased in pancreatic adenocarcinoma compared to normal pancreas. OATP1B3 expression was highest in pancreatic hyperplasia and stage one adenocarcinomas compared to stage two and three adenocarcinomas. CONCLUSION: OATP1B3, OATP2A1, OATP3A1 and OATP4A1 are up-regulated in pancreatic adenocarcinoma and could potentially be used to target anticancer drugs to pancreatic cancer. Additionally, because expression of OATP1B3 is highest in pancreatitis and stage one adenocarcinoma, which leads to pancreatic cancer, OATP1B3 is a potential marker to diagnose patients with early stage pancreatic adenocarcinomas.

Beta-catenin-activated hepatocellular adenoma showing hyperintensity on hepatobiliary-phase gadoxetic-enhanced magnetic resonance imaging and overexpression of OATP8.

We report a male case of beta-catenin-activated hepatocellular adenoma (HCA) focusing on findings of gadoxetic-acid-enhanced magnetic resonance imaging (EOB-MRI) and discussing the molecular background and possible clinical significance. The patient was a 31-year-old man in whom computed tomography (CT) showed a large nodule of 14 cm in diameter in the right liver lobe. On dynamic contrast-enhanced CT, heterogeneous and slight to moderate enhancement was observed during the early phase, with washout in the late phase. Focal fat deposits and a scar-like portion in the lesion were also seen. Most of the lesion was slightly hyperintense compared with the background liver on the hepatobiliary phase of EOB-MRI. After operation, this patient was confirmed pathologically as having beta-catenin-activated HCA with a portion suggestive of malignant transformation. In addition, intense organic anion transporter polypeptide 8 expression was observed throughout the tumor by immunohistochemical staining.

Gene expression analysis of membrane transport proteins in normal and lipopolysaccharide-inflamed rat dental pulp.

INTRODUCTION: Membrane transport proteins (transporters) play a crucial role in the transmembrane uptake and/or efflux of various compounds such as inorganic ions, endogenous bioactive substances such as prostaglandins (PGs), and drugs such as nonsteroidal anti-inflammatory drugs. This study aimed to analyze mRNA expression of selected transporters related to drug disposition and PG transport in normal and lipopolysaccharide (LPS)-inflamed rat incisor pulp. METHODS: Pulp tissues were subjected to reverse transcription-polymerase chain reaction (PCR) detection for transporter isoforms belonging to organic anion transporting polypeptide (Oatp), organic anion transporter (Oat), organic cation transporter (Oct), multidrug resistance-associated protein (Mrp), and multidrug resistance protein (Mdr) families. The levels of mRNA expression for PG transporters (Oatp1a5, Oatp1b2, Oatp2a1, Oatp2b1, and Oatp3a1) were compared in normal and LPS-inflamed pulps by using real-time PCR. RESULTS: The pulp tissue expressed mRNAs for various transporters belonging to the Oatp, Oat, Oct, Mrp, and Mdr families. LPS inflammation caused significant up-regulation of Oatp2a1 (P < .01) and significant down-regulation of Oatp1a5, Oatp2b1 (P < .01), and Oatp3a1 (P < .05). CONCLUSIONS: Rat incisor dental pulp expressed mRNAs for various transporter isoforms. The levels of mRNA expression for PG transporters were significantly up-regulated or down-regulated in LPS-inflamed dental pulp.

Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues.

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 µg mL(-1) (0.040 µg mg(-1)) to 20 µg mL(-1) (4.0 µg mg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.

The HMG-CoA reductase inhibitor pravastatin stimulates insulin secretion through organic anion transporter polypeptides.

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin has been reported to have a beneficial effect on reducing the new onset of diabetes as well as lowering plasma lipids. Because pravastatin is a water-soluble organic anion, it cannot easily penetrate the lipid bilayer of the cell membrane. As the precise mechanisms of the effect of pravastatin on glucose metabolism and diabetes have not been clarified, we examined the roles of the organic anion transporter family on pravastatin-treated islet and adipocyte functions. Rat oatp1/slco1a1, oatp2/slco1a4 and oatp3/slco1a5 were expressed in the pancreas, and rat oatp3/slco1a5 was also detected in rat insulinoma cell line INS-1e. Pravastatin was transported not only by oatp1/slco1a1 and oatp2/slco1a4, but also by rat oatp3/slco1a5. Pravastatin uptake into INS-1e cells was detected and this transport was inhibited by sulfobromophthalein and rifampicin, both of which are known to inhibit oatp family-mediated uptake. In addition, pravastatin enhanced the glucose-stimulated insulin secretion from INS-1e cells. When fat-loaded db/db mice were treated with pravastatin, glucose intolerance and insulin resistance were prevented. In addition, insulin secretion from isolated islets was enhanced by pravastatin. These data suggest that pravastatin has pleiotropic effects on islets through membrane transport under high fat/glucose conditions.

