Iwata Ozaki

Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis

The programmed cell death 4 gene (PDCD4), a newly identified transformation suppressor, was analysed in lung tumour cell lines and primary lung carcinomas. Reduced PDCD4 mRNA expression was observed in two immortalized lung cell lines and 18 cancer cell lines by northern blot analysis. In the survey of primary lung tumours, PDCD4 cDNA was poorly represented in 47 lung tumours compared with normal lung tissue by cDNA microarray analysis and this poor representation was significantly associated with high‐grade (G3) adenocarcinomas (p = 0.012). Immunohistochemical analysis of 124 primary carcinomas comprising all subtypes demonstrated that PDCD4 protein expression was widely lost in tumour samples (83%) and was negatively related to poor prognosis (p = 0.013). The loss of PDCD4 expression correlated with higher grade and disease stage (p = 0.045 and 0.034, respectively), but not tumour size and nodal status. Similarly to the cDNA data, lack of PDCD4 expression was significantly linked to tumour grade in adenocarcinoma (n = 59, p = 0.048), while in squamous cell carcinoma (n = 58), no relationship between PDCD4 expression and clinicopathological parameters was established. These data suggest that the loss of PDCD4 expression is a prognostic factor in lung cancer and may correlate with tumour progression. Copyright

Evaluation of acoustic radiation force impulse elastography for fibrosis staging of chronic liver disease: a pilot study

Background: Acoustic radiation force impulse (ARFI) is a new technology integrated into conventional B‐mode ultrasonography. ARFI is used to evaluate tissue stiffness in several organs, but this method has not been applied for liver fibrosis.

Visceral fat accumulation and insulin resistance are important factors in nonalcoholic fatty liver disease

BackgroundNonalcoholic fatty liver diseases are often associated with obesity, insulin resistance, and excessive visceral fat accumulation. The aims of this study were (1) to evaluate the relationship between the severity of fatty liver and visceral fat accumulation in nonalcoholic fatty liver diseases, and (2) to investigate the relationships of fatty liver with biochemical data and insulin resistance.MethodsOne hundred twenty-nine subjects (63 women) with fatty liver diagnosed by ultrasonography were enrolled. Subjects positive for hepatitis B virus, hepatitis C virus, or autoimmune antibodies and those whose alcohol intake was over 20 g/day were excluded. The visceral fat area at the umbilical level and the liver–spleen ratio were evaluated by computed tomography.ResultsThe severity of fatty liver evaluated by ultrasonography showed a significant positive relationship with the visceral fat area and waist circumstance (fatty liver severity: mild, 92.0 ± 30.9 cm2; moderate, 122.1 ± 32.6 cm2; severe, 161.0 ± 48.4 cm2; P < 0.0001). The visceral fat area and liver–spleen ratio were negatively correlated (r = −0.605, P < 0.0001). The severity of fatty liver showed strong positive relationships with serum aspartate aminotransferase, alanine aminotransferase, fasting plasma glucose, fasting plasma insulin, and insulin resistance. The severity of fatty liver was positively related to the visceral fat area in 49 nonobese subjects (body mass index <25).ConclusionsThe severity of fatty liver was positively correlated with visceral fat accumulation and insulin resistance in both obese and nonobese subjects, suggesting that hepatic fat infiltration in nonalcoholic fatty liver disease may be influenced by visceral fat accumulation regardless of body mass index.

Correlations between trait anxiety, personality and fatigue: Study based on the Temperament and Character Inventory

OBJECTIVE In our study, we explored the associations among anxiety, the dimensions of Cloningers theoretically based and empirically validated psychobiological model of personality (Temperament and Character Inventory, TCI) and fatigue in order to clarify the personality risk factors for fatigue. METHODS Fifth-year students (n=89) and freshmen (n=162) at Saga Medical School and psychiatric outpatients of Saga Medical School Hospital (n=101) were investigated with the State-Trait Anxiety Inventory (STAI, Japanese version), the TCI (Japanese version), the General Health Questionnaire (GHQ-30) and the self-rating Fatigue Symptom Checklist (FSC), which describe fatigue along three subscales (general, physical and psychological fatigue). Correlation and ANOVA analyses were performed in this study. RESULTS The analysis identified a significant relation (P<.0005) between trait anxiety and fatigue. The TCI dimension of harm avoidance (HA) is positively correlated with both trait anxiety and fatigue (general fatigue, psychological fatigue and physical fatigue). The character dimension of self-directedness is negatively correlated with both trait anxiety and fatigue. CONCLUSIONS There is an inherent relationship among trait anxiety, the temperament dimension of harm avoidance, character dimension of self-directedness and fatigue. The TCI dimensions, harm avoidance and self-directedness, might be considered as predictors for fatigue-related disorders.

