Hiroyasu Imanishi

Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Emodin

Previous experiments have shown that emodin is highly active in suppressing the proliferation of several tumor cell lines. However, it is not clear that emodin can induce growth inhibition of hepatoma cells. We have found that emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC/PRF/5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time‐and dose‐dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (ΔΨm), followed by the activation of caspase–9 and caspase–3, leading to DNA fragmentation and apoptosis. Our findings demonstrate that ROS and the resulting oxidative stress play a pivotal role in apoptosis. Preincubation of hepatoma cell lines with the hydrogen peroxide‐scavenging enzyme, catalase (CAT) and cyclosporin A (CsA), partially inhibited apoptosis. These results demonstrate that enhancement of generation of ROS, ΔΨmdisruption and caspase activation may be involved in the apoptotic pathway induced by emodin.

NS-398, a selective cyclooxygenase 2 inhibitor, inhibited cell growth and induced cell cycle arrest in human hepatocellular carcinoma cell lines

Cyclooxygenase 2 (COX‐2) has been suggested to be associated with liver carcinogenesis. Several reports have shown that NSAIDs inhibit the growth of hepatocellular carcinoma cell lines. There is little evidence of how COX‐2 inhibitors regulate the proliferation of hepatocellular carcinoma cells or the mechanism involved. In our study, we investigated the growth‐inhibitory mechanism of a selective COX‐2 inhibitor, NS‐398, in 4 hepatocellular carcinoma cell lines by studying cell growth, COX‐2 and proliferating cell nuclear antigen (PCNA) expression, cell cycle distribution and the evidence of apoptosis. NS‐398 inhibited the growth of all 4 cell lines in a time‐ and dose‐dependent manner and the inhibitory effects were independent of the level of COX‐2 protein expression. PCNA expression was downregulated by NS‐398 in a dose‐independent manner. NS‐398 caused cell cycle arrest in the S phase with a reduction in cell numbers and cell accumulation in the G0/G1 phase, for all 4 cell lines. No evidence of apoptosis was observed in our present study. Our findings suggest that a selective COX‐2 inhibitor might serve as an effective tool for the chemoprevention and treatment of hepatocellular carcinomas. A reduction in cell number in the S phase may be an important event in cell cycle arrest caused by NS‐398 in hepatocellular carcinoma cell lines.

Inhibition of the expression of α-smooth muscle actin in human hepatic stellate cell line, LI90, by a selective cyclooxygenase 2 inhibitor, NS-398

Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS-398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC.

Involvement of cell cycle regulatory proteins and MAP kinase signaling pathway in growth inhibition and cell cycle arrest by a selective cyclooxygenase 2 inhibitor, etodolac, in human hepatocellular carcinoma cell lines.

Recent studies have shown that selective cyclooxygenase‐2 (COX‐2) inhibitors induce growth inhibition and cell cycle arrest in hepatocellular carcinoma (HCC) cell lines. However, the mechanism by which COX‐2 inhibitors regulate the cell cycle and whether or not growth signal pathways are involved in the growth inhibition remain unclear. In this study, we investigated the mechanisms of growth inhibition and cell cycle arrest by etodolac, a selective COX‐2 inhibitor, in HCC cell lines, HepG2 and PLC/PRF/5, by studying cell cycle regulatory proteins, and the MAP kinase and PDK1‐PKB/AKT signaling pathways. Etodolac inhibited growth and PCNA expression and induced cell cycle arrest in both HCC cell lines. Etodolac induced p21WAF1/Cip1 and p27Kip1 expression and inhibited CDK2, CDK4, CDC2, cyclin A and cyclin B1 expression, but did not affect cyclin D1 or cyclin E. HGF and 10% FBS induced ERK phosphorylation, but phosphorylation of p38, JNK and AKT was down‐regulated by etodolac. PD98059, a selective inhibitor of ERK phosphorylation, induced growth inhibition, the expression of p27Kip1 and cell cycle arrest. In conclusion, p21WAF1/Cip1 p27Kip1, CDK2, CDK4, CDC2, cyclin A, cyclin B1 and the MAP kinase signaling pathway are involved in growth inhibition and cell cycle arrest by a selective COX‐2 inhibitor in HCC cell lines.

A NOVEL POINT MUTATION ASSOCIATED WITH ALKAPTONURIA

To the Editor: Alkalptonuria (AKU) is a rare autosomal recessive disorder resulting from loss of homogentisate 1,2dioxygenase (HGO; EC 1.13.1 1.5) activity and has existed for more than 3000 years in humans (1, 2). The locus (symbol AKU) was assigned to chromosome 3q2 (2-4). The HGO complementary DNA (cDNA) was cloned by Fernandez-Canon et al. ( 5 , 6). They revealed for the first time that AKU is caused by loss of HGO activity at the molecular level. One point mutation, Pro230Ser in exon 10, has been determined responsible for this disease either because HGO is not correctly expressed or because the encoded polypeptide lacks enzymatic activity. Another point mutation, Va1300Gly in exon 12, may also be responsible for this disease (1). Now, we report a novel point mutation which is associated with a Japanese AKU family. A 40-year-old woman was admitted to our hospital because of ‘lumbago and black urine’. Degenerative arthritis in the spine was observed on a roentgenogram and a high level of urinary homogentisic acid (HGA: 5.5 mmol/l, normal value: 0 mmol/I) was detected by high performance liquid chromatography. The patient was diagnosed as having alkaptonuria. Her parents had been healthy and had no history of having any medications. They had died of cerebrovascular diseases. but their marriage was not consanguineous. The patient was married and had three healthy children. The patient, her three children and five healthy volunteers were selected for this study. The DNA of peripheral white blood cells from the four family members and five healthy volunteers was extracted using an IsoQuick nucleic acid extraction kit (ORCA Research Inc., Bothell, WA, USA). The entire sequence of the HGO gene from exon 1-14 was amplified using a series of primers and a polymerase chain reaction (PCR) amplification kit (Takara, Shiga, Japan). After confirming the sizes and homogeneity of the PCR products by agarose gel electrophoresis, direct sequencing of the entire coding region of the HGO gene was performed using an ABI PRISMTM dye terminator cycle sequencing core kit (Perkin-Elmer, Foster

