Takahisa Sakai

Cellular FLICE/Caspase-8–Inhibitory Protein as a Principal Regulator of Cell Death and Survival in Human Hepatocellular Carcinoma

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8–inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R–mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-κB and cFLIP down-regulation attenuated NF-κB activation induced by TNF-α or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-κB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-α, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-κB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor–mediated apoptosis but also by regulating NF-κB activation in human HCCs.

Atherosclerosis and ω-3 fatty acids in the populations of a fishing village and a farming village in Japan

The effect of different dietary habits on atherosclerosis was investigated by examining the content of ordinary diets and relevant risk factors through a mass health survey on two village populations in Japan. In total, 261 inhabitants in the fishing village and 209 in the farming village were examined for body build, blood pressure, and blood chemistry. Information on smoking habits and food consumption was obtained using a semi-quantitative item-frequency questionnaire. Pulse wave velocity of the aorta, intima-media thickness of the carotid artery, and atherosclerotic plaques as obtained by ultrasonography were used as measures of atherosclerosis. All measures of atherosclerosis are lower in the fishing village than in the farming village in both men and women. There is a striking 5-8-fold difference in the number of atherosclerotic plaques (P < 0.0001) between the populations. The observed differences in atherosclerosis parallels differences in dietary habits and differences in the serum essential fatty acids. Evaluation of the omega-3 fatty acids over the combined populations reveals a negative association with the number of plaques in the common carotid while the omega-6 fatty acids shows a weak positive association with plaques.

Complications of Partial Splenic Embolization in Cirrhotic Patients

In recent years, partial splenic embolization (PSE) has been widely used in patients with cirrhosis and hypersplenism caused by portal hypertension. We investigated the complications associated with PSE cases seen in our hospital. Seventeen cases of liver cirrhosis that had undergone PSE were examined to investigate the complications associated with it. Mean infarcted area of the spleen was 66.2%. Leukocyte and platelet counts in 16 of 17 patients were seen to improve after PSE and persisted for at least one year. The most frequent side effects were abdominal pain (82.4%) and fever (94.1%). Severe side effects were seen in two of those 17 patients. One patient died from acute on chronic liver failure. The other patients contracted bacterial peritonitis and splenic abscess and needed drainage of splenic abscess before recovery. These two cases were in Child-Pugh class B. In conclusions, PSE is a useful treatment for patients with cirrhosis and hypersplenism caused by portal hypertension. However, the possibility of severe complications, especially in patients with noncompensated cirrhosis, should be kept in mind.

Expression of survivin during liver regeneration.

Survivin functions to suppress cell death and regulate cell division, and is observed uniquely in tumor cells and developmental cells. However, the expression and regulation of survivin in non-transformed cells are not well elucidated. Therefore, we investigated the expression of survivin in a murine liver regeneration model after partial hepatectomy and intraperitoneal carbon tetrachloride (CCl(4)) injection. We found that the expression of survivin transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases during liver regeneration. In a normal mouse liver cell line, over-expression of survivin resulted in a decrease in the G0/G1 phase and an increase in the S and G2/M phases, resulting in Rb phosphorylation. These findings suggest that survivin is dramatically expressed in a cell cycle-dependent manner during liver regeneration and provide a new insight into the regulation of cell proliferation and differentiation.

Septic endophthalmitis and meningitis associated with Klebsiella pneumoniae liver abscess

We report a female case of septic endophthalmitis and meningitis associated with Klebsiella pneumoniae liver abscess which was thought to be caused by duodeno-biliary reflux related to choledochoduodenostomy. We treated this patient by ultrasonography-guided percutaneous abscess drainage and intravenous administration of third generation antibiotics. However, the visual function of her left eye was eventually lost. Reports of liver abscess with metastatic lesions are rare, and our experience suggests that more physicians should be alert to septic metastatic lesions such as K. pneumoniae liver abscess or bacteremia with complaints of ocular or central nervous system symptoms.

Functional Expression of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Human Colonic Adenocarcinoma Cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. Therefore, we investigated TRAIL sensitivity, TRAIL-induced nuclear factor-κB (NF-κB) activation, and expression of TRAIL in human colonic adenocarcinoma cell lines (HT-29, LS180, SK-CO-1). All four TRAIL receptors (TRAIL-R1 through TRAIL-R4) are expressed in these cell lines. TRAIL sensitivity was assessed by assay of cell viability. Cancer cell viabilities were 83 ± 3.1% (HT-29), 90 ± 4.3% (LS180), and 88 ± 6.3% (SK-CO-1) at 24 hours after the addition of 100 ng/ml TRAIL, indicating that these cell lines were relatively resistant to TRAIL. Activation of NF-κB was variably influenced by TRAIL administration, with no consistent tendency among the cell lines, indicating that TRAIL-induced NF-κB activation might be cell-type dependent. In contrast, TRAIL was expressed in the human colonic adenocarcinoma cell lines by Western blotting and RT-PCR. Increased expression of TRAIL on tumor cells was observed by flow cytometry after cytokine stimulation (IFN-γ, TNF-α) or the addition of chemotherapeutic agents (camptothecin, doxolubicin hydrochloride). TRAIL on HT-29 cells was functional and able to induce apoptosis in Jurkat cells. Jurkat cell viability was increased by the addition of TRAILR1-R4-Fc. In the presence of various cytokines or chemotherapeutic agents, functional TRAIL is expressed on the surface of tumor cells, and this expressed TRAIL might contribute to tumor immune privilege by inducing apoptosis of activated human lymphocytes.

