Seiichi Ishida

Decreased Expression of Cytochromes P450 1A2, 2E1, and 3A4 and Drug Transporters Na+-Taurocholate-Cotransporting Polypeptide, Organic Cation Transporter 1, and Organic Anion-Transporting Peptide-C Correlates with the Progression of Liver Fibrosis in Chronic Hepatitis C Patients

Patients with chronic hepatitis C viral infection underwent liver biopsies and laboratory studies for evaluation and to determine subsequent treatment. Changes in status of drug metabolism and disposition may vary with chronic hepatitis C stage and should be assessed. Total RNA was extracted from liver biopsy specimens (n = 63) and reverse transcribed to yield cDNA. Relative mRNA levels of drug-metabolizing enzymes, transporters, nuclear receptors, and proinflammatory cytokines were analyzed with normalization to glyceraldehyde 3-phosphate dehydrogenase expression. mRNAs encoding cytochromes P450 1A2, 2E1, and 3A4, and drug transporters, Na+-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1 showed remarkable decreases, and tumor necrosis factor-α showed an increase according to fibrosis stage progression. HepG2 cells and primary hepatocytes of two human individuals were treated with interleukin 1β, interleukin 6, or tumor necrosis factor-α. CYP1A2 and Na+-taurocholate-cotransporting polypeptide mRNA levels significantly decreased in HepG2 cells with interleukin 1β and interleukin 6 treatments. CYP2E1 and organic cation transporter 1 mRNA levels significantly decreased with tumor necrosis factor-α treatment only in HepG2. These results suggested that down-regulation of CYP1A2, 2E1, and 3A4, and drug transporters, Na+-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1, manifested in livers of patients with chronic hepatitis C viral infection, was associated, at least in part, with the elevated production of proinflammatory cytokines, including tumor necrosis factor-α.

Inflammatory cytokines promote the retrodifferentiation of tumor‐derived hepatocyte‐like cells to progenitor cells

Human hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and resistance to therapies. Recent studies have reported that HCC may be derived not only from adult hepatocytes and hepatoblasts but also hepatic stem/progenitors. In this context, HepaRG cells may represent a suitable cellular model to study stem/progenitor cancer cells and the retrodifferentiation of tumor‐derived hepatocyte‐like cells. Indeed, they differentiate into hepatocyte‐ and biliary‐like cells. Moreover, tumor‐derived HepaRG hepatocyte‐like cells (HepaRG‐tdHep) differentiate into both hepatocyte‐ and biliary‐like cells through a hepatic progenitor. In this study we report the mechanisms and molecular effectors involved in the retrodifferentiation of HepaRG‐tdHep into bipotent progenitors. Gene expression profiling was used to identify genomic changes during the retrodifferentiation of HepaRG‐tdHep into progenitors. We demonstrated that gene expression signatures related to a poor‐prognosis HCC subclass, proliferative progenitors, or embryonic stem cells were significantly enriched in HepaRG progenitors derived from HepaRG‐tdHep. HepaRG‐tdHep retrodifferentiation is mediated by crosstalk between transforming growth factor beta 1 (TGFβ1) and inflammatory cytokine pathways (e.g., tumor necrosis factor alpha [TNFα] and interleukin 6 [IL6]). Signatures related to TNFα, IL6, and TGFβ activation pathways are induced within the first hour of retrodifferentiation. Moreover, specific activation or inhibition of these signaling pathways allowed us to determine that TNFα and IL6 contribute to the loss of hepatic‐specific marker expression and that TGFβ1 induces an epithelial‐to‐mesenchymal transition of HepaRG‐tdHep. Interestingly, the retrodifferentiation process is blocked by the histone deacetylase inhibitor trichostatin A, opening new therapeutic opportunities. Conclusion: Cancer progenitor cells (or metastasis progenitors) may derive from tumor‐derived hepatocyte‐like cells in an inflammatory environment that is frequently associated with HCC. (Hepatology 2014;60:2076–2089)

Polymorphic tandem repeat sequences of the thymidylate synthase gene correlates with cellular-based sensitivity to fluoropyrimidine antitumor agents

