Indirect activation of constitutive androstane receptor in three-dimensionally cultured HepG2 cells.

Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, is retained as an inactive form phosphorylated at threonine in the cytoplasm of hepatocytes. Upon activation, CAR is dephosphorylated to move into the nucleus and induces the transcription of genes. Thus, nuclear translocation is a key step for CAR activation in hepatocytes. However, this nuclear translocation has not been demonstrated in conventional two-dimensionally-cultured immortalized cell lines such as HepG2, in which CAR spontaneously accumulates in the nucleus. In this study, we showed that treatment with the indirect CAR activator phenobarbital activated transcription of the CYP3A4 gene in three-dimensionally (3D)-cultured HepG2 cells. CAR was retained as its phosphorylated form in the cytoplasm and was translocated to the nucleus in 3D-cultured HepG2 cells in response to treatment with phenobarbital. Moreover, okadaic acid and epidermal growth factor, were found to repress phenobarbital-induced CAR nuclear translocation and subsequent activation of the CYP3A4 gene promoter. These results suggested that 3D-cultured HepG2 cells properly regulated CAR activation as has been observed in hepatocytes.

Differentiated HASTR/ci35 cells: A promising in vitro human astrocyte model for facilitating CNS drug development studies.

Astrocytes have shown longstanding promise as therapeutic targets for various central nervous system diseases. To facilitate drug development targeting astrocytes, we have recently developed a new conditionally immortalized human astrocyte cell line, termed HASTR/ci35 cells. In this study, in order to further increase their chances to contribute to various astrocyte studies, we report on the development of a culture method that improves HASTR/ci35 cell differentiation status and provide several proofs related to their astrocyte characteristics. The culture method is based on the simultaneous elimination of serum effects and immortalization signals. The results of qPCR showed that the culture method significantly enhanced several astrocyte marker gene expression levels. Using the differentiated HASTR/ci35, we examined their response profiles to nucleotide treatment and inflammatory stimuli, along with their membrane fatty acid composition. Consequently, we found that they responded to ADP or UTP treatment with a transient increase of intracellular Ca2+ concentration, and that they could show reactive response to interleukin-1ß treatments. Furthermore, the membrane phospholipids of the cells were enriched with polyunsaturated fatty acids. To summarize, as a unique human astrocyte model carrying the capability of a differentiation induction properties, HASTR/ci35 cells are expected to contribute substantially to astrocyte-oriented drug development studies.

Cancer-type organic anion transporting polypeptide 1B3 is a target for cancer suicide gene therapy using RNA trans-splicing technology.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) has been identified as a cancer-specific transcript in various solid cancers, including colorectal cancer. Given its excellent cancer-specific expression profile, we hypothesized that Ct-OATP1B3 could represent a promising target for cancer-specific expression of the suicide gene, herpes simplex virus 1 thymidine kinase (HSV-tk), via a spliceosome-mediated RNA trans-splicing (SMaRT) approach. SMaRT technology is used to recombine two RNA molecules to generate a chimeric transcript. In this study, we engineered an RNA trans-splicing molecule carrying a translation-defective HSV-tk sequence (RTM44), which was capable of inducing its own trans-splicing to the desired Ct-OATP1B3 pre-mRNA target. RTM44 expression in LS180 cells resulted in generation of Ct-OATP1B3/HSV-tk fusion mRNA. A functional translation start site contributed by the target pre-mRNA restored HSV-tk protein expression, rendering LS180 cells sensitive to ganciclovir treatment in vitro and in xenografted mice. The observed effects are ascribed to accurate and efficient trans-splicing, as they were absent in cells carrying a splicing-deficient mutant of RTM44. Collectively, our data highlights Ct-OATP1B3 as an ideal target for the HSV-tk SMaRT suicide system, which opens up new translational avenues for Ct-OATP1B3-targeted cancer therapy.

Development of a Caco-2 Cell Line Carrying the Human Intestine-Type CES Expression Profile as a Promising Tool for Ester-Containing Drug Permeability Studies.

