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金沢大学医薬保健研究域医学系, The identification of genes involved in tumor growth is crucial for the development of inventive anticancer treatments. Here, we have cloned a 17-kDa secretory protein encoded by c19orf10 from hepatocellular carcinoma (HCC) serial analysis of gene expression libraries. Gene expression analysis indicated that c19orf10 was overexpressed in approximately two-thirds of HCC tissues compared to the adjacent noncancerous liver tissues, and its expression was significantly positively correlated with that of alpha-fetoprotein (AFP). Overexpression of c19orf10 enhanced cell proliferation of AFP-negative HLE cells, whereas knockdown of c19orf10 inhibited cell proliferation of AFP-positive Hep3B and HuH7 cells along with G1 cell cycle arrest. Supplementation of recombinant c19orf10 protein in culture media enhanced cell proliferation in HLE cells, and this effect was abolished by the addition of antibodies developed against c19orf10. Intriguingly, c19orf10 could regulate cell proliferation through the activation of Akt/mitogen-activated protein kinase pathways. Taken together, these data suggest that c19orf10 might be one of the growth factors and potential molecular targets activated in HCC. © 2010 UICC.

Background & Aims The mechanisms of treatment resistance to interferon (IFN) and ribavirin (Rib) combination therapy for hepatitis C virus (HCV) infection are not known. This study aims to gain insight into these mechanisms by exploring hepatic gene expression before and during treatment. Methods Liver biopsy was performed in 50 patients before therapy and repeated in 30 of them 1week after initiating combination therapy. The cells in liver lobules (CLL) and the cells in portal areas (CPA) were obtained from 12 patients using laser capture microdissection (LCM). Results Forty-three patients were infected with genotype 1 HCV, 20 of who were viral responders (genotype 1-Rsp) with treatment outcome of SVR or TR, while 23 were non-responders (genotype 1-nonRsp) with NR. Only seven patients were infected with genotype 2. Before treatment, the expression of IFN and Rib-stimulated genes (IRSGs), apoptosis-associated genes, and immune reaction gene pathways was greater in genotype 1-nonRsp than in Rsp. During treatment, IRSGs were induced in genotype 1-Rsp, but not in nonRsp. IRSG induction was irrelevant in genotype 2-Rsp and was mainly impaired in CLL but not in CPA. Pathway analysis revealed that many immune regulatory pathways were induced in CLL from genotype 1-Rsp, while growth factors related to angiogenesis and fibrogenesis were more induced in CPA from genotype 1-nonRsp. Conclusions Impaired IRSGs induction in CLL reduces the sensitivity to treatment for genotype 1 HCV infection. CLL and CPA in the liver might be differentially involved in treatment resistance. These findings could be useful for the improvement of therapy for HCV infection.

Aim: Host genetic variants leading to inosine triphosphatase (ITPA) deficiency, a condition not thought to be clinically important, protect against hemolytic anemia in chronic hepatitis C patients receiving ribavirin. In this study, we evaluated the clinical significance of ITPA variants in Japanese hepatitis C patients who were treated with pegylated interferon plus ribavirin. Methods: In this multicenter retrospective cross-sectional study, 474 hepatitis C patients were enrolled who were treated with pegylated interferon plus ribavirin in four geographically different hospitals in Japan. Patients were grouped according to hemoglobin decline of more than 3 g/dL at week 4. Two single nucleotide polymorphisms (SNP) within or adjacent to the ITPA gene (rs6051702, rs1127354) were genotyped. Results: A functional SNP, rs1127354, within the ITPA exon was strongly associated with protection against anemia with only one (0.8%) in 129 patients with the ITPA minor variant A developing severe anemia (P = 5.9 × 10−20). For rs6051702, which had significant association in European-Americans, significant but weak association with severe hemoglobin reduction was found in Japanese (P = 0.009). In patients excluding genotype 1b and high viral load, those with the ITPA minor variant A achieved significantly higher sustained viral response rate than those with the major variant (CC) (96% vs 70%, respectively, P = 0.0066). Conclusion: ITPA SNP, rs1127354, is confirmed to be a useful predictor of ribavirin-induced anemia in Japanese patients. Patients with the ITPA minor variant A (∼27%) have an advantage in pegylated interferon plus ribavirin-based therapies, due to expected adherence of ribavirin doses, resulting in a higher viral clearance rate.

Hepatocyte nuclear factor 4 alpha (HNF4α) is one of the most ancient nuclear receptors known as a master regulator of hepatocytes in lipids, carbohydrates, and drug metabolism. It also works as a tumor suppressor to regulate cell cycle in hepatocellular carcinoma (HCC), but the ligand activating HNF4α has not yet been discovered thus far. Here we evaluated the effect of polyprenoic acid (PA), formerly known as an acyclic retinoid, on HNF4α activation in dysplastic nodule and HCC in vitro and in vivo. PDGF-C transgenic (Tg) mice was evaluated by Gd-EOB-DTPA-enhanced MRI to examine the effect of PA on HCC development in vivo. Whole exome sequence analysis was performed using Illumina Hiseq 2000 system. Contrast enhanced CT scan data of patients enrolled in previous phase II/III randomized placebo-controlled study were collected and analyzed (n = 124 for PA and 127 for placebo). PA treatment regressed the liver dysplastic nodule and a subset of HCCs in PDGF-C Tg mice evaluated by MRI. This tumor regression was accompanied with the reduction of accumulated gene mutations, inactivation of cell cycle, and transcriptional activation of HNF4α-target genes. The binding of PA on ligand binding domain of HNF4α was shown by docking simulation in silico and immunoprecipitation-mass spectrometry analysis in vitro. Transcriptional activation of HNF4α by PA was also verified using a luciferase reporter assay and binding of HNF4α on DNA binding elements. PA treatment in HCC cells immediately resulted in the degradation of HNF4α by ubiquitin-proteasome system with activation of DNA damage responses when cultured in lipid depleted medium. In vivo knockdown of HNF4A cancelled the effect of PA on regression of liver tumor growth in PDGF-C Tg mice. PA treatment (600 mg/day) suppressed the HCC development from dysplastic nodule compared with placebo in human with statistical significance (HR 0.41; 95% CI, 0.17~0.98, P = 0.046). Taken together, our data indicated that PA is the first-discovered HNF4α ligand to activate its function. PA activates the DNA damage responses and suppresses the accumulation of gene mutations especially in dysplastic nodule, warranting the future prospective study to evaluate the effect of PA on suppression of HCC development from dysplastic nodules in human. Citation Format: Taro Yamashita, Hikari Okada, Masao Honda, Shuichi Kaneko. Polyprenoic acid, the first-discovered hepatocyte nuclear factor 4 alpha agonist, inhibits multistep liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1904.

