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The prognosis of patients with hepatocellular carcinoma (HCC) is poor and the development of effective treatments for this malignancy, including combination chemotherapy, is required. This study examined the possible combined inhibitory effects of bevacizumab, an anti-vascular endothelial growth factor monoclonal antibody, and acyclic retinoid (ACR), which can prevent the development of HCC, on the growth of Huh7 human HCC cells. Xenograft tumors were produced by subcutaneously injecting Huh7 cells into nude mice. Starting 1 wk after the tumor cell injection, the mice were treated with bevacizumab alone (5 mg/kg body weight, subcutaneous injection, twice a week), ACR alone (given in a diet containing 0.03%), or their combination for 6 wk, and the effects of these regimens on xenograft growth were examined. Combined treatment with bevacizumab plus ACR significantly suppressed the growth of Huh7 xenografts. The combination of these agents significantly inhibited the phosphorylation of the Akt protein in tumor tissues. With combination therapy, the population of Ki-67-positive cells in xenografts decreased, while that of TUNEL-positive cells increased. The combination of bevacizumab and ACR exerts growth-suppressing effects on HCC cells by inhibiting cell proliferation and inducing apoptosis. This combination might be an effective regimen for the treatment of HCC.

Obesity and related metabolic abnormalities, including adipocytokine dysbalance, are risk factors for hepatocellular carcinoma (HCC). Visfatin, an adipocytokine that is highly expressed in visceral fat, is suggested to play a role in the progression of human malignancies. Branched-chain amino acids (BCAA) reduce the incidence of HCC in obese patients with liver cirrhosis and prevent obesity-related liver carcinogenesis in mice. In this study, we investigated the possible role of visfatin on HCC progression and the effects of BCAA on visfatin-induced proliferation of HCC cells. In patients with HCCs, serum visfatin levels were significantly correlated with stage progression and tumor enlargement. Visfatin preferentially stimulated the proliferation of HepG2, Hep3B, and HuH7 human HCC cells compared with Hc normal hepatocytes. Visfatin phosphorylated extracellular signal-regulated kinase (ERK), Akt, and GSK-3β proteins in HepG2 cells. LY294002 [a phosphoinositide-3-kinase (PI3K) inhibitor], PD98059 [a MAP/ERK 1 kinase (MEK1) inhibitor], CHIR99021 (a GSK-3β inhibitor), and BCAA significantly inhibited visfatin-induced proliferation in HepG2 cells. BCAA also inhibited phosphorylation of GSK-3β, increased cellular levels of p21(CIP1), caused cell-cycle arrest in G(0)/G(1) phase, and induced apoptosis in HCC cells in the presence of visfatin. These findings suggest that visfatin plays a critical role in the proliferation of HCC cells and may be associated with the progression of this malignancy. In addition, BCAA might inhibit obesity-related liver carcinogenesis by targeting and, possibly, by overcoming the stimulatory effects of visfatin., (2011 AACR)

Pancreatic cancer is a highly lethal disease and gemcitabine is considered to be the standard of care for the treatment of advanced pancreatic cancer. However, the outcome of the patients treated with gemcitabine is still unstatisfactory and further development of new treatments is required. We recently found that short wavelength ultra-violet (UV-C) suppresses cell proliferation with downregulation of epidermal growth factor receptor (EGFR) in human pancreatic cancer cells, but not in normal pancreatic epithelial (PE) cells. In this study, we investigated the effect of UV-C on apoptosis in several cell lines derived from the pancreas. UV-C induced poly(ADP-ribose) polymerase (PARP) cleavage, which is a marker of cells undergoing apoptosis, in Panc1, MiaPaca2, KP3 and BxPC3 pancreatic cancer cells, but not in PE cells. We also observed similar effects in Hoechst 33258 staining, which shows DNA fragmentation. While p53, a tumor suppressor protein, plays a critical role in UV-C-induced cell damage, we did not observe the correlation between the sensitivity to UV-C and p53 status. Thapsigargin, an agent that promotes endoplasmic reticulum (ER) stress by depletion of lumenal calcium stores, as well as cis-diamineplatinum (II) dichloride, a classical anti-cancer drug that causes DNA damage, induced PARP cleavage even in PE cells. Moreover, UV-C-induced apoptosis in Panc1 and KP3 cells was associated with the release of cytochrome c, indicating that it was mediated via mitochondrial pathway. Taken together, UV-C has a potent anti-cancer effect on pancreatic cancer cells without adverse effect on normal cells and it could be useful for the treatment of human pancreatic cancers.