Carboxylesterase 2 (CES2), which is a member of the serine hydrolase superfamily, is primarily expressed in the human small intestine, where it plays an important role in the metabolism of ester-containing drugs. Therefore, to facilitate continued progress in ester-containing drug development, it is crucial to evaluate how CES2-mediated hydrolysis influences its intestinal permeability characteristics. Human colon carcinoma Caco-2 cells have long been widely used in drug permeability studies as an enterocyte model. However, they are not suitable for ester-containing drug permeability studies due to the fact that Caco-2 cells express CES1 (which is not expressed in human enterocytes) but do not express CES2. To resolve this problem, we created a new Caco-2 cell line carrying the human small intestine-type CES expression profile. We began by introducing short-hairpin RNA for CES1 mRNA knockdown into Caco-2 cells to generate CES1-decifient Caco-2 cells (Caco-2CES1KD cells). Then, we developed Caco-2CES1KD cells that stably express CES2 (CES2/Caco-2CES1KD cells) and their control Mock/Caco-2CES1KD cells. The results of a series of functional expression experiments confirmed that CES2-specific activity, along with CES2 mRNA and protein expression, were clearly detected in our CES2/Caco-2CES1KD cells. Furthermore, we also confirmed that CES2/Caco-2CES1KD cells retained their tight junction formation property as well as their drug efflux transporter functions. Collectively, based on our results clearly showing that CES2/Caco-2CES1KD cells carry the human intestinal-type CES expression profile, while concomitantly retaining their barrier properties, it can be expected that this cell line will provide a promising in vitro model for ester-containing drug permeability studies.

Cancer-Type OATP1B3 mRNA in Extracellular Vesicles as a Promising Candidate for a Serum-Based Colorectal Cancer Biomarker.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA is a variant isoform of the liver-type OATP1B3. Because Ct-OATP1B3 mRNA shows an excellent cancer-specific expression profile in colorectal cancer (CRC), and that its expression levels are associated with CRC prognosis, it holds the potential to become a useful CRC detection and diagnosis biomarker. While the potential is currently justified only at the tissue level, if existence of Ct-OATP1B3 mRNA in CRC-derived extracellular vesicles (EVs) is validated, the findings could enhance its translational potential as a CRC detection and diagnosis biomarker. Therefore, this study aims at proving that Ct-OATP1B3 mRNA exists in CRC-derived EVs, and can be detected using serum specimens. To examine the possibility of Ct-OATP1B3 mRNA being existed in extracellular milieu, we isolated EVs from the human CRC (HCT116, HT-29, and SW480) cell lines, and prepared their cDNAs. The RT-PCR results showed that Ct-OATP1B3 mRNA was clearly present in EVs derived from the human CRC cell lines. Then, in order to further explore the possibility that Ct-OATP1B3 mRNA in CRC-derived EVs can be detected in serum, we isolated serum EVs derived from human CRC xenograft mice, and then performed RT-PCR. The results showed that Ct-OATP1B3 mRNA could be found in all serum EV and CRC tissue samples of the mice examined. Collectively, our findings, which show that Ct-OATP1B3 mRNA exists in EVs and can be detected in (at least) mouse serum, strengthen the potential use of Ct-OATP1B3 mRNA as a serum-based CRC biomarker.

Indirect activation of pregnane X receptor in the induction of hepatic CYP3A11 by high-dose rifampicin in mice.

Rifampicin (RIF), a typical ligand of human pregnane X receptor (PXR), powerfully induces the expression of cytochrome P450 3A4 (CYP3A4) in humans. Although it is thought that RIF is not a ligand of rodent PXR, treatment with high-dose RIF (e.g. more than 20 mg/kg) increases the expression of CYP3A in the mouse liver. In this study, we investigated whether the induction of CYP3A by high-dose RIF in the mouse liver is mediated via indirect activation of mouse PXR (mPXR). The results showed that high-dose RIF increased the expression of CYP3A11 and other PXR-target genes in the liver of wild-type mice but not PXR-knockout mice. However, the results of reporter gene and ligand-dependent assembly assays showed that RIF does not activate mPXR in a ligand-dependent manner. In addition, high-dose RIF stimulated nuclear accumulation of mPXR in the mouse liver, and geldanamycin and okadaic acid attenuated the induction of Cyp3a11 and other PXR-target genes in primary hepatocytes, suggesting that high-dose RIF triggers nuclear translocation of mPXR. In conclusion, the present study suggests that high-dose RIF stimulates nuclear translocation of mPXR in the liver of mice by indirect activation, resulting in the transactivation of Cyp3a11 and other PXR-target genes.

Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method.

Pregnane X receptor (PXR) is localized in the cytoplasm of liver cells, whereas it is localized in the nucleus of monolayer-cultured HepG2 cells. Since cultured cells are affected by the microenvironment in which they are grown, we studied the effect of three-dimensional (3D) culture on the localization of PXR in HepG2 cells using the hanging drop method. The results showed that PXR was retained in the cytoplasm of HepG2 cells and other human hepatocarcinoma cell lines (FLC5, FLC7 and Huh7) when they were cultured by the hanging drop method. Treatment with rifampicin, a ligand of PXR, translocated PXR from the cytoplasm to nucleus and increased expression levels of CYP3A4 mRNA in HepG2 cells cultured by the hanging drop method. These findings suggest that 3D culture is a key factor determining the intracellular localization of PXR in human hepatocarcinoma cells and that PXR that becomes retained in the cytoplasm of HepG2 cells with 3D culture has functions of nuclear translocation and regulation of target genes in response to human PXR ligands. Three-dimensionally cultured hepatocarcinoma cells would be a useful tool to evaluate induction potency of drug candidates and also to study mechanisms of nuclear translocation of PXR by human PXR ligands.

Cancer-type OATP1B3 mRNA has the potential to become a detection and prognostic biomarker for human colorectal cancer.

AIM: We aimed to clarify diagnostic and prognostic biomarker potentials of cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA in colorectal cancer (CRC) patients. PATIENTS & METHODS: Ct-OATP1B3 mRNA levels in 97 CRC and adjacent normal colon tissues were measured by real-time PCR. The receiver operating characteristic curve analysis and the Kaplan-Meier curve analysis were performed to characterize its biomarker potentials. RESULTS: Ct-OATP1B3 mRNA showed noticeable diagnostic power (the area under the receiver operating characteristic = 0.91) in the CRC patients. Additionally, the higher/lower mRNA expression was clearly associated with better/poorer overall survival in the CRC patients (p < 0.05). CONCLUSION: Ct-OATP1B3 mRNA has the potential to be a tissue-based biomarker for definitive diagnosis and prognostic stratification in CRC.

Identification of novel glutathione adducts of benzbromarone in human liver microsomes.

Benzbromarone (BBR) is a potent uricosuric drug that can cause serious liver injury. Our recent study suggested that 1'-hydroxy BBR, one of major metabolites of BBR, is metabolized to a cytotoxic metabolite that could be detoxified by glutathione (GSH). The aim of this study was to clarify whether GSH adducts are formed from 1'-hydroxy BBR in human liver microsomes (HLM). Incubation of 1'-hydroxy BBR with GSH in HLM did not result in the formation of GSH adducts, but 1',6-dihydroxy BBR was formed. In addition, incubation of 1',6-dihydroxy BBR with GSH in HLM resulted in the formation of three novel GSH adducts (M1, M2 and M3). The structures of M1 and M2 were estimated to be GSH adducts in which the 1-hydroxyethyl group at the C-2 position and the hydroxyl group at the C-1' position of 1',6-dihydroxy BBR were substituted by GSH, respectively. We also found that the 6-hydroxylation of 1'-hydroxy BBR is mainly catalyzed by CYP2C9 and that several CYPs and/or non-enzymatic reaction are involved in the formation of GSH adducts from 1',6-dihydroxy BBR. The results indicate that 1'-hydroxy BBR is metabolized to reactive metabolites via 1',6-dihydroxy BBR formation, suggesting that these reactive metabolites are responsible for BBR-induced liver injury.

Human organic anion transporter 2 is an entecavir, but not tenofovir, transporter.