Background & aims Many individuals are infected with hepatitis B virus (HBV) worldwide, and this virus is commonly controlled by treatments with interferon (IFN)-alpha and nucleoside analogues (NA). However, the complete elimination of HBV by these treatments is difficult and, thus, the development of new treatments is needed. Host immune responses are closely involved in the elimination of HBV, suggesting the usefulness of immunotherapy. In the present study, we attempted to identify novel cytotoxic T-lymphocyte (CTL) epitopes that are useful for immunotherapy against HBV. Methods CTL epitopes were predicted using computer software. Immune responses to each peptide were evaluated by IFN-γ ELISPOT and cytotoxic assays. The relationships between the immune responses to these newly identified CTL epitopes and the clinical backgrounds of patients and administration of NA were analyzed. Peptides were administered to mice as vaccines and peptide-specific T-cell induction was measured in vivo. Results Positive reactions to 10 synthesized peptides were detected in 3 or more patients using the IFN-γ ELISPOT assay, and concentration-dependent cytotoxicity against 2 of these peptides was observed in the cytotoxic assay. Some peptides that correlated with serum ALT, HBsAg, and HBV core-related antigen (HBcrAg) levels were identified. Immune reactions against some peptides were enhanced by the administration of NA. Regarding their effects as a vaccine, peptide-specific T-cells were induced by four peptides in vivo. Conclusions Novel HBV epitopes that correlated with HBsAg and HBcrAg levels were identified. These newly identified epitopes may be useful in the analysis of immune responses to HBV and development of immunotherapy against HBV.

金沢大学医薬保健研究域医学系, To elucidate the molecular feature of human hepatocellular carcinoma (HCC), we performed 5'-end serial analysis of gene expression (5'SAGE), which allows genome-wide identification of transcription start sites in addition to quantification of mRNA transcripts. Three 5'SAGE libraries were generated from normal human liver (NL), non-B, non-C HCC tumor (T), and background non-tumor tissues (NT). We obtained 226,834 tags from these libraries and mapped them to the genomic sequences of a total of 8,410 genes using RefSeq database. We identified several novel transcripts specifically expressed in HCC including those mapped to the intronic regions. Among them, we confirmed the transcripts initiated from the introns of a gene encoding acyl-coenzyme A oxidase 2 (. ACOX2). The expression of these transcript variants were up-regulated in HCC and showed a different pattern compared with that of ordinary ACOX2 mRNA. The present results indicate that the transcription initiation of a subset of genes may be distinctively altered in HCC, which may suggest the utility of intronic RNAs as surrogate tumor markers. © 2010 Elsevier Inc.

Background— Acute coronary syndrome is sometimes accompanied by accelerated coagulability, lipid metabolism, and inflammatory responses, which are not attributable to the cardiac events alone. We hypothesized that the liver plays a pivotal role in the pathophysiology of acute coronary syndrome. We simultaneously analyzed the gene expression profiles of the liver and heart during acute myocardial ischemia in mice. Methods and Results— –Mice were divided into 3 treatment groups: sham operation, ischemia/reperfusion, and myocardial infarction. Mice with liver ischemia/reperfusion were included as additional controls. Marked changes in hepatic gene expression were observed after 24 hours, despite the lack of histological changes in the liver. Genes related to tissue remodeling, adhesion molecules, and morphogenesis were significantly upregulated in the livers of mice with myocardial ischemia/reperfusion or infarction but not in those with liver ischemia/reperfusion. Myocardial ischemia, but not changes in the hemodynamic state, was postulated to significantly alter hepatic gene expression. Moreover, detailed analysis of the signaling pathway suggested the presence of humoral factors that intervened between the heart and liver. To address these points, we used isolated primary hepatocytes and showed that osteopontin released from the heart actually altered the signaling pathways of primary hepatocytes to those observed in the livers of mice under myocardial ischemia. Moreover, osteopontin stimulated primary hepatocytes to secrete vascular endothelial growth factor-A, which is important for tissue remodeling. Conclusions— Hepatic gene expression is potentially regulated by cardiac humoral factors under myocardial ischemia. These results provide new insights into the pathophysiology of acute coronary syndrome.

Hepatocellular carcinoma (HCC) frequently develops from hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA) were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA), and all-trans-retinoic acid (ATRA), had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1) to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC.

Background & aims The modest consumption of alcohol has been reported to decrease the incidence of fatty liver or prevalence of steatohepatitis. In this study, we investigated the effect of light alcohol consumption on liver function and gene expression in patients with non-alcoholic fatty liver disease (NAFLD). Methods The study group was formed of 178 patients diagnosed with non-alcoholic fatty liver disease, subclassified into two groups for analysis based on the daily alcohol consumption: non-alcohol group and light alcohol consumer group (≤20 g of ethanol/day). Clinical characteristics, liver histological features, gene expression, comprehensively analyzed using microarrays (BRB-Array tools), and molecular network were evaluated and compared between the two groups. Results No significant differences in steatosis or inflammation score were noted among the groups. However, the ballooning and fibrosis scores were significantly lower in the light alcohol consumer group than in the non-alcohol group. Gene expression analysis revealed a marked inhibition of the pathways involved in the immune response in the light alcohol group compared to that in the non-alcohol group. Conclusions Light alcohol consumption might suppress activity of non-alcoholic steatohepatitis by reducing gene expression levels involved in the immune response. This inhibition in gene expression was associated with a lowering of liver fibrosis and hepatocellular injury.

OBJECTIVE Although patients with diabetes suffer from increased infections and a higher incidence of cancer due to impaired immune function, details on diabetes-induced decrease in immunity are lacking. We assessed how immune-mediating peripheral blood mononuclear cells (PBMCs) are affected in diabetes. RESEARCH DESIGN AND METHODS From 33 patients with type 2 diabetes and 28 healthy volunteers, we obtained PBMCs and investigated their susceptibility to apoptosis and functional alteration. RESULTS In a subpopulation of PBMCs, monocytes derived from patients with diabetes were more susceptible to apoptosis than monocytes from healthy volunteers. Monocytes from patients with diabetes had decreased phagocytotic activity and were less responsive to Toll-like receptor (TLR) ligands, although the expression of TLRs did not differ significantly between the two groups. Furthermore, monocytes from patients with diabetes had a distinctly different gene expression profile compared with monocytes from normal volunteers as assessed with DNA microarray analysis. Specifically, quantitative real-time detection PCR measurements showed an elevated expression of the markers of endoplasmic reticulum (ER) stress in diabetic monocytes, and electron microscopic examination of monocytes revealed morphologic alterations in the ER of cells derived from patients with diabetes. Consistently, the ER stress inducer tunicamycin increased apoptosis of otherwise healthy monocytes and attenuated the proinflammatory responses to TLR ligands. CONCLUSIONS These data suggest that monocytes comprise a substantially impaired subpopulation of PBMCs in patients with diabetes and that ER stress is involved in these pathologic changes mechanistically. This implies that the affected monocytes should be investigated further to better understand diabetic immunity.