Hepatocyte growth factor induces collagenase (matrix metalloproteinase-1) via the transcription factor Ets-1 in human hepatic stellate cell line

BACKGROUND/AIMS Although hepatocyte growth factor recently has been shown to decrease hepatic fibrosis in animal models, the molecular mechanisms of this effects remain to be elucidated. We investigated regulation of collagenase expression by hepatocyte growth factor in hepatic stellate cells. METHODS A human hepatic stellate cell line, LI90, was treated with hepatocyte growth factor. Expression of collagenase, 72 kDa gelatinase, procollagen alpha 1(I), tissue inhibitor of matrix metalloproteinase-1, transforming growth factor-beta 1, or Ets-1, and carboxyterminal telopeptide of type I collagen was examined. Ets-1 binding activity was determined by gel mobility shift assay, collagenase promoter activity was evaluated by reporter gene assay. LI90 cells were also transfected with Ets-1 antisense oligonucleotides with or without hepatocyte growth factor. RESULTS Hepatocyte growth factor increased expression of collagenase mRNA and protein, and an increase in Ets-1 mRNA preceded the increase in collagenase mRNA. Collagenase activity and protein, and a degradation product of type I collagen were increased in the medium. Nuclear extracts from treated LI90 cells also showed increased Ets-1 binding activity. Hepatocyte growth factor and cotransfection of Ets-1 enhanced promoter activity of collagenase gene. Furthermore, treatment of LI90 cells with Ets-1 antisense oligonucleotides downregulated basal and hepatocyte growth factor-induced Ets-1 and collagenase mRNA expression. CONCLUSIONS Collectively, the results suggest that hepatocyte growth factor increases collagenase expression in hepatic stellate cells via the Ets-1 transcription factor-dependent manner.

Inverse association between coffee drinking and the risk of hepatocellular carcinoma: a case‐control study in Japan

Coffee use has consistently been associated with lower serum liver enzyme levels and a reduced risk of liver cirrhosis. A limited number of cohort and case‐control studies also suggest a decreased risk of hepatocellular carcinoma (HCC) among coffee drinkers, but mostly without consideration of hepatitis virus infection. In the present case‐control study, we recruited 209 incident HCC cases and three different controls (1308 community controls, 275 hospital controls, and 381 patients with chronic liver disease [CLD] without HCC), all of whom were aged 40–79 years and residents of Saga Prefecture, Japan. A questionnaire survey elicited information on coffee use during the last 1–2 years and 10 years before, and plasma hepatitis B surface antigen and antibodies to hepatitis C virus were tested for all but community controls. After adjustment for sex, age, heavy alcohol use, smoking status and hepatitis virus markers (except for community controls), coffee use during the last 1–2 years was associated with a decreased risk against any control group. For coffee use 10 years before, comparison between HCC cases and either community controls or CLD patients revealed a decreased risk; adjusted odds ratios for occasional use, 1–2 cups/day and ≥3 cups/day compared with no use were 0.33, 0.27 and 0.22 (P trend < 0.001), respectively, against community controls, and 0.86, 0.62 and 0.53 (P trend = 0.05), respectively, against CLD patients. These results suggest that coffee may protect against the development of HCC, yet further elaborate studies (hopefully, intervention studies) are warranted to corroborate these findings. (Cancer Sci 2007; 98: 214–218)

Menatetrenone, a Vitamin K2 Analogue, Inhibits Hepatocellular Carcinoma Cell Growth by Suppressing Cyclin D1 Expression through Inhibition of Nuclear Factor κB Activation

Purpose: Menatetrenone, a vitamin K2 analogue, plays an important role in the production of blood coagulation factors. Menatetrenone has also bee shown to have antineoplastic effects against several cancer cell lines including hepatocellular carcinoma (HCC) cells. However, the mechanisms by which vitamin K2 inhibits HCC cell growth have not bee fully clarified, and we therefore investigated the molecular basis of vitamin K2–induced growth inhibition of HCC cells. Experimental Design: HCC cells were treated with vitamin K2 and the expression of several growth-related genes including cyclin-dependent kinase inhibitors and cyclin D1 was examined at the mRNA and protein levels. A reporter gene assay of the cyclin D1 promoter was done under vitamin K2 treatment. The regulation of nuclear factor κB (NF-κB) activation was investigated by a NF-κB reporter gene assay, an electrophoretic mobility shift assay, a Western blot for phosphorylated IκB, and an in vitro kinase assay for IκB kinase (IKK). We also examined the effect of vitamin K2 on the growth of HCC cells transfected with p65 or cyclin D1. Results: Vitamin K2 inhibited cyclin D1 mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-κB binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)–, TNF-α–, and interleukin (IL)-1–induced activation of NF-κB binding and transactivation. Concomitant with the suppression of NF-κB activation, vitamin K2 also inhibited the phosphorylation and degradation of IκBα and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1 and p65 became resistant to vitamin K2 treatment. Conclusion: Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1 expression through the IKK/IκB/NF-κB pathway and might therefore be useful for treatment of HCC.

