Katsuhiko Fujikawa

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.

p21WAF1/CTP1 expression and hepatitis virus type.

Since p21WAF1/CIP1 (p21) is a universal inhibitor of cyclin-dependent kinases and is regulated transcriptionally by p53, which is activated by DNA stress, its expression reflects DNA stress in chronic hepatitis. Recently an association with both hepatitis B and C virus and the expression of p53 or p21 was reported. We analyzed p21 expression in 18 cases of HBV-associated chronic liver diseases and 32 cases of HCV-associated chronic liver diseases by immunohistochemical analysis, and investigated the possible association between hepatocyte p21 expression and hepatic inflammation, fibrosis, and especially hepatitis virus type. The p21-positive hepatocytes were more numerous in areas of intense inflammation and spotty necrosis and areas close to fibrosis, and they increased according to the degrees of grading and staging. The p21 labeling index (LI) in patients with liver cirrhosis was significantly higher than that in patients with chronic hepatitis of both hepatitis viral types (5.84 ± 0.61 vs 12.0 ± 0.83, P < 0.0001 in hepatitis B, 10.28 ± 0.80 vs 15.6 ± 1.09, P = 0.0004 in hepatitis C), Furthermore, the p21 LI was significantly higher in HCV-associated liver disease than in HBV-associated liver disease in every group (4.02 ± 0.48 vs 7.74 ± 0.96, P = 0.021 in low grade group, 7.35 ± 0.46 vs 12.8 ± 0.57, P < 0.0001 in high grade, 12.0 ± 0.83 vs 15.6 ± 1.09, P = 0.034 in liver cirrhosis). In, conclusion, p21 expression was up-regulated by the stress of inflammation and fibrosis and might be influenced by viral proteins in human chronic liver disease.

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.