The recommended treatment for patients with chronic hepatitis C, pegylated interferon-alpha (PEG-IFN-alpha) plus ribavirin (RBV), does not provide sustained virologic response (SVR) in all patients. We report a genome-wide association study (GWAS) to null virological response (NVR) in the treatment of patients with hepatitis C virus (HCV) genotype 1 within a Japanese population. We found two SNPs near the gene IL28B on chromosome 19 to be strongly associated with NVR (rs12980275, P = 1.93 x 10(-13), and rs8099917, 3.11 x 10(-15)). We replicated these associations in an independent cohort (combined P values, 2.84 x 10(-27) (OR = 17.7; 95% CI = 10.0-31.3) and 2.68 x 10(-32) (OR = 27.1; 95% CI = 14.6-50.3), respectively). Compared to NVR, these SNPs were also associated with SVR (rs12980275, P = 3.99 x 10(-24), and rs8099917, P = 1.11 x 10(-27)). In further fine mapping of the region, seven SNPs (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, rs8099917 and rs7248668) located in the IL28B region showed the most significant associations (P = 5.52 x 10(-28)-2.68 x 10(-32); OR = 22.3-27.1). Real-time quantitative PCR assays in peripheral blood mononuclear cells showed lower IL28B expression levels in individuals carrying the minor alleles (P = 0.015).

Metabolic dysregulation is one of the risk factors for the development of hepatocellular carcinoma (HCC). We investigated the activated metabolic pathway in HCC to identify its role in HCC growth and mortality.Gene expression profiles of HCC tissues and non-cancerous liver tissues were obtained by serial analysis of gene expression. Pathway analysis was performed to characterize the metabolic pathway activated in HCC. Suppression of the activated pathway by RNA interference was used to evaluate its role in HCC in vitro. Relation of the pathway activation and prognosis was statistically examined.A total of 289 transcripts were up- or down-regulated in HCC compared with non-cancerous liver (P0.005). Pathway analysis revealed that the lipogenic pathway regulated by sterol regulatory element binding factor 1 (SREBF1) was activated in HCC, which was validated by real-time RT-PCR. Suppression of SREBF1 induced growth arrest and apoptosis whereas overexpression of SREBF1 enhanced cell proliferation in human HCC cell lines. SREBF1 protein expression was evaluated in 54 HCC samples by immunohistochemistry, and Kaplan-Meier survival analysis indicated that SREBF1-high HCC correlated with high mortality.The lipogenic pathway is activated in a subset of HCC and contributes to cell proliferation and prognosis.

Proteome analysis of human hepatocellular carcinoma was conducted using two-dimensional difference gel electrophoresis, and the protein expression profiles were compared to the mRNA expression profiles made from serial analysis of gene expression (SAGE) in identical samples from a single patient. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the protein expression profiles. A total of 188 proteins were identified, and the expression profiles of 164 proteins which had the corresponding SAGE data were compared to the mRNA expression profiles. Among them, 40 proteins showed significant differences in the mRNA expression levels between non HCC and HCC. We compared expression changes of proteins with those of mRNAs. We found that the expression tendency of 24 proteins were similar to that of mRNA, whereas 16 proteins showed different or opposite tendency to the mRNA expression.

Multidrug resistance-associated protein 3 (MRP3) is a carrier-type transport protein belonging to the ABC transporters. It is expressed in normal tissues, and enhanced expression in many cancers has been reported. In this study, we investigated the usefulness of MRP3 as a target antigen in immunotherapy for hepatocellular carcinoma (HCC).The MRP3 expression level in HCC tissue was measured by quantitative PCR. MRP3-specific T cell responses were investigated by several immunological techniques using peripheral blood mononuclear cells or tumor-infiltrating lymphocytes.The MRP3 expression level in HCC tissue was significantly higher than that in non-cancerous tissue (P0.05). MRP3-specific cytotoxic T cells (CTLs) could be induced regardless of liver function, the presence or absence of HCV infection, the blood AFP level, and the stage of HCC. The CTLs showed cytotoxicity against HCC cells overexpressing MRP3. A negative correlation was present between the MRP3 expression level in HCC tissue and the frequency of MRP3-specific CTLs. The frequency of MRP3-specific CTLs increased after HCC treatment, such as transcatheter arterial embolization and radiofrequency ablation.Our study demonstrates that MRP3 is a potential candidate for tumor antigen with strong immunogenicity in HCC immunotherapy.

Background/Aims Specific markers are required for early detection and diagnosis of intrahepatic cholangiocarcinoma (ICC); however, the tumour markers currently in use are not specific for ICC. Methods We compared an ICC cDNA library with that of hepatocellular carcinoma (HCC) by serial analysis of gene expression (SAGE). The expression patterns in each were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunohistochemical analysis of 74 samples including 16 ICC samples. Results A comparison of the two libraries revealed distinct gene expression patterns for each type of liver cancer. In addition to the known tumour markers, we detected nine novel genes associated with ICC. By comparing the mean transcript abundance in the ICC library with those in other libraries, including gastric, colon, prostate and breast cancer, together with our RT-PCR results, we identified three genes as specific markers of ICC: biglycan, insulin-like growth factor-binding protein 5 and claudin-4. Immunoblotting and immunohistochemical analyses showed that claudin-4 was highly expressed in ICC. Moreover, discrimination analysis revealed that a combination of these genes could be used to distinguish ICC from HCC or metastatic adenocarcinoma. Conclusions We identified novel marker genes of ICC that are potentially useful for the diagnosis of liver cancer.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high α-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.