Purpose: Thymidylate synthase (TS) is one of the target molecules for the antitumor effects of fluoropyrimidine drugs. The cellular thymidylate synthase level is one of the determining factors for the antitumor activity of fluoropyrimidines. TYMS, which encodes TS, has been reported to possess 28-bp tandem repeat sequences in its 5′-untranslated region, the number of which varies. In addition, single nucleotide polymorphisms have also been shown in a triple repeat sequence. In this study, correlation between the polymorphic tandem repeat sequences of the TYMS gene and the antitumor activities of 5-fluorouracil (5-FU) and 5-fluoro-2′-deoxyuridine (FUdR) were investigated with 30 established human cell lines derived from solid tumors. Methods: A reporter assay system was developed in order to compare the ability of the transactivation mediated by the double (2R) and triple (c- or g-type, 3Rc or 3Rg, respectively) repeat sequences using a human colon cancer cell line, DLD-1. The 50% inhibitory concentration (IC50) of cell growth by 5-FU and FUdR was measured with 30 different established cell lines of human solid tumors. Genotypes based on the number of the 28-bp TYMS tandem repeat for the above cell lines were determined by electrophoretical analysis of PCR products containing the repeat sequences and nucleotide sequencing. Results: The reporter activity mediated by the 3Rg sequence was significantly higher than that by the 2R and 3Rc sequences. Activities mediated by the 2R and 3Rc sequences were comparable. According to the reporter assay, 2R and 3Rc were judged as low TS expression alleles and 3Rg as a high TS expression allele. On the basis of IC50 values, cells possessing the 2R/2R and 2R/3R repeat of TYMS were significantly more sensitive to FUdR than those with the 3R/3R repeat. Cells possessing 3Rg/3Rg (a high TS expression genotype) were significantly less sensitive to FUdR than cells with 2R/2R, 2R/3Rc, and 3Rc/3Rc (low TS expression genotypes). Conclusions: Our results of the reporter assays using 2R, 3Rc, and 3Rg repeat sequences prompted us to classify 3Rg as a high TS expression allele, and 2R and 3Rc as low TS expression alleles. The cells with low TS expression alleles were shown to exhibit significantly higher FUdR sensitivity than the cells with high TS expression alleles for the first time. These results were consistent with numerous previous in vitro and in vivo findings that tumors showing high TS expression were less sensitive to fluoropyrimidines. These results support the idea that genotyping the tandem repeat sequences of TYMS in the 5′-untranslated region is useful for individualized therapy involving fluoropyrimidine antitumor drugs.

Global gene expression changes including drug metabolism and disposition induced by three-dimensional culture of HepG2 cells-Involvement of microtubules.

Constitutive upregulation and a higher degree of induction of drug metabolism and disposition-related genes were found in a three-dimensional HepG2 culture. The upregulated genes are believed to be regulated by different regulatory factors. Global gene expression analysis using the Affymetrix GeneChip indicated that altered expression of microtubule-related genes may change the expressed levels of drug metabolizing and disposition genes. Stabilization of microtubule molecules with docetaxel, a tubulin-stabilizing agent, in the two-dimensional culture showed gene expression patterns similar to those found in the three-dimensional culture, indicating that the culture environment affects drug metabolism functions in HepG2 cells.

Increase of β2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. β-actin, β-catenin, and β1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but β2-integrin expression markedly increased. Graphical abstract THP-1 cells were seeded in T-75, or CVM, or CVM added PMA 10 ng/mL; and β-actin(A), β-catenin(B), β1-integrin(C) and β2-integrin(D) expression were measured (N = 3).

Substitution of arginine for cysteine 643 of the glucocorticoid receptor reduces its steroid-binding affinity and transcriptional activity

To investigate the mechanism for glucocorticoid resistance in leukemic cells, we sequenced the coding region of glucocorticoid receptor (GR) gene in ten Japanese leukemic cells. We identified a novel heterozygous mutation (C643R) in the ligand-binding domain in P30/OHK cells. Western blot analysis for COS-7 cells transfected with the wild-type or C643R mutant GR plasmid revealed similar protein expression levels. In the ligand-binding assay, the dissociation constant of the C643R GR was six-fold higher than that of the wild-type GR. Moreover, the C643R GR showed no transcriptional activity in the luciferase reporter assay.

Characterization of human CYP1A1/1A2 induction by DNA microarray and α-naphthoflavone

Abstract DNA microarrays and real time PCR were used to analyze the mechanism of gene induction by CYP1A1 inducers, β-naphthoflavone, and omeprazole, in the human hepatocellular carcinoma HepG2 cells. Reproducible and significant inductions were observed in a limited number of genes including CYP1A1 and CYP1A2 . Genes induced by omeprazole included several protein tyrosine kinase targets. This result confirmed that omeprazole could modulate gene expressions through protein tyrosine kinase-mediated pathway. Induction ratios were considerably different from CYP1A1 and CYP1A2 (>10-fold) to other induced genes ( p -dioxin, inhibited the inductions of heme oxygenase 1, glutamate-cysteine ligase (modifier unit), and thioredoxin reductase by β-naphthoflavone but not those of CYP1A1 and CYP1A2 . It unexpectedly enhanced the β-naphthoflavone-mediated CYP1A1 and CYP1A2 induction. These results suggest that the CYP1A1 and CYP1A2 genes, which share their 5 ′ enhancer regions, are regulated differently from the other genes.

Upregulations of metallothionein gene expressions and tolerance to heavy metal toxicity by three dimensional cultivation of HepG2 cells on VECELL 3-D inserts.

The VECELL 3-D insert is a new culture scaffold consisting of collagen-coated ePTFE (expanded polytetrafluoroethylene) mesh. We analyzed the effects of VECELL 3-D inserts on the functionality of HepG2, a human hepatocellular carcinoma cell line. HepG2 cells cultured on VECELL 3-D inserts maintained a round shape, while those cultured on a standard culture plate or collagen-coated cell culture plate showed a flattened and cubic epithelial-like shape. HepG2 cells cultured on VECELL 3-D inserts had showed upregulated expression of metallothionein genes and in turn a higher tolerance to toxicity induced by heavy metals. These results suggest that HepG2 cell functions were changed by the cell morphology that is induced by culturing on a VECELL 3-D insert.

A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.