Intrinsic resistance to TNF-alpha-induced hepatocyte apoptosis in ICR mice correlates with expression of a short form of c-FLIP.

The administration of tumor necrosis factor-alpha (TNF-alpha) or the anti-Fas antibody (Jo-2) to mice causes acute liver failure, which is lethal within hours as a result of the induction of apoptosis in hepatocytes. It was recently reported that nonobese diabetic (NOD) mice are less sensitive to TNF-alpha/D-galactosamine (GalN)-induced liver failure than C57BL/6J (B6) mice, whereas both NOD and B6 mice were sensitive to the lethal effect of Jo-2. In the present study, we investigated the differences between the apoptotic liver cell death induced by TNF-alpha/GalN and that induced by Jo-2. B6, NOD, and Jcl-Imperial Cancer Research (ICR) mice were injected intravenously with TNF-alpha/GalN or Jo-2. ICR mice were less sensitive to TNF-alpha/GalN-induced liver failure than NOD and B6 mice (P<0.0001). In contrast, ICR mice were more sensitive to Jo-2-induced liver failure than B6 mice (P=0.0003). The liver caspase-3, -8 activity, serum transaminase levels, and the number of apoptotic liver nuclei all decreased in ICR in comparison to B6 mice treated with TNF-alpha/GalN. The mRNA expression of TNFR-associated death domain, Fas associated protein with death domain, and Bcl family and nuclear factor-kappaB activation induced by TNF-alpha/GalN were similar in both mice. Interestingly, the short form of cellular FLICE/caspase-8-inhibitory protein (c-FLIP(S)) was constitutively upregulated in ICR mice. In conclusion, these results suggest that ICR mice have an intrinsic resistance to TNF-alpha-induced hepatocyte apoptosis, and that c-FLIP(S) may play a role in TNF-alpha/GalN-induced liver failure, but not in Fas-induced liver failure.

Dual effects of heat stress on tumor necrosis factor-alpha-induced hepatocyte apoptosis in mice.

The major heat shock protein, HSP70, plays a critical role in cell survival in response to stress, possibly by inhibiting a number of antisurvival pathways. However, heat stress (HS) and HSPs also sensitize cells to certain apoptotic stimuli, such as TNF-alpha. To clarify the relations between HS and apoptosis, we examined the differential effects of the intensity of HS on liver injury and apoptosis induced by TNF-alpha in mice. TNF-alpha was injected into D-galactosamine (GalN)-sensitized mice that were pretreated with or without HS. Liver injury was assessed biochemically and histologically. In GalN-sensitized mice, application of HS for 7 days led to significant enhancement of TNF-alpha-induced hepatotoxicity, despite upregulation of HSP70 in the liver. In contrast, application of HS for 1 day led to attenuation of TNF-alpha-induced liver injury. Repeated HS decreased the levels of the FLICE inhibitory protein short (FLIP(S)) and activated caspase-8 in the liver. The caspase-8 inhibitor Z-IETD-FMK effectively protected both the nontreated and HS-pretreated mice from the hepatotoxicity induced by GalN/TNF-alpha. HS shows dual effects on TNF-alpha-induced hepatocyte apoptosis. Exposure to repeated HS, but not to single HS, leads to enhancement of TNF-alpha-induced hepatocyte apoptosis via the interaction of FLIP and caspase-8.

Differential caspase-9-dependent signaling pathway between tumor necrosis factor receptor- and Fas-mediated hepatocyte apoptosis in mice.

BACKGROUND/AIMS: Two apoptosis signaling pathways, which are used by different cell types, are identified. The activation of caspases is critical for the apoptosis process. The aim of this study was to investigate the effects of the caspase-9 inhibitor Ac-LEHD-CHO on tumor necrosis factor receptor (TNFR)- and Fas-mediated hepatocyte apoptosis in vivo, in order to evaluate the similarities and distinctions between TNFR- and Fas-mediated signaling pathways. METHODS: BALB/c mice were intravenously injected with d-galactosamine (GalN, 20 mg/mouse)/tumor necrosis factor-alpha (TNF-alpha, 0.5 microg/mouse), or alphaFas (10 microg/mouse) 30 min after treatment with the caspase-9 inhibitor Ac-LEHD-CHO or pan-caspase inhibitor Z-VAD-fmk. Liver injury was assessed biochemically and histologically. Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using a fluorogenic peptide substrate. RESULTS: Pretreatment with Z-VAD-fmk prevented liver injury and hepatocyte apoptosis induced by either GalN/TNF-alpha or alphaFas. On the other hand, pretreatment with Ac-LEHD-CHO prevented GalN/TNF-alpha-induced hepatotoxicity and hepatocyte apoptosis but not alphaFas-induced liver injury and apoptosis. Both inhibitors reduced the activities of caspase-9 and -3 in the livers of mice administered by GalN/TNF-alpha. However, unlike Z-VAD-fmk, Ac-LEHD-CHO did not inhibit caspase-3 activation in alphaFas-treated mice, although this inhibitor attenuated caspase-9. CONCLUSION: Fas may rely on both caspase-8 activation (extrinsic pathway) and mitochondria (intrinsic pathway) to activate caspase-3. If the mitochondria-dependent pathway is blocked, the other pathway can compensate. In contrast, TNFR may mediate hepatocellular apoptosis mainly through the mitochondria-mediated caspase-9 activation pathway alone.