Proteome analysis of human hepatocellular carcinoma (HCC) was done using two-dimensional difference gel electrophoresis. To gain an understanding of the molecular events accompanying HCC development, we compared the protein expression profiles of HCC and non-HCC tissue from 14 patients to the mRNA expression profiles of the same samples made from a cDNA microarray. A total of 125 proteins were identified, and the expression profiles of 93 proteins (149 spots) were compared to the mRNA expression profiles. The overall protein expression ratios correlated well with the mRNA ratios between HCC and non-HCC (Pearson’s correlation coefficient: r = 0.73). Particularly, the HCC/non-HCC expression ratios of proteins involved in metabolic processes showed significant correlation to those of mRNA ( r = 0.9). A considerable number of proteins were expressed as multiple spots. Among them, several proteins showed spot-to-spot differences in expression level and their expression ratios between HCC and non-HCC poorly correlated to mRNA ratios. Such multi-spotted proteins might arise as a consequence of post-translational modifications.

We hypothesized that systemically circulating peripheral blood mononuclear cells (PBMCs) reflect the pathophysiology of type 2 diabetes. PBMCs were obtained from 18 patients with type 2 diabetes and 16 non-diabetic subjects. The expression of genes in the PBMCs was analyzed by using a DNA chip followed by statistical analysis for specific gene sets for biological categories. The only gene set coordinately up-regulated by the existence of diabetes and down-regulated by glycemic control consisted of 48 genes involved in the c-Jun N-terminal kinase (JNK) pathway. In contrast, the only gene set coordinately down-regulated by the existence of diabetes, but not altered by glycemic control consisted of 92 genes involved in the mitochondrial oxidative phosphorylation (OXPHOS) pathway. Our findings suggest that genes involved in the JNK and OXPHOS pathways of PBMCs may be surrogate transcriptional markers for hyperglycemia-induced oxidative stress and morbidity of type 2 diabetes, respectively.

Consensus has been reached that diabetes is a risk factor for development of HCC, but the impact on postoperative recurrence is still controversial. To clarify this point, we analyzed the relationship of postoperative recurrence rate of HCC and coexistence of diabetes in the patients with viral hepatitis.A total of 90 patients who had undergone curative resection for HCC were analyzed. They were divided into two groups with and without diabetes, and the recurrence-free survival rates after surgical treatment and the factors contributing to recurrence were examined.Kaplan-Meier survival analysis showed the recurrence-free survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P= 0.005) and overall survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P= 0.005). These results were emphasized in the analysis of patients infected with hepatitis C virus. Univariate and multivariate analyses showed diabetes was a significant factor contributing to HCC recurrence after treatment. Furthermore, multivariate analysis in HCC patients with diabetes showed Child-Pugh classification B (P= 0.001) and insulin therapy (P= 0.049) were significant factors contributing to HCC recurrence after treatment.The results of the present study suggest that diabetes is a risk factor for the recurrence of HCV-related HCC and decreases the overall survival rates after surgical treatment. HCV-related HCC patients with diabetes should be closely followed for postoperative recurrence.

症例は，57歳，女性．2002年全身倦怠感を主訴に，近医を受診，血液検査と肝生検にて，原発性胆汁性肝硬変Scheuer分類III期と診断された．その後ウルソデオキシコール酸による内服加療を続けていたが，2004年1月頃より血清ビリルビン値の上昇傾向を認め，2005年2月頃より動悸，手指振戦，労作時呼吸困難の出現とともに，ビリルビン値の急激な上昇を認め，2005年5月精査加療目的に当科へ入院となった．入院後，甲状腺機能検査，甲状腺自己抗体検査，甲状腺ラジオアイソトープ検査にてBasedow病の合併を診断した．その後抗甲状腺剤による治療を開始し，甲状腺機能の速やかな正常化とともに，ビリルビン値の改善を認めた． 原発性胆汁性肝硬変例でのBasedow病の合併の報告例は極めて稀ではあるが，原発性胆汁性肝硬変の経過観察中に急激な黄疸の進行を認めた場合は，Basedow病の合併の可能性も考慮する必要があると考えられた．

症例は56歳男性．B型肝炎ウイルス（hepatitis B virus, HBV）キャリア状態かつ非ホジキンリンパ腫（Non-Hodgikin lymphoma, NHL）と診断され，ステロイドを含まないリツキシマブ併用化学療法が施行された．治療中HBV-DNA量が増加したため，ラミブジンを予防投与し，HBV-DNA量は速やかに低下した．地固め療法としてリツキシマブを単独投与した後よりHBV-DNA量が増加し，肝炎を発症し，YVDD変異株を検出したためadefovir dipivoxil（アデフォビル）を追加投与した．肝炎はやや遷延したものの徐々に軽快し，約21カ月経過した現在も肝炎の再燃はみられていない．

症例は71歳，男性．労作時息切れと食道胃静脈瘤の精査・加療のため入院となった．右心カテーテル検査で平均肺動脈圧は36mmHgと上昇しており，肺動脈性高血圧症を認めた．基礎疾患にC型肝硬変症と門脈圧亢進症があり，他の肺動脈性高血圧症の原因となる疾患を除外し，門脈肺高血圧症と診断した． 食道胃静脈瘤に対してバルーン下逆行性経静脈的塞栓術を4カ月前に施行しており，心エコーでの推定平均肺動脈圧は9.3%改善していた．労作時息切れや低酸素血症を依然として認めたために在宅酸素療法を導入した．心エコーでの推定平均肺動脈圧は4カ月後にさらに20.5%の改善を認め，両治療法の有効性が示唆された．門脈肺高血圧症の発症機序と治療法を考える上で示唆に富む症例と考えられ報告した．

症例は49歳男性．B型慢性肝炎にて他院通院中，高度脈管浸潤を伴う多発肝細胞癌を認めたため，インターフェロン併用動注化学療法を施行したところ，奏効し，外来通院していた．経過観察中に施行した腹部CTにて胆嚢内にポリープ状の腫瘤を認めたため，当科に入院した．各種画像検査の結果，胆嚢ポリープと診断したが，胆嚢癌の可能性も考えられたため，開腹下胆嚢摘出術を施行した．病理組織では腫瘍細胞が索状や胞巣形成性に増生し，胆嚢腫瘍基部のリンパ管および静脈に腫瘍の浸潤を認め，化学療法前の腫瘍生検組織と類似しており，肝細胞癌の胆嚢転移再発と診断した．肝細胞癌の胆嚢転移はこれまでにほとんど報告がなく，きわめてまれであると考えられ報告した．