Background: Hepatocellular carcinoma has a high mortality rate due to its rate of recurrence. Acyclic retinoid prevents recurrence of hepatocellular carcinoma in patients after surgical removal of their primary tumors by inducing apoptosis in hepatocellular carcinoma cells, although the molecular mechanisms of action are not understood., Methods: Human hepatocellular carcinoma cells in culture, as well as nude mice transplanted with hepatocellular carcinoma cells and rats given with N-diethylnitrosamine were treated with acyclic retinoid. Changes in activated caspase 3 and transglutaminase 2 (TG2) levels, Sp1 cross-linking and its activities, expression of epidermal growth factor receptor, and apoptotic levels were measured., Results: Acyclic retinoid simultaneously stimulated the activation of caspase 3, and the expression, nuclear localization and crosslinking activity of TG2, resulting in crosslinking and inactivation of the transcription factor, Sp1, thereby reducing expression of epidermal growth factor receptor and cell death in three hepatocellular carcinoma cell lines. These effects were partially restored by a caspase inhibitor, transfection of antisense TG2, restoration of functional Sp1, or an excess of epidermal growth factor. Nuclear expression of TG2 and crosslinked Sp1, as also activated caspase 3 were found in both hepatocellular carcinoma cells transplanted into nude mice and cancerous regions within the liver in N-diethylnitrosamine-induced hepatocarcinogenesis model in rats, following treatment of animals with acyclic retinoid., Conclusions: Treatment with acyclic retinoid produces a dual activation of caspase 3 and TG2 induced apoptosis of hepatocellular carcinoma cells via modification and inactivation of Sp1, resulting in reduced expression of epidermal growth factor receptor.

We investigated the priming effect and mechanism of granulocyte colony-stimulating factor (G-CSF) in chemotherapy with low-dose Ara-C and VP-16 for acute myeloid leukemia. We analyzed cell proliferation, apoptosis, and cell cycle in vitro using leukemia cell lines 32Dcl3, U937, HL-60, and Ba/F3. Cell proliferation assays were performed using the Trypan Blue dye exclusion method. For detection of apoptosis, the Annexin V-binding capacity of treated cells was examined by flow cytometry. To evaluate the cell cycle, we used an FITC BrdU Flow kit and conducted analysis by flow cytometry. The combination of Ara-C and VP-16 significantly enhanced the observed effects compared with those of Ara-C or VP-16 alone. Concurrent administration of G-CSF further reduced the cell number and viability of 32Dcl3 and U937 cells, but not of HL-60 and Ba/F3 cells. Apoptotic cells were significantly increased in number by the addition of G-CSF to 32Dcl3 and U937 cells, while G-CSF had no significant effect on HL-60 and Ba/F3 cell lines. The addition of G-CSF significantly decreased the percentage of G0/G1-phase cells and significantly increased that of S-phase cells among 32Dcl3 and U937 cells. No significant effect was observed for HL-60 and Ba/F3 cells. An enhancement was confirmed for the combination of Ara-C, VP-16, and G-CSF for 32Dcl3 and U937 cells but not for HL-60 and Ba/F3 cells. It was thought that this difference was a result of different responses to G-CSF. G-CSF potentiates Ara-C- and VP-16-induced cytotoxicities through apoptosis induction by mobilizing resting G0-G1-phase cells into S phase.

