Tomoyuki Kawakita

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.

15-Deoxy-Δ-12-14-PGJ2 regulates apoptosis induction and nuclear factor-κB activation via a peroxisome proliferator-activated receptor-γ-independent mechanism in hepatocellular carcinoma

The peroxisome proliferator-activated receptor-γ (PPARγ) high-affinity ligand, 15-deoxy-Δ-12,14-PGJ2 (15d-PGJ2), is toxic to malignant cells through cell cycle arrest and apoptosis induction. In this study, we investigated the effects of 15d-PGJ2 on apoptosis induction and expression of apoptosis-related proteins in hepatocellular carcinoma (HCC) cells. 15d-PGJ2 induced apoptosis in SK-Hep1 and HepG2 cells at a 50 μm concentration. Pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (2-VAD-fmk), only partially blocked apoptosis induced by 40 μm 15d-PGJ2. This indicated that 15d-PGJ2 induction of apoptosis was associated with a caspase-3–independent pathway. 15d-PGJ2 also induced down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bclx, and apoptotic protease-activating factor-1 in SK-Hep1 cells but not in HepG2 cells. However, 15d-PGJ2 sensitized both HCC cell lines to TNF-related apoptosis-induced ligand–induced apoptosis. In SK-Hep1 cells, cell toxicity, nuclear factor-κB (NF-κB) suppression, and XIAP down-regulation were induced by 15d-PGJ2 treatment under conditions in which PPARγ was down-regulated. These results suggest that the effect of 15d-PGJ2 was through a PPARγ-independent mechanism. Although cell toxicity was induced when PPARγ was down-regulated in HepG2 cells, NF-κB suppression and XIAP down-regulation were not induced. In conclusion, 15d-PGJ2 induces apoptosis of HCC cell lines via caspase-dependent and -independent pathways. In SK-Hep1 cells, the ability of 15d-PGJ2 to induce cell toxicity, NF-κB suppression, or XIAP down-regulation seemed to occur via a PPARγ-independent mechanism, but in HepG2 cells, NF-κB suppression by 15d-PGJ2 was dependent on PPARγ.

Ticlopidine induced acute cholestatic hepatitis complicated with pure red cell aplasia.

To the Editor: A 69-year-old man was admitted to our hospital because of general malaise, anorexia, jaundice and pruritus. He was given ticlopidine because of a cerebral infarction, after which, he presented with the above symptoms. Blood chemistries showed liver dysfunction and hyperbilirubinemia alanine transaminase (ALT), 455IU/L; asparate transaminase (AST), 190IU/l; total bilirubin, 8.2mg/dl; -glutamyltranspeptidase ( -GTP), 806IU/L. Serological analysis was negative for HBsAg, HBsAb, HBeAg, HBeAb, IgMHBc Ab, HCV Ab, and IgM-HA Ab. Anti-nuclear antibody was negative. An abdominal ultrasound scan showed normal liver and no evidence of obstructive jaundice. The patient was diagnosed with acute intrahepatic cholestasis induced by ticlopidine. Ticlopidine was discontinued, and the liver enzymes and Tbilirubin levels improved. However, a normocytic, normochronic anemia gradually developed, and reticulocytes disappeared. On the 16th day after admission, bone marrow smears showed marked depletion in the erythroblastic series (0.7%), with no abnormality in the granulocytic and megakaryocytic series. Based on these findings, the patient was diagnosed as having pure red cell aplasia (PRCA). As his hemoglobin concentration decreased rapidly, 5 units (2000 ml) of packed red cells were administered. Although no other treatment was given for anemia, hypoplasia of the erythroblastic series was transient and the anemia gradually improved. On the 35th day after admission, bone marrow smears showed improved eryhroblastic bone marrow series .Reticulocytes in the peripheral blood (5.2%) were present. On the 43rd day after admission, laparoscopy and biopsy of the liver was performed. Intrahepatic cholestasis was observed. The patient was diagnosed with acute intrahepatic cholestasis associated with pure red cell aplasia due to ticlopidine. Ticlopidine is widely used for cerebrovascular or cardiovascular disease. Adverse effects of ticlopidine include allergic skin rash, gastrointestinal symptoms (nausea etc), bone marrow toxicity, and elevation of liver function tests. Ticlopidine induced mild elevations in serum liver enzymes are observed in some studies with a 1% to 2% incidence. The incidence of severe hepatitis is estimated at 0.0013%. Only a few cases of a severe cholestatic pattern of liver injury have been reported. In such cases, liver biopsies demonstrated centro-acinar cholestasis. In our case, liver biopsy showed intrahepatic cholestasis, compatible with a druginduced liver damage. PRCA is an uncommon disease. It is almost always associated with a recent viral disease, although multiple causes have been described as a result of exposure to drugs or chemicals, infections, thymoma, or Adult Still Disease. Reports associated PRCA with lamivudine, HCV infection, and acute hepatitis A. The exact mechanism that causes the disorder is unknown, possibly associated with the immune system. Possible mechanisms for PRCA include IgGmediated destruction in the presence of the offending drug and a direct effect on DNA synthesis. Autoantibody mechanisms also were proposed for the druginduced blood cell dyscrasias. The diagnosis of PRCA in this patient was based on the progression of normocytic, normochromic anemia with normal megakaryocyte and leukocyte counts and an absence of erythroid precursors. Ticlopidine induced acute cholestatic hepatitis complicated with pure red cell aplasia is rare.

Retroperitoneal perforation of a pancreatic cyst during endoscopic retrograde pancreatography

A 70‐year‐old man was admitted to Ueno Municipal Hospital, Ueno, Japan, for evaluation of abdominal distension. Computed tomography showed a 1 × 1 cm cyst at the pancreas tail. Endoscopic retrograde pancreatography (ERP) showed a normal pancreatic duct after the first gentle injection and an enhanced cyst at the pancreas tail. Extravasation of the contrast medium occurred from the pancreatic duct to the superior‐dorsal extrapancreas at the same time of the next low‐pressure manual injection. Computed tomography showed extravasation of the contrast medium from the pancreas cyst to the retroperitoneal space after ERP. It was considered that the cyst wall weakness, in addition to slight elevated pancreatic duct pressure, caused the disruption of the cyst wall.