Entecavir (ETV) and tenofovir (TFV) are essential nucleoside analogues in current hepatitis B virus (HBV) treatments. Since these drugs target the HBV polymerase that is localized within human hepatocytes, determining of their cellular uptake process is an important step in fully understanding their pharmacological actions. However, the human hepatic transporters responsible for their uptake have remained unidentified. Therefore, this study aimed at identifying the primary ETV and TFV uptake transporter(s) in human hepatocytes. In transport assays, temperature-sensitive ETV and TFV uptake by human hepatocytes were observed, and their uptake were strongly inhibited by bromosulfophthalein, which is an inhibitor of organic anion transporters/organic anion transporting polypeptides (OATs/OATPs). Given these results, ETV and TFV uptake activities in several human OAT/OATP expression systems were examined. The results showed that, among the transporters tested, only OAT2 possessed ETV transport activity. On the other hand, none of the transporters showed any TFV uptake activity. To summarize, our results identify that human OAT2 is an ETV transporter, thereby suggesting that it plays an important part in the mechanisms underlying ETV antiviral activity. Furthermore, although the hepatic TFV transporters remain unknown, our results have, at least, clarified that these two anti-HBV drugs have different hepatocyte entry routes.

Involvement of ESE-3, epithelial-specific ETS factor family member 3, in transactivation of the ABCB1 gene via pregnane X receptor in intestine-derived LS180 cells but not in liver-derived HepG2 cells.

Pregnane X receptor (PXR) is involved in the transactivation of ABCB1 gene by rifampicin (RIF). However, we found that increase in ABCB1 mRNA by RIF was observed in LS180 cells but not in HepG2 cells. Since both cell lines expressed PXR equally, we hypothesized that a factor(s) other than PXR is responsible for PXR-mediated transactivation of the ABCB1 gene. Reporter activities of a distal enhancer module containing direct repeat 4 (DR4) motifs were increased by RIF in LS180 cells but not in HepG2 cells. Mutation of the DR4 motifs diminished the increase in reporter activities in LS180 cells. Gene subtraction showed that epithelial-specific ETS factor 3 (ESE-3) is a transcription factor enriched in LS180 cells compared to HepG2 cells. When ESE-3 and PXR were co-expressed in HepG2 cells, reporter activities were increased by RIF, which were completely abolished by mutation of DR4 motifs. Chromatin immunoprecipitation assays showed specific binding of ESE-3 to the region containing the DR4 motifs of the ABCB1 gene. Finally, knock-down of ESE-3 in LS180 cells resulted in a decrease in the induction of ABCB1 mRNA. These results suggest that ESE-3 is a factor responsible for PXR-mediated transactivation of the ABCB1 gene by RIF in LS180 cells.

Establishment and characterization of a new conditionally immortalized human astrocyte cell line.

Astrocytes are the most abundant cell types in mammalian brains, within which they participate in various neuronal activities, partly by utilizing the numerous transporters expressed at their plasma membranes. Accordingly, detailed characterization of astrocytic functions, including transporters, are essential for understanding of mechanistic basis of normal brain functions, as well as the pathogenesis and treatment of various brain diseases. As a part of overall efforts to facilitate such studies, this study reports on the establishment of a new human astrocyte cell line, which is hereafter referred to as human astrocyte/conditionally immortalized, clone 35 (HASTR/ci35). This line, which was developed utilizing a cell immortalization method, showed excellent proliferative ability and expressed various astrocyte markers, including glial fibrillary acidic protein. When co-cultured with neuronal cells, HASTR/ci35 cells could facilitate their dendritic network formation. Furthermore, HASTR/ci35 cells not only possessed significant glutamate and adenosine transporter activities but also exhibited organic ion transporter activities. To summarize, HASTR/ci35 cells possess several key astrocytic characteristics, including various transporter functions, while simultaneously showing infinite proliferation and scalability. Based on these findings, HASTR/ci35 cells can be expected to contribute significantly to various human astrocyte study fields. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. Here, we report the establishment of a new human astrocyte cell line, HASTR/ci35, which show various key astrocyte properties, including astrocytic transporter activities, glycogen storage and facilitation of neuronal cell differentiation. Thus, HASTR/ci35 is expected to significantly contribute to advances toward detailed understanding of human astrocyte functions.

Cancer-type Organic Anion Transporting Polypeptide 1B3: Current Knowledge of the Gene Structure, Expression Profile, Functional Implications and Future Perspectives.