Although sorafenib is a standard drug for advanced hepatocellular carcinoma (HCC), little is known about a patient's clinical course after treatment. We investigated the effect of post-progression survival (PPS) and progression-free survival (PFS) on overall survival (OS) in patients whose advanced HCC was treated by sorafenib.We searched in the PubMed database for reports with survival data of patients with HCC treated with sorafenib monotherapy, and selected reports with 20 or more patients each that provided data for both OS and PFS or time to progression (TTP). Median PPS (mPPS) was defined as the period obtained by subtracting median PFS or TTP (mPFS/TTP) from median OS (mOS). We identified 56 reports with 5803 patients. We investigated the correlation of mOS and either mPPS or mPFS/TTP using weighted linear regression.Median PPS correlated with mOS (r = 0.834) very strongly, whereas mPFS/TTP did not correlate with mOS as highly as PPS did (r = 0.546). When we stratified survival data by Child-Pugh classification, a significantly greater average percentage of mPPS to mOS was seen in Child-Pugh class A (54.4 ± 17.6%) than in Child-Pugh class B (32.0 ± 11.6%) (P = 0.015).PPS highly correlated with OS, and its importance should be more emphasized for advanced HCC patients treated after sorafenib therapy, whereas we need to take more care in interpreting the results of PFS to evaluate treatment efficacy in clinical trials of advanced HCC.

TSU-68 is a multikinase inhibitor that targets platelet-derived growth factor receptors (PDGFRs). In the present study, we evaluated the effect of TSU-68 on the tumor-microenvironment interaction in hepatocellular carcinoma (HCC).HCC and fibroblast cell lines (HuH7, Hep3B, HuH1 and WI-38) were used to evaluate their interactions. Cancer characteristics were evaluated by spheroid formation and tumorigenicity in immunodeficient mice. Time-lapse image analysis was performed to monitor cell motility.Although PDGFA was abundantly expressed, PDGFR-α was predominantly located in the cytoplasm and was not functional in HuH7 cells. Co-culture experiments demonstrated that HCC cells induced phosphorylation of PDGFR-α in WI-38 fibroblasts and that stimulated fibroblasts, in turn, boosted the spheroid formation capacity of HCC cells. TSU-68 inhibited phosphorylation of PDGFR-α in WI-38 cells and suppressed the growth of subcutaneously co-injected HuH7/WI-38 tumor xenografts.TSU-68 inhibits stromal PDGF signaling activated by cancer cells and suppresses HCC growth.

A 62-year-old female was admitted to our hospital for examination of icterus and thrombocytopenia. She had a history of diabetes mellitus (under treatment), and liver cirrhosis was evident on abdominal CT. Because she was clinically obese and had no past history of alcohol consumption, the initial diagnosis was NASH. However, subsequent MRI findings and normal serum transaminase levels were not consistent with this diagnosis. We then performed additional examinations, including liver biopsy, measurements of serum Cu and ceruloplasmin concentrations, and measurement of urinary Cu secretion, which resulted in a diagnosis of Wilson's disease. It is necessary to include Wilson's disease in the differential diagnosis of NASH in cases of unidentified liver disease even among elderly patients.

金沢大学先進予防医学研究センター / 金沢大学医学部附属病院内科, Purpose: In patients with chronic genotype 1b hepatitis C and a high viral load, the viral load was reduced by double filtration plasmapheresis (DFPP), followed by combined interferon and ribavirin therapy. The safety and virological effects of this treatment method were preliminarily investigated. Methods: In nine patients with chronic hepatitis C, DFPP was performed three times on days 1, 2, and 4, and the administration of interferon and ribavirin was initiated immediately after DFPP on day 1. Result: The HCV RNA was undetectable in all patients after the plasma was passed through a plasma fractionator (second filter) in the DFPP circuit. After 2 weeks, the HCV RNA tended to decrease in the DFPP group more than in the control group (-2.45 ± 1.12 versus -1.57 ± 0.95, P = 0.073). However, this decrease was not attributable to a sustained virological response (SVR) (22.2% versus 18.2%, P = 0.822). Most of the adverse events were caused by the interferon and ribavirin combination therapy. Conclusion: DFPP can be safely performed concomitantly with interferon and ribavirin combination therapy in chronic hepatitis C patients. The combination may contribute to an early virological response. The effect of DFPP on the SVR and its significance remain to be clarified. © 2006 Elsevier Ireland Ltd. All rights reserved.

Objectives: Splenic artery aneurysm (SAA) is usually asymptomatic, but can be fatal if it ruptures. Portal hypertensive patients with varix or splenomegaly are sometimes complicated by SAA. However, there have been no large-scale clinical studies regarding whether liver cirrhosis itself is associated with splenic aneurysm regardless of varix or splenomegaly. Methods: In the present study, we retrospectively analyzed 303 cirrhotic patients examined with arteriography. The diagnosis and characteristics of SAAs were determined, and the relation with splenic artery diameter was evaluated. Results: Nine patients (2.97%) had 12 complicated SAAs. The aneurysms, which measured 4–22 mm in diameter, were all saccular, and occurred commonly in the splenic hilum (50.0%). A correlation was noted between splenic artery diameter and aneurysm diameter (R2=0.706). Aneurysm growth was strongly associated with an increase in diameter of the splenic artery trunk (R2=0.705), which is closely related to arterial flow. Conclusions: SAA is considered a complication of cirrhosis. The increase in splenic artery diameter may result in SAA enlargement and rupture. Elective procedures should be considered based on the follow-up of main trunk or diameter of the splenic artery in addition to SAA size, a known risk factor of aneurysmal rupture.

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

医薬保健研究域保健学系, Differentially regulated microRNA (miRNA) are associated with hepatic fibrosis; however, their potential usefulness for blocking hepatic fibrosis has not been exploited fully. We examined the expression of miRNA in the liver of a transgenic mouse model in which platelet-derived growth factor C (PDGF-C) is overexpressed (Pdgf-c Tg), resulting in hepatic fibrosis and steatosis and the eventual development of hepatocellular carcinoma (HCC). Robust induction of miR-214 correlated with fibrogenesis in the liver of Pdgf-c Tg mice, atherogenic high-fat diet-induced NASH mice, and patients with chronic hepatitis B or C. Pdgf-c Tg mice were injected with locked nucleic acid (LNA)-antimiR-214 via the tail vein using Invivofectamine 2.0 and the degree of hepatic fibrosis and tumor incidence were evaluated. Pdgf-c Tg mice treated with LNA-antimiR-214 showed a marked reduction in fibrosis and tumor incidence compared with saline or LNA-miR-control-injected control mice. In vitro, LNA-antimiR-214 significantly ameliorated TGF-β1-induced pro-fibrotic gene expression in Lx-2 cells. MiR-214 targets a negative regulator of EGFR signaling, Mig-6. Mimic-miR-214 decreased the expression of Mig-6 and increased the levels of EGF-mediated p-EGFR (Y1173 and Y845) and p-Met (Tyr1234/1235) in Huh-7 cells. Conversely, LNA-antimiR-214 repressed the expression of these genes. In conclusion, miR-214 appears to participate in the development of hepatic fibrosis by modulating the EGFR and TGF-β signaling pathways. LNA-antimiR-214 is a potential therapy for the prevention of hepatic fibrosis. MiR-214 appears to participate in the development of hepatic fibrosis by modulating EGFR and TGF-β signaling pathways. LNA-anti-miR-214 may be a potentially therapy in the prevention of hepatic fibrosis. © 2015 Japanese Cancer Association.