We examined the effects of 9-cis retinoic acid (RA) on the expression levels of retinoid X receptor alpha (RXR alpha) and its phosphorylated form (p-RXR alpha) in HL-60 and HL-60R cells. 9-cis RA reduced both RXR alpha and p-RXR alpha in HL-60 cells, but did neither in HL-60R cells. However, when the HL-60R cells were treated with the combination of 9-cis RA plus PD98059, MEK inhibitor, the p-RXR alpha and RXR alpha proteins all markedly decreased. Moreover, the combination of those agents induced apoptosis in HL-60R cells. Phosphorylation of RXR alpha might be associated with RA-resistance in HL-60R cells.

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.

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.

Acyclic retinoid (ACR), a novel synthetic retinoid, has been demonstrated by us to inhibit the in vitro growth of human hepatoma cells, and this effect was associated with modification of cell cycle control molecules, suggesting that this agent may be useful in the chemoprevention and therapy of various types of malignancies. However, whether or not ACR exerts anticancer activities on human colon carcinoma cells has not yet been elucidated. The purpose of this study was to examine the inhibitory effects of ACR in human colon carcinoma cells and to characterize the molecular mechanism of action of this agent. ACR inhibited the growth of the HCT116 and SW480 human colon carcinoma cell lines with IC50 values of about 30 and 60 microM, respectively. ACR also induced G1-phase cell cycle arrest and apoptosis in these cell lines. When the HCT116 cells were treated with 5-25 microM ACR, there was a marked decrease in the cellular levels of cyclin D1 mRNA and an approximate 2.5- to 3-fold increase in those of p21CIP1 mRNA, and this induction occurred via a p53-independent mechanism. Furthermore, ACR induced a dose-dependent mRNA elevation of differentiation markers at concentrations of ACR that affect the levels of expression of p21CIP1. These novel results suggest that ACR inhibits cell proliferation by inducing G1 arrest and apoptosis and that cyclin D1 and p21CIP1 play critical roles in the molecular mechanisms of growth inhibition and differentiation induced by ACR. Collectively, these findings provide further evidence that ACR may be a potential agent for the chemoprevention and therapy of human colon cancer.

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.

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.

Background/aims: Tumor necrosis factor (TNF)-alpha itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF-alpha/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)-kappaB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF-alpha. Here, we focused on the roles of NF-kappaB and phosphatidylinositol 3-kinase (PI3K)-regulated serine/threonine kinase Akt., Methods: TNF-alpha was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IkappaBalpha (Ad5IkappaB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl-2 family members and caspase activity were examined., Results: In the mice livers, treatment with Ad5IkappaB or the wortmannin suppressed the activation of NF-kappaB or Akt, respectively. Suppression of either NF-kappaB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF-alpha administration. However, in mice treated with both Ad5IkappaB and wortmannin, TNF-alpha administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IkappaB, wortmannin, and TNF-alpha markedly increased the activation of caspase-3 and -9, and activated caspase-8 to a lesser degree, suggesting that TNF-alpha-induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF-kappaB and PI3K/Akt pathways had no effect on expression of Bcl-2 families., Conclusion: The inducible activation of NF-kappaB and constitutive activation of Akt regulate hepatocyte survival against TNF-alpha, which occurs independent of Bcl-2 families.