A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line

Alpha 1-antitrypsin (α1AT) deficiency disease is one of the more common hereditary disorders that affects the liver and lung. The liver disease of α1AT deficiency is generally thought to be caused by the accumulation of an abnormal α1AT protein in hepatocytes, whereas the lung disease is thought to be due to a relative lack of the normal protein in the circulation. Therefore, one possible approach to prevent and treat α1AT disease is to both inhibit the expression of the mutated α1AT gene, and to provide a means of synthesizing the normal protein. To do this, we designed specific hammerhead ribozymes that were capable of cleaving the α1AT mRNA at specific sites, and constructed a modified α1AT cDNA not susceptible to ribozyme cleavage. Ribozymes were effective in inhibiting α1AT expression in a human hepatoma cell line using a newly developed simian virus (SV40) vector system. In addition, the hepatoma cell line was stably transduced with a modified α1AT cDNA that was capable of producing wild-type α1AT protein, but was not cleaved by the ribozyme that decreased endogenous α1AT expression. These results suggest that ribozymes can be employed for the specific inhibition for an abnormal α1AT gene product, the first step in designing a gene therapy for the disease. The findings also suggest that the novel SV40-derived vector may represent a fundamental improvement in the gene therapeutic armarmentarium.

Gln27Glu β2-adrenergic receptor variant is associated with hypertriglyceridemia and the development of fatty liver

BACKGROUND Nonalcoholic steatohepatitis (NASH) is associated with the metabolism of lipid, glucose and energy. Beta-adrenergic receptors play an important role in the regulation of energy expenditure, in part, by stimulating lipid mobilization through lipolysis. METHODS To assess whether it is common for the beta2-adrenergic receptor (B2AR) gene polymorphisms in codons 16 and 27 to play a role in the development of fatty liver, we investigated 251 unrelated healthy Japanese males who were drug-free and showed no signs of heavy drinking. RESULTS The allelic frequency of B2AR gene mutation in codons 16 and 27 did not differ between obese subjects (BMI>25.0 kg/m(2), n=151) and non-obese subjects (BMI</=25.0 kg/m(2), n=100). The Gly16 homozygotes had a lower high-density lipoprotein cholesterol (HDL-C) level than the Arg16 homozygotes (1.50+/-0.4 vs. 1.32+/-0.3 mmol/l, p=0.014). However, no significant association with fatty liver was observed in the Gly16 allele frequency. The Gln27Glu27 heterozygotes showed higher concentrations of serum triglycerides (TG) than the Gln27Gln27 homozygotes (1.62+/-0.93 vs. 2.21+/-1.67 mmol/l, p=0.013). This correlation was also observed in all subjects regardless of weight classification. Univariate analysis indicated that subjects with the heterozygous Gln27Glu mutant alleles had a significantly higher prevalence of fatty liver vs. those without the mutation (Glu27 allele frequency, 0.07 vs. 0.12, p=0.047; odds ratio, 1.92; 95% confidence interval, 1.01-3.68). However, multivariate logistic regression models showed the prevalence of fatty liver to be significantly related to the homeostasis model assessment (HOMA) index, BMI, triglyceride and HDL-cholesterol. CONCLUSIONS These results suggest that the amino-terminal polymorphisms of the beta2-adrenergic receptor gene in codon 27 were associated with hypertryglyceridemia and independent of obesity, and thereby could be involved in the molecular pathogenesis of fatty liver.

Influence of alcohol consumption and gene polymorphisms of ADH2 and ALDH2 on hepatocellular carcinoma in a Japanese population.

Although alcohol intake as well as hepatitis viruses has been associated with hepatocellular carcinoma (HCC), gene–alcohol interactions on HCC risk remain to be elucidated. We conducted a case‐control study to examine whether polymorphisms of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) modified the HCC risk depending on the amount of alcohol intake. ADH2 and ALDH2 genotyping was performed by a duplex polymerase chain reaction with confronting two‐pair primers in 209 newly diagnosed HCC cases and 2 different controls [275 hospital controls and 381 patients with chronic liver disease (CLD)]. Multiple logistic regression analyses revealed that heavy drinkers consuming ≥3 “go”s/day of sake (69 g of ethanol/day) showed an increased risk of HCC based on comparison of HCC cases with hospital controls [adjusted odds ratio (OR) = 13.5; 95% confidence interval (CI) 3.3–54.3] or CLD patients (adjusted OR = 7.0; 95% CI 2.5–19.2), whereas the overall risk was not elevated among light to moderate drinkers consuming <3 “go”s/day. Interestingly, light to moderate drinking was associated with an increased risk among those with ALDH2*1/*2 (adjusted OR = 4.5 or 2.0), but not among those with ALDH2*1/*1 (adjusted OR = 0.8 or 1.0; p interaction = 0.03 or 0.13). However, this gene–alcohol interaction was not observed for heavy drinking. Among light to moderate drinkers, people with the combination of ALDH2*1/*2 and ADH2*2/*2 revealed the highest risk of HCC. These findings indicate that the ALDH2 polymorphism may modify HCC risk among light to moderate drinkers.