OBJECTIVES: Primary biliary cirrhosis (PBC) is a progressive disease. However, little is understood about the molecular mechanisms underlying its features. METHODS: We analyzed gene expression profiles of liver biopsy samples from 16 patients with PBC, seven with autoimmune hepatitis, eight with chronic hepatitis C, and eight normal control livers. In addition to whole liver samples, we selectively analyzed chronic nonsuppurative destructive cholangitis (CNSDC) lesions by laser capture microdissection. RESULTS: Hierarchical clustering analysis using only early-stage liver disease demonstrated 85 genes were upregulated in stage I PBC specifically. Surprisingly, the expression of these genes was not maintained in advanced-stage PBC, while other gene clusters were upregulated. Expression analysis of CNSDC lesions in stage I PBC showed the presence of active inflammatory changes, characterized by the significant elevation of interferon-gamma and the development and maturation of lymphocytes. Expression of these genes was diminished in lymphoid cells aggregation in stage III PBC, and genes reflecting hepatocyte damage were upregulated with disease progression. CONCLUSION: Gene expression patterns in stage I PBC are different from others. There are distinct changes in molecular pathology from early- to late-stage PBC, which might be a clue to reveal the etiology and progression of PBC.

Background and aims: α-Fetoprotein (AFP), a tumour marker for hepatocellular carcinoma (HCC), is associated with poor prognosis. Using cDNA microarray analysis, we previously found that ephrin-A1, an angiogenic factor, is the most differentially overexpressed gene in AFP producing hepatoma cell lines. In the present study, we investigated the significance of ephrin-A1 expression in HCC. Methods: We examined ephrin-A1 expression and its effect on cell proliferation and gene expression in five AFP producing hepatoma cell lines, three AFP negative hepatoma cell lines, and 20 human HCC specimens. Results: Ephrin-A1 expression levels were lowest in normal liver tissue, elevated in cirrhotic tissue, and further elevated in HCC specimens. Ephrin-A1 expression was strongly correlated with AFP expression ( r = 0.866). We showed that ephrin-A1 induced expression of AFP. This finding implicates ephrin-A1 in the mechanism of AFP induction in HCC. Ephrin-A1 promoted the proliferation of ephrin-A1 underexpressing HLE cells, and an ephrin-A1 antisense oligonucleotide inhibited the proliferation of ephrin-A1 overexpressing Huh7 cells. Thus ephrin-A1 affects hepatoma cell growth. cDNA microarray analysis showed that ephrin-A1 induced expression of genes related to the cell cycle (p21), angiogenesis (angiopoietin 1 and thrombospondin 1), and cell-cell interactions (Rho, integrin, and matrix metalloproteinases) in cultured hepatoma cells. These ephrin-A1 induced genes are also activated in HCC tissues that overexpress AFP. Conclusion: These findings suggest that the poor prognosis of patients with AFP producing HCC is partially caused by ephrin-A1 expression, which induces expression of genes related to tumour cell growth, angiogenesis, invasion, and metastasis.

We assayed chromosomal abnormalities in hepatoma cell lines using the microarray-based comparative genomic hybridization (array-CGH) method and investigated the relationship between genomic copy number alterations and expression profiles in these hepatoma cell lines. We modified a cDNA array-CGH assay to compare genomic DNAs from seven hepatoma cell lines, as well as DNA from two non-hepatoma cell lines and from normal cells. The mRNA expression of each sample was assayed in parallel by cDNA microarray. We identified small amplified or deleted chromosomal regions, as well as alterations in DNA copy number not previously described. We predominantly found alterations of apoptosis-related genes in Hep3B and HepG2, cell adhesion and receptor molecules in HLE, and cytokine-related genes in PLC/PRF/5. About 40% of the genes showing amplification or loss showed altered levels of mRNA (p < 0.05). Hierarchical clustering analysis showed that the expression of these genes allows differentiation between alpha-fetoprotein (AFP)-producing and AFP-negative cell lines. cDNA array-CGH is a sensitive method that can be used to detect alterations in genomic copy number in tumor cells. Differences in DNA copy alterations between AFP-producing and AFP-negative cells may lead to differential gene expression and may be related to the phenotype of these cells.

Background & Aims: Translation of hepatitis C virus is an essential step of viral replication and is mediated by an internal ribosome entry site. We previously reported that the hepatitis C virus internal ribosome entry site is most active during the synthetic (S) or mitotic (M) phases and lowest during quiescent (G 0 ) phase. Here, we investigated host factors responsible for the regulation of the hepatitis C virus internal ribosome entry site. Methods: We synchronized the cell-cycle progression and evaluated gene-expression dynamics of host factors and kinetics of hepatitis C virus internal ribosome entry site activity in cells at various points during the cell cycle by using a complementary DNA microarray. We also validated the significance of identified host factors on hepatitis C virus replication in vivo. Results: Hepatitis C virus internal ribosome entry site activity correlated with a gene cluster induced in the S and G 2 /M phases. It is interesting to note that most initiation factors known to bind or interact with the hepatitis C virus internal ribosome entry site [poly(rC)-binding protein 2, polypyrimidine tract binding protein, eukaryotic initiation factor 3, eukaryotic initiation factor 2γ, eukaryotic initiation factor 2β, La protein, and heterogenous nuclear ribonucleoprotein L] were induced during the S and G 2 /M phases. Expression of La protein, polypyrimidine tract binding protein, and eukaryotic initiation factor 3 (p116, p170) were predominantly repressed in G 0 phase and induced in S and G 2 /M phases. Suppression or overexpression of La protein and polypyrimidine tract binding protein in RCF-26 significantly changed hepatitis C virus internal ribosome entry site activity. In the livers of patients with chronic hepatitis C, expression of La protein was significantly increased and correlated with the amount of hepatitis C virus RNA. Conclusions: Hepatitis C virus uses host factors induced during cell division but not during quiescence for replication. Of these, La protein is a potent regulator and enhances hepatitis C virus replication in regenerating hepatocytes in patients with chronic hepatitis C.