Using a cDNA microarray analysis, we identified x-ray-inducible immediate early response factor-1 (IEX-1) as a proapoptotic gene which was induced by TNF-alpha and also depend on NF-kappaB activation in Hc human hepatocytes. In these cells only the original form of IEX-1, termed IEX-1S, but not its longer transcript IEX-1L, was expressed. Overexpression of IEX-1S resulted in promotion of TNF-alpha-induced apoptosis in Hc cells expressing a mutant form of IkappaB. This proapoptotic action can be explained by its inhibitory findings on survival signals; inhibition of TNF-alpha-induced activation and expression of phosphatidylinositol 3-kinase (PI3K)/Akt, and also blockage of expression of Mcl-1, an antiapoptotic Bcl-2 family member which is located downstream of Akt, was inhibited by IEX-1S. LY 294002, an inhibitor of PI3K, increased IEX-1S expression induced by TNF-alpha and accelerated TNF-alpha-induced apoptosis in IkappaB-treated Hc cells. Overexpression of the dominant-negative Akt enhanced, but the constitutively active Akt suppressed, TNF-alpha-induced IEX-1S expression, suggesting that PI3K/Akt negatively regulated IEX-1S expression. These results demonstrate that NF-kappaB-dependent recruitment of IEX-1S may play a proapoptotic role in TNF-alpha-stimulated hepatocytes through blockage of the PI3K/Akt pathway. Moreover, the reciprocal cross-talk between IEX-1S and PI3K/Akt may closely be involved in the regulation of TNF-alpha-induced hepatocyte apoptosis.

Background/aims: The Fas-mediated apoptosis pathway has been implicated in liver diseases. The aim of the study was to investigate the role of this system in alcoholic hepatitis., Methodology: The expression of Fas, Fas ligand, and intracellular signaling molecules for apoptosis were determined by immunoblot analysis in fresh frozen liver samples from 19 patients with alcoholic liver disease., Results: Fas and Fas ligand expression was significantly increased in the liver of patients with alcoholic hepatitis (n = 11) as compared with alcoholic liver disease patients without hepatitis (n = 8). Similarly, there were significant differences in the expression of FADD, ICE, and CPP32 in the liver between the two groups. There were significant positive correlations between the Fas ligand and the FADD, ICE, or CPP32 levels in the liver. The expression of Fas, Fas ligand, FADD, ICE, or CPP32 correlated with serum markers of hepatic injury. Plasma soluble Fas levels in patients with alcoholic hepatitis (median: 15.1 U/mL, range: 9.7-19.2 U/mL) were significantly higher than in normal controls (n = 9) (median: 2.8 U/mL, range: 1.9-3.7 U/mL; p < 0.001). There was a significant positive correlation between plasma soluble Fas levels and the hepatic expression of FADD in these patients., Conclusions: These results indicate that Fas-mediated apoptosis may play an important role in alcoholic hepatitis.

Tumor necrosis factor alpha (TNF-alpha) not only induces apoptotic signals but also causes antiapoptotic and regenerative responses in the liver. However, the molecular mechanism(s) of the latter events remains unclear. In the present study, we examined TNF-alpha-induced genes in Hc human normal (unsensitized) hepatocytes by cDNA microarray analysis. Interleukin-8 (IL-8) induction was the most pronounced of the upregulated genes. The IL-8 protein level was also increased. IL-8 belongs to the ELR-CXC chemokine family and appears to exert mitogenic and antiapoptotic functions in other cell systems. IL-8 expression by TNF-alpha was inhibited when two survival signals, nuclear factor kappaB (NF-kappaB) and phosphatidylinositol 3-kinase (PI3K)/Akt, were inhibited by a mutant form of inhibitor of NF-kappaB (IkappaB); by dominant negative (kinase-dead) Akt; or by treatment with LY 294002, an inhibitor of PI3K. TNF-alpha induced apoptosis in Hc cells that were sensitized by inhibition of NF-kappaB and PI3K activation. IL-8 administration protected mice against concanavalin A-induced hepatitis in vivo. IL-8 also rescued the sensitized Hc cells, at least in part, from TNF-alpha-induced apoptosis in vitro. TNF-alpha inhibited DNA synthesis in unsensitized Hc cells in the absence of serum. Exogenous IL-8 reversed, though anti-IL-8 neutralization antibody enhanced, growth inhibition by TNF-alpha. These results indicate that IL-8, the production of which is stimulated by TNF-alpha, inhibits apoptosis of sensitized hepatocytes and releases normal (unsensitized) hepatocytes from growth inhibition induced by TNF-alpha.