[A case of lung adenocarcinoma of the lung with disappearance of brain metastasis by re-treatment with gefitinib].

BACKGROUND: Tumor response rate to gefitinib by previously treated patients with advanced non-small-cell lung cancer was approximately 20%. However, there are few reports about effect of re-treatment with gefitinib. CASE: A 40-year-old man was given a diagnosis of adenocarcinoma of lung (c-T2N3M1). Response was not obtained with chemotherapy (paclitaxel + carboplatin, vinorelbine + gencitabin). He developed brain metastasis and received whole brain radiation therapy. He was then given gefitinib. Reduction of the primary tumor, brain metastasis, pulmonary metastasis and liver metastasis was seen. Recurrence of pulmonary metastasis and liver metastasis was discovered 8 months after treatment with gefitinib. Therefore, treatment by gefitinib was stopped. Response was not achieved with chemotherapy (docetaxel and amrubicin). At the request of the patient, retreatment with gefitinib was resumed. Disappearance of brain metastasis was achieved. However, primary tumor, pulmonary metastasis and liver metastasis progressed. CONCLUSION: We reported a case whose brain metastases disappeared with re-treatment with gefitinib. This report is thought to be useful in terms of information about the acquired resistance of gefitinib. Further investigations are needed to determine the indication of re-treatment with gefitinib.

[A case of pulmonary infection due to Mycobacterium simiae].

A 61-year-old woman presented to our hospital with a chronic cough. She had been diagnosed with pulmonary infection due to non-tuberculous mycobacteria by her previous doctor and had received antimycobacterial chemotherapy for 1 year. Chest radiography and computed tomography on the first visit to our hospital revealed nodular shadows with a cavity in the right upper lung field and infiltrative shadows with bronchiectasis in the lingular segment. Mycobacterium simiae was identified using DNA hybridization methods. Analysis of base sequences from sputum samples using 16S rRNA confirmed the identity of all tested isolates as Mycobacterium simiae, and the organism was isolated repeatedly from sputum mycobacterial tests. Pulmonary infection due to Mycobacterium simiae was diagnosed. Rifampicin, Ethambutol and Clarithromycin were administered to the patient, but clinical symptoms have continued, and findings on chest radiography have deteriorated. Cases of pulmonary infection due to Mycobacterium simiae are rare, and this represents the first such case reported in Japan.

Normal liver regeneration and liver cell apoptosis after partial hepatectomy in tumor necrosis factor-alpha-deficient mice.

AIMS/BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF-alpha also influences liver repair following hepatotoxic damage, and regeneration following partial hepatectomy (PH). The aim of this study was to assess the mechanism by which TNF-alpha influences liver cell apoptosis and regeneration following PH in TNF-alpha-deficient (TNF-alpha(-/-)) mice. METHODS: PH was performed in wild mice and TNF-alpha(-/-) mice. RESULTS: In both groups, serum alanine aminotransferase and serum total bilirubin levels comparably peaked at 6 and 48 h after PH, respectively. No differences were observed in hepatocyte proliferation, as determined by mitotic and the proliferating cell nuclear antigen labeling indices, between TNF-alpha(+/+) and TNF-alpha(-/-) mice. Few terminal deoxynucleotidyl transferase nick end-labeling-positive hepatocytes were seen in either type of mice. Nuclear factor-kappa B DNA binding activity in the remaining liver of TNF-alpha(-/-) mice after PH was similar to that of control mice. Ribonuclease protection assay showed that transforming growth factor beta1 mRNA was up-regulated comparably in the livers of the two groups, and that other cytokines were hardly seen in either. Interleukin-6/ signal transducer and activator of transcription-3-dependent pathway was not affected in TNF-alpha(-/-) mice. CONCLUSIONS: These findings suggest that TNF-alpha has little influence on liver regeneration and liver cell apoptosis after PH in mice.

Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB.

Leflunomide is a novel immunosuppressive and anti-inflammatory agent for the treatment of autoimmune disease. The aim of this study was to investigate whether leflunomide protects from liver injury induced by concanavalin A (Con A), a T-cell-dependent model of liver damage. BALB/c mice were injected with 25 mg/kg Con A in the presence or absence of 30 mg/kg leflunomide. Liver injury was assessed biochemically and histologically. Levels of circulating cytokines and expressions of cytokine messenger RNA (mRNA) in the liver and the spleen were determined. Treatment with leflunomide markedly reduced serum transaminase activities and the numbers of dead liver cells. Leflunomide significantly inhibited increases in plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 2 concentrations, and also reduced TNF-alpha mRNA expression in the liver after administration of Con A. These findings were supported by the results in which leflunomide administration decreased the number of T lymphocytes infiltrating the liver as well as inhibiting their production of TNF-alpha. Activation of nuclear factor kappaB (NF-kappaB), which regulates TNF-alpha production, was inhibited in the liver of mice treated with leflunomide, resulting in a reduction of TNF-alpha production from lymphocytes infiltrating the liver. In conclusion, leflunomide is capable of regulating T-cell-mediated liver injury in vivo and that this event may depend on the decrease of TNF-alpha production in the liver through inhibition of NF-kappaB activation caused by leflunomide.