Human organic anion transporting polypeptide 1B3 (OATP1B3) is a hepatocyte drug transporter that facilitates uptake of various therapeutic drugs from the circulatory system. Shortly after its initial identification in the liver, OATP1B3 expression was also reported in various solid cancer tissues. In the years since that time, it has been presumed that the OATP1B3 expressed in cancer tissues is identical to that expressed in the liver. However, we have recently identified a new OATP1B3 mRNA variant in cancer tissues, which we have named cancer-type OATP1B3 (Ct-OATP1B3). Given that the identification of Ct-OAT1B3 as a bona fide cancer-associated isoform revises a longstanding study premise, it is essential to fully elucidate the molecular function of Ct-OATP1B3 in cancer cells. Based on the predicted Ct-OATP1B3 protein structure, it is reasonable to assume that it functions as a transporter, but there are a number of ongoing arguments regarding Ct-OATP1B3 protein expression and its functions. With the above points in mind, this review will summarize current knowledge of Ct-OATP1B3 mRNA expression features in cancer tissues and its proposed, yet currently controversial, functions. Based on that background, our future perspectives related to Ct-OATP1B3 studies will also be presented.

Differential inhibition features of direct-acting anti-hepatitis C virus agents against human organic anion transporting polypeptide 2B1.

Simeprevir (SMV), asunaprevir (ASV), daclatasvir (DCV) and sofosbuvir (SOF), which are direct-acting antiviral (DAA) agents, are expected to become essential pharmaceutical tools in the fight against the hepatitis C virus (HCV). However, because DAAs are taken orally, there is a potential risk of drug-drug interactions (DDIs) at the absorption step with co-administered drugs in the small intestine. Since it is known that organic anion transporting polypeptide 2B1 (OATP2B1) is one of the key transporters contributing to intestinal drug absorption, it is important to thoroughly understand the inhibition profiles of various DAAs in relation to OATP2B1 function in order to avoid unexpected DDIs. Therefore, using a cell-based transport assay, this study aimed at clarifying such DAA inhibition characteristics towards OATP2B1 function. The results of co-incubation inhibition assays showed that SMV and ASV strongly inhibited estrone sulfate (5 nM) uptake by OATP2B1, with half maximal inhibitory concentrations of 0.49 ± 0.12 µM and 0.16 ± 0.06 µM, respectively. Furthermore, it was found that SMV and ASV imposed long-lasting pre-incubation inhibitory effects on OATP2B1 function that enhanced their co-incubation inhibition potencies. On the other hand, no (or much less significant) inhibitory effects were observed for SOF or DCV. To summarise, these results show that SMV and ASV are co-incubation, as well as long-lasting pre-incubation, inhibitors of OATP2B1 function and therefore these inhibitions may lead to clinically relevant DDIs when used with OATP2B1 substrates.

Effects of cytokines on CYP3A4 expression and reversal of the effects by anti-cytokine agents in the three-dimensionally cultured human hepatoma cell line FLC-4.

The expression of hepatic cytochrome P450 (CYP) enzymes is altered under pathological conditions with increased levels of cytokines. In this study, we analyzed the effects of cytokines (interleukin [IL]-1ß, IL-6 and tumor necrosis factor α) on the expression of CYP3A4 using newly introduced three-dimensionally cultured human hepatocarcinoma FLC-4 cells. The mRNA level of CYP3A4 was significantly decreased by IL-1ß, IL-6 and tumor necrosis factor-α. Formation of α-hydroxytriazolam catalyzed by CYP3A was decreased by IL-1ß and IL-6. Pre-treatment with IL-6 enhanced the cytotoxic effects of gefitinib and paclitaxel. In addition, tocilizumab and IL-1 receptor antagonist restored the decreased expression of CYP3A4 mRNA by IL-6 and IL-1ß, respectively. These results obtained by using three-dimensionally cultured FLC-4 cells are consistent with results obtained by using primary human hepatocytes and results of clinical studies. Therefore, three-dimensionally cultured FLC-4 cell system may be a promising cellular tool to assess the effects of cytokines on CYP3A4 expression.

Different interaction profiles of direct-acting anti-hepatitis C virus agents with human organic anion transporting polypeptides.