Acyclic retinoid, a synthetic retinoid analog, as well as interferon alfa (IFN-alpha) and IFN-beta induce apoptosis in hepatocellular carcinoma (HCC) cells and are used clinically in the prevention of HCC. Here, we show that acyclic retinoid acts synergistically with IFNs in suppressing the growth and inducing apoptosis (as characterized by DNA fragmentation and chromatin condensation) in 5 human HCC cell lines (JHH7, HuH7, PLC/PRF/5, HLE, and HLF). This synergism was only observed when cells were pretreated with the acyclic retinoid, whereas natural retinoic acids (all-trans and 9-cis retinoic acid) were ineffective. This promotion may be due to up-regulation of type 1 IFN receptor (IFNR) expression by the retinoid. Accordingly, incubation with antitype 1 IFNR antibody abolished the synergy. Enhanced IFNR expression was accompanied by increased expression and DNA-binding activity of STAT1, an intracellular signal transducing molecule of IFNR, and increased induction of 2', 5'-oligoadenyl-5'-triphosphate synthetase, which is a target gene of STAT1. Acyclic retinoid did not have any effects on the growth of normal human hepatocytes (Hc) probably because of a lack of IFNR and STAT1 up-regulation. In conclusion, these results provide a rationale for combined biochemoprevention of HCC using acyclic retinoid and IFN-beta.

Background/aims: Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level., Methods: Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining., Results: Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis., Conclusions: AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.

Background: A malfunction of RXRα due to phosphorylation is associated with liver carcinogenesis, and acyclic retinoid (ACR), which targets RXRα, can prevent the development of hepatocellular carcinoma (HCC). Activation of PI3K/Akt signaling plays a critical role in the proliferation and survival of HCC cells. The present study examined the possible combined effects of ACR and LY294002, a PI3K inhibitor, on the growth of human HCC cells.Methods: This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells.Results: ACR and LY294002 preferentially inhibited the growth of HLF cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR and 5 μM LY294002, in which the concentrations used are less than the IC₅₀ values of these agents, synergistically inhibited the growth of HLF, Hep3B, and Huh7 human HCC cells. These agents when administered in combination acted cooperatively to induce apoptosis in HLF cells. The phosphorylation of RXRα, Akt, and ERK proteins in HLF cells were markedly inhibited by treatment with ACR plus LY294002. Moreover, this combination also increased RXRE promoter activity and the cellular levels of RARβ and p21(CIP1), while decreasing the levels of cyclin D1.Conclusion: ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC. [ABSTRACT FROM AUTHOR]

Acute liver failure caused by viruses, drugs, or liver resection, is marked by a massive degree of hepatocyte apoptosis and impaired hepatocyte proliferation, the mechanisms of which, however, still remain to be understood. The choice between life and death is associated with events in regulation of the immune system. The liver is continuously exposed to a large antigenic load that includes pathogens, toxins and dietary antigens. Bacterial toxins, including endotoxin and staphylococcal enterotoxin, have been implicated in the pathogenesis of multi-organ failure associated with liver damage through production of cytokines and chemokines. Inflammation involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Among pro-inflammatory mediators, tumor necrosis factor-α (TNF-α)/TNF receptor (TNFR) systems play central roles in the physiological regulation of apoptosis as well as inflammation and immunity. These pleiotropic biological effects of TNF-α result from its ability to initiate different intracellular signaling pathways, which induce both pro-apoptotic and anti-apoptotic molecules. Hepatocytes appear to be poorly responsive to pro-apoptotic stimuli by TNF-α. Tumor necrosis factor-α, however, induces excessive hepatocyte apoptosis, once cells are sensitized by D-galactosamine or actinomycin D, suggesting that TNF-α itself also induces molecules that protect cells from apoptosis by TNF-α. Besides the apoptosis-inducing signal, the binding of TNF-α to TNFR1 triggers a series of intracellular events that result in the activation of nuclear factor-κB (NF-κB), phosphatidylinositol 3-kinase (PI3K)/Akt, and c-Jun NH2-terminal kinase (JNK). Inhibition of NF-κB may be a two-edged sword against liver injury, which inhibits pro-inflammatory gene expression in leukocytes and causes the sensitization of hepatocytes to TNF-α-induced apoptosis. A variety of mechanisms exist to modulate the activity of intracellular molecules and thereby affect the ultimate outcome of a liver cell's fate. [ABSTRACT FROM AUTHOR]