Molecular determinants in the transport of a bile acid-derived diagnostic agent in tumoral and nontumoral cell lines of human liver.

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

Immunocytochemical characterization of the incubated rat renal cortical slices.

The use of renal cortical slices in vitro and the data obtained in these studies have been subjects of controversy, largely due to uncertain viability, e.g., structural and functional integrity of the proximal and other tubules. However, detailed studies of tubule integrity have not been reported. To correlate functional and structural viability of the hand-cut rat renal cortical slices, incubated in optimally conditioned media for up to 25 h, we studied the time course of p-aminohippurate (PAH) uptake, the immunocytochemical distribution of several proteins that reside in the proximal tubule basolateral [Na/K-ATPase, organic anion transporters (OAT)1 and OAT3], or brush border [megalin, sodium-proton exchanger (NHE)3] membrane, as well as the general integrity of the tubule epithelium and its cytoskeleton (actin filaments, microtubules). PAH uptake in slices was proportional to time within 1 h of incubation and gradually declined thereafter. The immunostaining experiments indicated a fast, time-dependent loss of basolateral transporters, at a rate of OAT1 > Na/K-ATPase > OAT3. In the brush border membrane, the loss of megalin was faster than that of NHE3, and a partial redistribution of NHE3 into the basolateral domain indicated the loss of cell polarity. The loss of intracellular actin and tubulin cytoskeleton in the proximal tubule was already visible after 15 min of incubation and gradually increased with time, whereas a partial redistribution of actin to the basolateral domain indicated a compromised polarity of the cells. The data also revealed very early (after 15 min) necrotic events in the proximal tubule epithelium, with sloughing of brush border and cell debris into the tubule lumen, detachment of cells from the basal membrane, and opening and widening of the tubule lumen. We conclude that the loss of cellular structure, cytoskeleton, and cell membrane transporters in the nephron epithelium is a very early event in the incubated rat renal cortical slices.

Detection of the human organic anion transporters SLC21A6 (OATP2) and SLC21A8 (OATP8) in liver and hepatocellular carcinoma.

Transport proteins mediating the selective uptake of organic anions into human hepatocytes include the organic anion transporters SLC21A6 (also termed OATP2, OATP-C, or LST-1) and SLC21A8 (OATP8). Both transporters are localized to the basolateral membrane of human hepatocytes. Because of the importance of these transporters for hepatobiliary elimination, including the removal of bilirubin and its conjugates from the blood circulation, we have generated monoclonal antibodies for studies on the expression and localization of these transport proteins. We describe two antibodies, designated monoclonal antibody MDQ (mMDQ) and monoclonal antibody ESL (mESL), directed against the amino terminus and the carboxyl terminus of human SLC21A6, respectively. Both antibodies have been characterized by immunoblot analysis, immunoprecipitation, and immunofluorescence microscopy. While mESL reacted specifically with SLC21A6, mMDQ detects both SLC21A6 and SLC21A8. Neither of the two antibodies reacted with other human, or with dog, rat, or mouse liver SLC21A family members. Antibody mMDQ may be used for the simultaneous detection of SLC21A6 and SLC21A8 in immunoblotting because of its immunoreactivity with both molecules and because of the different molecular masses of both glycosylated proteins in human hepatocytes. This is exemplified in hepatocellular carcinomas where SLC21A6 and SLC21A8 were differentially synthesized and showed an irregular staining pattern. Both transport proteins have not been detected in human hepatoma HepG2 cells. In routine paraffin sections, 10 of 12 hepatocellular carcinomas were focally positive with antibody mMDQ. In contrast, cholangiocarcinomas and liver metastases of colorectal and pancreatic adenocarcinoma were negative without exception. This suggests the usefulness of SLC21A6/SLC21A8 within a panel of tumor markers for hepatocellular carcinomas. Moreover, both antibodies should be useful in studies on the expression and localization of two important uptake transporters of human hepatocytes under physiologic and pathophysiologic conditions.