症例は60歳男性. 1990年より糖尿病と肝機能障害を指摘され, 他院にて経過観察されていた. 2003年10月に心窩部痛のため当院初診, 上部消化管内視鏡検査で十二指腸乳頭部からの出血を認め入院となった. 背景にアルコール性肝硬変があり, AFP, PIVKA-II, AFP-L3分画の上昇に加え肝右葉に多発する腫瘤と門脈腫瘍塞栓を認めることから, 胆道出血を合併した肝細胞癌と診断した. インターフェロン併用肝動注化学療法を開始し, 2クール目からは外来にて化学療法を継続した. 4クール施行途中から胆道感染の悪化を認め, 胆道出血・閉塞性黄疸を最初に発症してから約8カ月後に死亡した. 胆道出血を契機に発見される肝細胞癌は比較的少なく, その予後は不良とされているが, 本症例は画像上腫瘍縮小効果はないものの, 他の報告に比較してインターフェロン併用肝動注化学療法によって延命が得られた1例と考えられた.

The Human Genome Project has revealed that there about 32,000 protein-encoding genes, which are distributed throughout the genome. It is unclear, however, whether genes are distributed on the chromosomes according to patterns linked to organ specificity. To explore the relationship between genes actively transcribed in normal tissues and their chromosomal locations, we analyzed serial analysis of gene expression libraries of normal human liver, brain, breast, and colon tissues. Transcriptome mapping analysis revealed that transcriptional activity in each tissue varied according to the chromosomal domains, and a weak positive correlation was observed between transcription density and gene density. We identified six liver-related and five colon-related chromosomal domains highly transcribed in each tissue, whereas no brain-related or breast-related chromosomal domains were identified. Representative genes located on these chromosomal domains were associated with the function of each organ and were highly conserved in both mouse and rat genomes. These data revealed that the transcriptional activities of normal human tissues are well orchestrated at chromosomal levels, suggesting that highly expressed genes may share physical proximity.

Type 2 diabetes is characterised by excessive hepatic glucose production and frequently leads to systemic vascular complications. We therefore analysed the relationship between the gene expression profile in the liver and the pathophysiology of Type 2 diabetes.Liver biopsy samples were obtained from twelve patients with Type 2 diabetes and from nine non-diabetic patients. To assay gene expression globally in the livers of both groups, we made complementary DNA (cDNA) microarrays consisting of 1080 human cDNAs. Relative expression ratios of individual genes were obtained by comparing cyanine 5-labelled cDNA from the patients with cyanine 3-labelled cDNA from reference RNA from the liver of a non-diabetic patient.On assessing the similarities of differentially expressed genes, the gene expression profiles of the twelve diabetic patients formed a separate cluster from those of the non-diabetic patients. Of the 1080 genes assayed, 105 (9.7%) were up-regulated and 134 (12%) were down-regulated in the diabetic livers (p0.005). The genes up-regulated in the diabetic patients included those encoding angiogenic factors such as vascular endothelial growth factor, endothelin and platelet-derived growth factor. They also included TGF superfamily genes such as TGFA and TGFB1 as well as bone morphogenetic proteins. Among the down-regulated genes in the diabetic patients were molecules defending against stress, e.g. flavin-containing monooxygenase and superoxide dismutase.These findings suggest that livers of patients with Type 2 diabetes have gene expression profiles indicative of an increased risk of systemic vascular complications.

We have developed a sensitive and quantitative assay using a DNA microarray for the detection of hepatitis B virus (HBV) DNA in serum. Fluorescently labeled target cDNA prepared from cloned HBV DNA or serum HBV DNA was hybridized to capture DNA on a slide. A linear relationship was obtained between the intensities of the array spot and the amount of the cloned or serum HBV DNA, indicating the quantitative accuracy of this assay system. In addition, there was a significant correlation between the number of molecules of serum HBV DNA determined by the DNA microarray and that determined by a branched-DNA assay ( n = 21, r = 0.89). Given these results, we conclude that the DNA microarray assay system may be useful as a diagnostic technique in the clinical laboratory.

We have developed a new analytical method to quantify the dl -homoalanine-4-yl(methyl)phosphinate ( dl -GLUF) enantiomers in biological specimens using a reversed-phase high-performance liquid chromatography system with a fluorescence detection system. The derivatization of dl -GLUF enantiomers with (+)-1-(9-fluorenyl)ethyl chloroformate was carried out under mild conditions (40 °C for 30 min) without inducing racemization. The lower limit of quantitation was 0.01 μg/ml for both d -GLUF and l -GLUF, and the detection limit was 5 ng/ml. When dl -GLUF enantiomers were added to serum to produce concentrations between 0.1 and 100 μg/ml, the mean recovery rate was at least 93.8%. The recovery rate from urine was also satisfactory.

The development of liver-directed virus vector may play a crucial role in hepatic gene therapy. Hepatitis B virus (HBV) is the only known DNA virus that has hepatocyte specificity. In order to construct an efficient HBV-based vector for targeting the liver, we studied the potential use of naturally occurring defective HBVs obtained from hepatitis patients. The enhanced green fluorescent protein (EGFP) gene or small tag sequences (Flag) were introduced in frame into the deleted sites of the defective HBVs. One HBV defective in site for putative T cell epitope and a part of the polymerase gene tolerated EGFP insertion and was successfully packaged. This defective recombinant HBV harboring 48 bp Flag tag sequence instead of EGFP (rHBV-7-Flag) replicated well. Human primary hepatocytes could uptake rHBV-7-Flag virions, though in a low frequency, when exposed to the virions at a high density in the culture medium, and also express Flag tag sequences. This defective HBV-based vector may have a potential application in liver targeting gene therapy.