The strategy to prevent liver carcinogenesis consists of: (i) antiviral modalities such as vaccination, lamivudin, and interferon; (ii) anti-inflammatory modality; and (iii) chemoprevention using such compounds as retinoid analog and vitamin K. Cancer chemoprevention is defined as an approach where natural or synthetic chemical compound works to arrest or reverse premalignant cells by using physiological pathways. As a consequence, such a clone of premalignant cells is eradicated (clonal deletion) by differentiation induction or apoptosis, and thus the process toward the development of clinically detectable cancer is disrupted. A particularly effective candidate target of chemoprevention in liver diseases is an advanced stage of chronic hepatitis, that is supposed to contain transformed hepatocyte clone(s); that is, primary prevention from liver cirrhosis and prevention of recurrent and second primary hepatocellular carcinoma following the treatment of the initial cancer. Retinoid is a collective term of vitamin A analog that binds to nuclear retinoid receptors;retinoic acid receptors (RAR) and retinoid X receptors (RXR). After ligand coupling, these receptors form homo- or heterodimers, bind to the response element (RARE or RXRE) upstream of the target gene, and regulate the gene expression as a transcriptional factor. Biological phenotypes of such transcriptional regulation by retinoid include cellular differentiation, tissue morphogenesis, and programmed cell death (apoptosis). Due to these functions, retinoid analogs are clinically tried to prevent/treat carcinoma in a wide variety of organs including head and neck cancer, uterine cervical cancer, certain leukemia and liver cancer. In this article, clinical trials of retinoid analog to inhibit second primary hepatoma, supposed molecular mechanism of the action of the compound, and aberrant metabolism of RXR and its role in liver carcinogenesis are briefly reviewed. [ABSTRACT FROM AUTHOR]

Nonobese diabetic (NOD) mice, a model of type I diabetes mellitus, harbor certain unique defects in their immune system. The aim of this study was to investigate how NOD mice show hepatic injury and subsequent fibrogenic responses. Methods: Hepatic fibrosis was induced by intraperitoneal injections of dimethylnitrosamine (DMN), and assessed biochemically and histologically. Expressions of cytokine messenger RNA (mRNA) in the liver were determined. Results: In a model of liver cirrhosis induced by dimethylnitrosamine (DMN), we found that NOD mice had lower levels of hepatic fibrosis and better survival than control ICR mice. The resistance to DMN-induced lethality in NOD mice was independent of apoptosis and necrosis of hepatocytes, but apparently due to the prevention of hepatic fibrosis. We also found increased inductions of interferon-γ (IFN-γ) mRNA in the liver of NOD mice and of intracellular IFN-γ from intrahepatic T cells following DMN administration. Treatment with neutralizing anti-IFN-γ-antibody cancelled the inhibition of hepatic fibrosis in NOD mice. Conclusions: These results suggest that IFN-γ is effective for inhibiting hepatic fibrosis and that genetic host factors may be important in determining differential responses to injury. [ABSTRACT FROM AUTHOR]