Involvement of hepatoma-derived growth factor in the growth inhibition of hepatocellular carcinoma cells by vitamin K2

BackgroundVitamin K2 has been reported to suppress the growth of human hepatocellular carcinoma (HCC) in vitro and hepatocarcinogenesis in hepatitis C virus (HCV)-related cirrhosis in vivo. Hepatoma-derived growth factor (HDGF) is a unique nuclear targeting growth factor that is highly expressed in HCC cells and is a possible prognostic factor for patients with HCC. We investigated the regulation of HDGF expression by vitamin K2.MethodsThree HCC-derived cell lines, HepG2, HuH-7, and SK-Hep-1, were used. Cell number was determined with the MTT assay. The expression levels of HDGF mRNA and protein were measured by the real-time reverse transcriptase-polymerase chain reaction (PCR) method and ELISA and Western blot analysis, respectively. The HDGF promoter activity was measured by a dual luciferase-reporter assay.ResultsVitamin K2 suppressed the growth of the three HCC cell lines in a dose-dependent manner. Vitamin K2 significantly suppressed the expression of the HDGF protein and mRNA in three cell lines. By a luciferase assay, vitamin K2 significantly suppressed the promoter activity of the HDGF protein. Based on some luciferase-reporter plasmids containing truncated promoter regions, the possible responsive site of vitamin K2 seems to reside in the region −1 to −150 bp of the HDGF gene.ConclusionsThese findings suggested that regulation of the HDGF gene expression is one of the crucial mechanisms of vitamin K2-induced cell growth suppression for HCC.

Development of a new in situ hybridization method for the detection of global bacterial DNA to provide early evidence of a bacterial infection in spontaneous bacterial peritonitis

BACKGROUND & AIMS Despite the importance of identifying the causative pathogen(s), ascitic fluid cultures are occasionally negative in patients with spontaneous bacterial peritonitis (SBP). A novel strategy using the in situ hybridization (ISH) method was introduced to detect the bacterial genomic DNA phagocytized in the blood of patients with sepsis. In the present study, we developed a new ISH probe to detect global bacterial DNA (named as GB probe) and evaluated its utility for detecting the phagocytized bacterial DNA in SBP ascites. METHODS Hybridization of bacterial DNA with the GB probe was examined by dot-blot and ISH tests. In addition, the utility of the ISH method to detect the bacterial DNA in the leukocytes of SBP ascites was evaluated. RESULTS The GB probe hybridized with the genomic DNA of all 59 bacterial strains tested (59 species of 36 genus). Eleven of 51 patients with ascites (out of total 542 cirrhotic inpatients) were categorized as SBP. The ISH tests showed positive results in 10 of 11 SBP cases. However, the ISH tests all showed negative results in the 40 non-SBP ascitic samples. Therefore, the ISH tests yielded highly sensitive and specific results for detecting the phagocytized bacterial DNA in the leukocytes of SBP ascites. Moreover, all of the ISH test results were obtained within only one day. CONCLUSIONS Our newly established ISH method was found to provide both a rapid and sensitive detection of bacterial DNA in SBP ascites, thus suggesting its utility for providing early and direct evidence of bacterial infection in SBP ascites.

Cost‐effectiveness analysis on the surveillance for hepatocellular carcinoma in liver cirrhosis patients using contrast‐enhanced ultrasonography

Aim:  Sonazoid is a new contrast agent for ultrasonography (US). Contrast‐enhanced ultrasonography (CEUS) using Sonazoid enables Kupffer imaging, which improves the sensitivity of hepatocellular carcinoma (HCC) detection. However, there are no studies on the cost‐effectiveness of HCC surveillance using Sonazoid.

Crucial role of impaired Kupffer cell phagocytosis on the decreased Sonazoid‐enhanced echogenicity in a liver of a nonalchoholic steatohepatitis rat model

Aims:  To evaluate the dynamics of Kupffer cell (KC) phagocytosis by performing both in vivo and in vitro studies using Sonazoid (GE Healthcare, Oslo) in a rat nonalcoholic steatohepatitis (NASH) model.

Novel mutation and multiple mutations found in the human butyrylcholinesterase gene

BACKGROUND Mutations in human butyrylcholinesterase (BChE) are linked to low BChE activity and abnormal response to muscle relaxants. METHODS Twenty Chinese patients with hepatic disease and low cholinesterase activity, and one Japanese patient and her mother were tested for BChE activity and BChE phenotype. The butyrylcholinesterase (BCHE gene) was amplified by polymerase chain reaction (PCR) and sequenced. Mutant BChE was expressed in 293 cells. RESULTS A novel mutation was found in one Chinese patient at nucleotide 943, where A was changed to T (943 A-->T), causing substitution of threonine 315 by serine (T315S). The T315S mutant had half of the normal BChE activity. One Japanese patient with low BChE activity had three nucleotide substitutions, 355 C-->T, 988 T-->A, and 1615 G-->A. The amino acid substitutions were Q119stop, L330I, and A539T, respectively. The single mutant L330I had low BChE activity, but the double mutant L330I/A539T had normal activity. CONCLUSIONS The L330I and the novel T315S mutation caused a decreased BChE activity. The T315S mutation is one of the first BChE mutations reported in the Chinese population. Multiple mutations in BChE may interact with each other in an intramolecular manner.