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5'-nontranslated region (5'NTR). We investigated the effect of interferon alfa (IFN-α) on the IRES-directed translation of HCV, using two stably transformed cell lines, RCF-1 and RCF-26, of Huh7 cells derived from human hepatocellular carcinoma that express dicistronic reporter proteins, Renilla luciferase (RL) and firefly luciferase (FL), separated by HCV-IRES. After the administration of IFN-α or poly(I)-poly(C), HCV-IRES–directed translation was inhibited in a dose-dependent manner. The relative HCV-IRES activity (F/L) decreased to 60% at 5,000 IU/mL of IFN-α and 45% at 40 μg/mL of poly(I)-poly(C). Thus, IFN-α or poly(I)-poly(C) inhibited HCV-IRES–directed translation more efficiently than a cellular cap–dependent translation. 2',5'–oligoadenylate synthetase (2',5'AS) protein level in cells analyzed significantly increased after the administration of IFN-α, but not upon poly(I)-poly(C). Overexpression of double-stranded RNA-activated protein kinase (PKR) gene did not mimic the selective inhibition of HCV-IRES–directed translation in the transformant cells, suggesting that neither the 2',5'AS nor the PKR system are involved in this selective inhibition. Interestingly, the expression of the autoantigen, La, which has been reported to enhance HCV-IRES–directed translation, was significantly reduced after the administration of IFN-α and poly(I)-poly(C) in a dose-dependent manner. Transient expression of La protein completely restored the selective inhibition of HCV-IRES–directed translation by IFN-α and poly(I)-poly(C). These findings suggested a new antiviral mechanism induced by IFN-α in that IFN-α or poly(I)-poly(C) selectively inhibited HCV-IRES–directed translation compared with the eukaryotic cap-dependent translation through the reduction of La protein. (H EPATOLOGY 2002;35:199-208.)

To obtain stereographic images of tissues containing both free and sectioned surfaces, backscattered electron (BS) imaging was tested with both resin and de-embedded specimens. Although the ultrastructure of several organelles in cells could be identified on a sectioned plane of the resin-embedded tissues, three-dimensional images of tissues were difficult to obtain. On the other hand, three-dimensional images showing both the free and sectioned surfaces of tissues could be obtained by scanning electron microscopic observation of the de-embedded specimens stained with heavy metals in BS mode. Organelles in the sectioned plane could be recognized with enough contrast to discriminate the different cellular components even after the de-embedding process. Our present method would make it easier to obtain images of rare structures barely detectable by the conventional cracking methods and will contribute to a three-dimensional analysis of biological specimens.

Hepatocellular carcinoma (HCC) develops with progressive accumulation of multiple genetic changes. An acyclic retinoid peretinoin is reported to suppress the development of HCC, but the molecular mechanism in detail remains to be obscure. Here we evaluated the effect of peretinoin on preneoplastic nodules and HCC developed in platelet derived growth factor-c (PDGF-C) transgenic mice liver by gadolinium ethoxybenzyl diethylenetriamide pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) and whole exome sequence analysis. We confirmed the progressive accumulation of somatic mutations in hypervascular liver cancer tissues compared with hypovascular precancerous tissues and non-cancerous background liver tissues in PDGF-C transgenic mice. Surprisingly, peretinoin treatment in HCC as well as preneoplastic nodules resulted in the accumulation of less gene mutations compared with vehicle. Gd-EOB-DTPA uptake capacity and its transporter OATP1 was confirmed to be re-activated in peretinoin treated nodules in mice. Gene and protein expression analysis indicated the inactivation of cell cycle with low Ki-67 labeling indexes in peretinoin treated nodules. The reversion of Gd-EOB-DTPA uptake capacity was confirmed in preneoplastic nodules and a subset of classical hypervascular HCCs. Notably, peretinoin supplementation induced the expression of OATP1B3 in mature hepatocyte-like HCC cell lines in vitro, whereas supplementation of tretinoin (all-trans retinoic acid) had no such effects and rather suppressed OATP1B3. Taken together, our data suggested the utility of peretinoin to revert the malignant transformation processes of hepatocytes by preventing accumulation of gene mutations especially in early stages of hepatocarcinogenesis. Peretinoin but not tretinoin can induce the expression of a mature hepatocyte marker OATP1B3, suggesting that peretinoin may exert its anti-tumor effect with hepatocytic differentiation capacity independently of the activation of retinoic acid signaling. Note: This abstract was not presented at the meeting. Citation Format: Taro Yamashita, Hikari Okada, Masao Honda, Shuichi Kaneko. Peretinoin prevents the accumulation of somatic gene mutations and reverts neoplastic lesions to normal liver-like in platelet-derived growth factor-c transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5245. doi:10.1158/1538-7445.AM2017-5245

An 81-year-old female was referred to our hospital for the examination of an S7 liver tumor. The tumor was suspected to be a hepatic adrenal rest tumor (HART) based on ultrasonography, dynamic CT, Gd-EOB-DTPA-enhanced MRI, and CT during abdominal angiography. After various hormonal tests, the tumor was confirmed as hormonally non-functional. The diagnosis of HART was confirmed based on (131)I-adosterol accumulation in the tumor by adrenocortical scintigraphy. The resected tumor was histologically compatible with HART, and it may have been able to produce cortisol based on the immunohistochemical findings of various adrenocortical hormone metabolic enzymes. Adrenocortical scintigraphy may thus be useful in diagnosing HART.

Inflammation plays a critical role in cancer. The aim of the present study was to investigate the impact of neutrophil to lymphocyte ratio (NLR) on patients with advanced hepatocellular carcinoma (HCC) treated with hepatic arterial infusion chemotherapy (HAIC).We retrospectively evaluated 266 patients with advanced HCC treated with HAIC between March 2003 and December 2012. NLR was calculated from the differential leukocyte count by dividing the absolute neutrophil count by the absolute lymphocyte count.The cut-off level of NLR was set as the median value of 2.87 among all patients in this study. The objective response rate in the patients with low NLR was 37.6%, which was significantly better than that of the patients with high NLR (21.1%; P 0.01). Multivariate analysis revealed that low NLR remained associated with the response to HAIC (P = 0.024). Median progression-free survival and median overall survival in patients with high NLR were 3.2 and 8.0 months, respectively, which were significantly shorter than that of the patients with low NLR (5.6 and 20.7 months; P 0.01 and P 0.01, respectively). High NLR was an independent unfavorable prognostic factor in multivariate analysis. The patient outcome was stratified more clearly by NLR calculated after HAIC added to calculations before HAIC. Serum platelet-derived growth factor-BB level was positively correlated with NLR.Results suggest that NLR is a useful predictor in patients with advanced HCC treated with HAIC. These findings may be useful in determining treatment strategies or in designing clinical chemotherapy trials in future.