Peroxisome proliferator-activated receptor gamma augments tumor necrosis factor family-induced apoptosis in hepatocellular carcinoma.

Proliferator-activated receptor &ggr; (PPAR &ggr;) is a nuclear receptor, which mainly associates with adipogenesis, but also appears to facilitate cell differentiation or apoptosis in certain malignant cells. This apoptosis induction by PPAR &ggr; is increased by co-stimulation with tumor necrosis factor (TNF)-&agr;-related apoptosis-inducing ligand (TRAIL), a member of the TNF family. In this study, we investigated the effect of PPAR &ggr; on Fas-mediated apoptosis in hepatocellular carcinoma (HCC) cell lines. PPAR &ggr; was expressed on all seven HCC cell lines and located in their nuclei. 15-Deoxy-&Dgr;-12,14-prostaglandin J2 (15d- PGJ2), a PPAR &ggr; ligand, inhibited cellular proliferation in HepG2, SK-Hep1 or HLE cells, unlike pioglitazone, another PPAR &ggr; ligand, which did not have a significant influence on proliferation of these cells. However, 15d-PGJ2 facilitated Fas-mediated HCC apoptosis that could not be induced by Fas alone. These results suggest that PPAR &ggr; can augment TNF-family-induced apoptosis.

Acute Pancreatitis in Patients with Ulcerative Colitis

Twenty-two patients (13 men and 9 women; median age, 34 years; range, 15–64 years) with ulcerative colitis (UC) were evaluated to determine the incidence of acute pancreatitis with UC at the First Department of Internal Medicine, Mie University School of Medicine, during 1989–2001. Among these, three patients (14%) were diagnosed as having had episodes of acute pancreatitis during the mean follow-up period of 6 years. One patient presented with acute pancreatitis and UC simultaneously. Two patients had drug-induced pancreatitis (one due to azathioprine and the other due to 5-ASA). In conclusion, acute pancreatitis is not a frequent, but an occasional extraintestinal manifestation of UC.

Difference in atherosclerosis between the populations of a fishing and a farming village in Japan.

Epidemiological studies from Greenland’ and Japan2 suggest that high dietary consumption of marine fats may prevent the development of atherosclerosis and thrombosis. Fish oils rich in n-3 polyunsaturated fatty acids suppress platelet aggregation, vascular prostaglandin production, hepatic triglyceride and very low density lipoprotein cholesterol formation, and intimal smooth muscle cell proliferation. These antiatherosclerotic effects may be important in preventing the development of cardiovascular diseases.

Histological features of cirrhosis with hepatitis C virus for prediction of hepatocellular carcinoma development; A prospective study

The histological features of pre-neoplastic lesions in HCV-associated cirrhosis remain uncertain. The aim of this prospective study was to elucidate histological features for predicting the development of hepatocellular carcinoma (HCC). A cohort of 72 consecutive patients with hepatitis C-associated cirrhosis, which was diagnosed by histology investigated for development of HCC. Seven histological features including small cell dysplasia (SCD) and large cell dysplasia (LCD) of liver cirrhosis were evaluated with regard to the development of HCC. In addition, proliferation and apoptosis were investigated using immunohistochemistry by proliferating cell nuclear antigen and TUNEL method, respectively. At enrollment, SCD was observed in the biopsy specimens of 18 out of 72 (25.0%) patients and LCD was observed in 20 out of 72 (27.8%). Twenty eight out of 72 patients (38.9%) developed HCC during a mean follow-up period of 72.4 months. Among the histological parameters, SCD, active inflammation and complete nodule were statistically significant factors for the cumulative probability of developing HCC. However, LCD did not appear to be important for HCC development. In multivariate analysis, SCD was the highest independent risk factor for HCC. Samples with SCD demonstrated a higher proliferative rate and a lower apoptotic rate than normal hepatocytes or samples with LCD. These results indicate that SCD is a major risk factor for HCC. Careful assessment of liver histology may be important in order to predict HCC development in patients with HCV-related cirrhosis.