Simeprevir (SMV), asunaprevir (ASV), daclatasvir (DCV), and sofosbuvir (SFV), which are newly developed direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) infection, are among the key components of anti-HCV regimens. Preclinical studies have identified inhibitory properties for some of these DAAs against organic anion transporting polypeptide 1B (OATP1B) functions. However, their details remain mostly uncharacterized. Because OATP1B1 and OATP1B3 play determinant roles in the pharmacokinetics of various drugs via their uptake into human hepatocytes, it is plausible that the inhibition of these OATP1Bs by a DAA would create a potential risk of drug-drug interaction, which has been an emerging concern in anti-HCV therapy. Accordingly, in the present study, we intended to clarify the inhibitory characteristics of newly developed DAAs toward OATP1B1 and -1B3 functions. The results of our coincubation inhibition assays have shown that all tested DAAs could inhibit OATP1B1 functions and that SMV, ASV, and DCV (to a lesser extent), but not SFV, exhibited long-lasting preincubation inhibitory effects on OATP1B1 functions. It was also found that the preincubation inhibitory effects of SMV and ASV could augment their coincubation inhibition potency. Furthermore, significant, but differential, inhibitory effects of the DAAs on the OATP1B3 function were identified. To summarize, our results clearly show that the newly developed DAAs are newly identified OATP1B1 and OATP1B3 inhibitors with distinctive interaction properties. It is believed that these inhibition profiles will provide essential information to all concerned parties with respect to the clinical significance of DAA-mediated inhibition of OATP1Bs in anti-HCV therapy.

Unique expression features of cancer-type organic anion transporting polypeptide 1B3 mRNA expression in human colon and lung cancers.

BACKGROUND: We have previously identified the cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA in several human colon and lung cancer tissues. Ct-OATP1B3 is a variant of the liver-type OATP1B3 (Lt-OATP1B3) mRNA, which is a hepatocyte plasma membrane transporter with broad substrate specificity. However, in cancer tissues, both the detailed characteristics of Ct-OATP1B3 mRNA expression and its biological functions remain unclear. With this point in mind, we sought to characterize Ct-OATP1B3 mRNA expression in colon and lung cancer tissues. In addition, we attempted to obtain functional implication of Ct-OATP1B3 in cancer cells. METHODS: Matched pairs of cancer and normal tissues were collected from 39 colon cancer and 28 lung cancer patients. The OATP1B3 mRNA expression levels in each of these tissues were separately determined by quantitative real-time polymerase chain reaction. Mann-Whitney U test and Fisher's exact test were used in statistical analysis. The Ct-OATP1B3 functional expression in colon cancer cells was then examined by Western blotting and transport analyses. RESULTS: Ct-OATP1B3 mRNA, but not Lt-OATP1B3 mRNA, was abundantly expressed in colon cancer tissues at a higher detection frequency (87.2%) than that of the adjacent normal tissues (2.6%). Furthermore, it was found that Ct-OATP1B3 mRNA expression was often detected in early colon cancer stages (88.9%, n = 18), and that its expression was associated with well-differentiated colon cancer statuses. On the other hand, Ct-OATP1B3 mRNA also showed a predominant and cancer-associated expression profile in lung tissues, although at frequencies and expression levels that were lower than those obtained from colon cancer. As for attempts to clarify the Ct-OATP1B3 functions, neither protein expression nor transport activity could be observed in any of the cell lines examined. CONCLUSIONS: Based on the unique characteristics of the Ct-OATP1B3 mRNA expression profile identified in this study, Ct-OATP1B3 mRNA can be expected to become a biomarker candidate for use in colon (and lung) cancer diagnosis. Simultaneously, our results advance the possibility that Ct-OATP1B3 might play yet unidentified roles, in addition to transporter function, in cancer cell biology.

Acrolein stimulates the synthesis of IL-6 and C-reactive protein (CRP) in thrombosis model mice and cultured cells.

Measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction with high sensitivity and specificity. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP in thrombosis model mice and cultured cells. In mice with photochemically induced thrombosis, acrolein produced at the locus of infarction increased the level of IL-6 and then CRP in plasma. This was confirmed in cell culture systems - acrolein stimulated the production of IL-6 in mouse neuroblastoma Neuro-2a cells, mouse macrophage-like J774.1 cells, and human umbilical vein endothelial cells (HUVEC), and IL-6 in turn stimulated the production of CRP in human hepatocarcinoma cells. The level of IL-6 mRNA was increased by acrolein through an increase in phosphorylation of the transcription factors, c-Jun, and NF-κB p65. Furthermore, CRP stimulated IL-6 production in mouse macrophage-like J774.1 cells and HUVEC. IL-6 functioned as a protective factor against acrolein toxicity in Neuro-2a cells and HUVEC. These results show that acrolein stimulates the synthesis of IL-6 and CRP, which function as protecting factors against acrolein toxicity, and that the combined measurement of PC-Acro, IL-6, and CRP is effective for identification of silent brain infarction. The combined measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP, and indeed acrolein increased IL-6 synthesis and IL-6 in turn increased CRP synthesis. Furthermore, IL-6 decreased acrolein toxicity in several cell lines.

Cytotoxic effects of benzbromarone and its 1'-hydroxy metabolite in human hepatocarcinoma FLC4 cells cultured on micro-space cell culture plates.

Treatment with benzbromarone (BBR), a potent uricosuric drug, can be associated with liver injury. Recently, we reported that culture of human hepatocellular carcinoma FLC-4 cells on micro-space cell culture plates could increase the functional expression of drug-metabolizing enzymes including CYP3A4 and CYP2C9, which are involved in 1'-hydroxylation and 6-hydroxylation of BBR, respectively. Therefore, we examined whether BBR and its two metabolites (1'-hydroxy BBR and 6-hydroxy BBR) have cytotoxic effects in FLC4 cells cultured on micro-space cell culture plates. The present study showed that BBR and 1'-hydroxy BBR, but not 6-hydroxy BBR, have cytotoxic effects in cells cultured on micro-space cell culture plates. BBR-induced cytotoxicity was decreased by CYP3A inhibitors (itraconazole and ketoconazole), an Nrf2 activator (tert-butylhydroquinone) and a GSH precursor (N-acetyl-L-cystein). In contrast, BBR-induced cytotoxicity was increased by a GSH biosynthesis inhibitor (buthionine sulfoximine) and an inhibitor of NAD(P)H quinone oxidoreductase 1 (dicoumarol). These results suggested that metabolic activation of 1'-hydroxy BBR via CYP3A, formation of quinone metabolites and the decrease in GSH levels were involved in the BBR-induced cytotoxicity observed in FLC4 cells cultured on micro-space cell culture plates.

Establishment of a new conditionally immortalized cell line from human brain microvascular endothelial cells: a promising tool for human blood-brain barrier studies.

The blood-brain barrier (BBB) is formed by brain microvascular endothelial cells (BMEC) working together with astrocytes and pericytes, in which tight junctions and various transporters strictly regulate the penetration of diverse compounds into the brain. Clarification of the molecular machinery that provides such regulation using in vitro BBB models has provided important insights into the roles of the BBB in central nervous system (CNS) disorders and CNS drug development. In this study, we succeeded in establishing a new cell line, hereinafter referred to as human BMEC/conditionally immortalized, clone ß (HBMEC/ciß), as part of our ongoing efforts to develop an in vitro human BBB model. Our results showed that HBMEC/ciß proliferated well. Furthermore, we found that HBMEC/ciß exhibited the barrier property of restricting small molecule intercellular penetration and possessed effective efflux transporter functions, both of which are essential to a functioning BBB. Because higher temperatures are known to terminate immortalization signals, we specifically examined the effects of higher temperatures on the HBMEC/ciß differentiation status. The results showed that higher temperatures stimulated HBMEC/ciß differentiation, marked by morphological alteration and increases in several mRNA levels. To summarize, our data indicates that the newly established HBMEC/ciß offers a promising tool for use in the development of a practical in vitro human BBB model that could make significant contributions toward understanding the molecular biology of CNS disorders, as well as to CNS drug development. It is also believed that the development of a specific culture method for HBMEC/ciß will add significant value to the HBMEC/ciß-based BBB model.