In recent years, diffuse large B-cell lymphoma (DLBCL) has been classified by DNA microarray analysis into the germinal center B-cell-like (GC) type, the activated B-cell-like (ABC) type and type 3. The latter two types can be collectively categorized as the non-GC (NGC) type. From the prognostic perspective, the GC type has a favorable clinical outcome when compared with the NGC type. The protein Fas induces apoptosis of lymphocytes by binding with the Fas ligand (FasL), and escape from such apoptosis is considered to lead to malignant transformation of the cells and unrestricted growth of lymphoma. We proposed a hypothesis that Fas/FasL expression could be possibly related with a better survival of GC type DLBCL and evaluated 69 DLBCL cases immunohistochemically with CD10, Bcl-6, MUM1, Fas and FasL. These lymphomas were classified as GC type (positive for CD10 or Bcl-6 and negative for MUM1) or NGC type. The GC type had a better overall survival rate than the NGC type ( P = 0.0723). Among markers as given above, positive CD10 was the most significant prognostic factor for overall survival in total DLBCL ( P < 0.05). In the GC type, Fas and FasL expressions were significantly associated with a favorable overall survival (Fas: P < 0.005; FasL: P < 0.05). Hence, Fas or FasL expression might contribute to a better prognosis of this type of DLBCL. [ABSTRACT FROM AUTHOR]

Abstract: Endotoxemia causes liver injury in which tumor necrosis factor (TNF)-α plays a significant role by inducing hepatic apoptosis. We here examined if such apoptosis is strictly dependent on TNF-α and which type of TNF receptor (TNFR) is involved, employing TNFR-1- and -2-knockout mice. Lipopolysaccharide (LPS) dose-dependently induced liver injury in both wild-type (WT) and TNFR-2-knockout mice as indicated by plasma ALT activities, whereas the injury was absent in TNFR-1-knockout mice. Similarly, apoptotic hepatocyte death was observed in WT and TNFR-2-knockout mice after LPS-injection, but not in TNFR-1-knockout mice. Plasma levels of TNF-α, interleukin (IL)-6, IL-10 and interferon-γ as well as hepatic TNF-α levels increased equally in mice with either genotype after LPS-injection. LPS also enhanced equally the mRNA expression of Fas but not Fas ligand irrespective of either genotype, as measured by RNase protection assay. These findings suggest that apoptotic liver injury induced by LPS depends on TNF-α signaling through TNFR-1 but not via TNFR-2 or Fas–Fas ligand pathway. [Copyright &y& Elsevier]

Hayashi H, Nagaki M, Imose M, Osawa Y, Kimura K, Takai S, Imao M, Naiki T, Kato T, Moriwaki H. Normal liver regeneration and liver cell apoptosis after partial hepatectomy in tumor necrosis factor-α deficient mice.Liver International 2005: 25: 162–170.© Blackwell Munksgaard 2005Tumor necrosis factor-α (TNF-α) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF-α 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-α influences liver cell apoptosis and regeneration following PH in TNF-α-deficient (TNF-α−/−) mice.PH was performed in wild mice and TNF-α−/− mice.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-α+/+ and TNF-α−/− mice. Few terminal deoxynucleotidyl transferase nick end-labeling-positive hepatocytes were seen in either type of mice. Nuclear factor-κB DNA binding activity in the remaining liver of TNF-α−/− mice after PH was similar to that of control mice. Ribonuclease protection assay showed that transforming growth factorβ1 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-α−/− mice.These findings suggest that TNF-α has little influence on liver regeneration and liver cell apoptosis after PH in mice. [ABSTRACT FROM AUTHOR]

Hara T, Ando K, Tsurumi H, Moriwaki H. Excessive production of tumor necrosis factor-alpha by bone marrow T lymphocytes is essential in causing bone marrow failure in patients with aplastic anemia. Eur J Haematol 2004: 73: 10–16. © Blackwell Munksgaard 2004. Aplastic anemia (AA) is regarded as an immunological disorder because of the clinical effect of immunosuppressive therapy. Recent studies have reported that cytokines play an important role in the development of AA. In the present study, we measured levels of T-cell derived intracellular cytokine production in peripheral blood and bone marrow (BM) of patients with AA. We demonstrated that BM lymphocytes, particularly CD4+ and CD8+ T cells, in patients with AA produced significantly higher amounts of tumor necrosis factor-alpha (TNF- α), compared with lymphocytes in normal controls. We have previously reported that expression of TNF receptor (R)1 and TNFR2 in the CD34+ CD38− and CD34+ CD38+ fractions of patients with AA is significantly higher than those in normal control. These results indicate that BM stem cells in patients with AA may possess high sensitivity to TNF- α. This in turn suggests that TNF- α affects hematopoiesis at an earlier stage in AA patients than in normal controls. We strongly support the hypothesis that a simultaneous increase in TNF- α production by BM lymphocytes and sensitivity of stem cells to TNF- α leads to BM failure in AA. [ABSTRACT FROM AUTHOR]

Abstract: Background/aims: Tumor necrosis factor (TNF)-α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro , is explained by the fact that the TNF-α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)-κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF-α. Here, we focused on the roles of NF-κB and phosphatidylinositol 3-kinase (PI3K)-regulated serine/threonine kinase Akt. Methods: TNF-α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl-2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF-κB or Akt, respectively. Suppression of either NF-κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF-α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF-α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF-α markedly increased the activation of caspase-3 and -9, and activated caspase-8 to a lesser degree, suggesting that TNF-α-induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF-κB and PI3K/Akt pathways had no effect on expression of Bcl-2 families. Conclusion: The inducible activation of NF-κB and constitutive activation of Akt regulate hepatocyte survival against TNF-α, which occurs independent of Bcl-2 families. [ABSTRACT FROM AUTHOR]

Abstract Hepatocellular carcinoma (HCC) often develops in patients with chronic liver diseases associated with hepatitis B (HBV) and hepatitis C (HCV) virus infections with high incidences. Particularly, post-therapeutic recurrence encountered after the curative treatment of the preceding HCC may limit the prognosis. Thus, prevention of HCC is of great significance. In the present review, immunopreventions with α-interferon and glycyrrhizin, as well as chemoprevention with acyclic retinoid, are discussed. α-Interferon prevents the development of HCC not only in patients with a long-term elimination of HCV (sustained virological responders), but in ones with normalized serum aminotransferases (sustained biochemical responders). Glycyrrhizin also suppresses serum aminotransferases and thereby prevents the tumor development, even though the compound does not have antiviral activity for HBV or HCV by itself. Therefore, suppression of hepatic necroinflammation by these drugs may serve to prevent hepatocarcinogenesis. In contrast, acyclic retinoid suppresses the post-therapeutic recurrence in cirrhotic patients who underwent curative treatment of preceding tumors. The retinoid induces the disappearance of serum lectin-reactive α-fetoprotein (AFP-L3), a tumor marker indicating the presence of unrecognizable tumors in the remnant liver, suggesting a deletion of such minute (pre)malignant clones (clonal deletion). As a molecular mechanism of the clonal deletion, a novel mechanism of apoptosis induction by the retinoid via tissue transglutaminase is implicated. In future, a combination of immunopreventive and chemopreventive therapies may give a clue to the further advances of cancer prevention, and thereby to the improvement of the prognosis of cirrhotic patients. [ABSTRACT FROM AUTHOR]

Background/Aims: To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor-α (TNF-α), caspase cascade and ceramide formation were investigated in the liver of D-galactosamine (GalN)-sensitized mice treated with TNF-α. Methods: Seven-week-old male BALB/c mice were intraperitoneally injected with 20 mg GalN 30 min prior to the intravenous injection of recombinant mouse TNF-α (0.5 μg/mouse). Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using chromogenic peptides as substrates. Ceramide content was determined using Escherichia coli diacylglycerol kinase. Results: Apoptosis of hepatocytes was observed in mice treated with both GalN and TNF-α (GalN/TNF-α), but not GalN or TNF-α alone. Activation of caspases-9 and -3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF-α. In a cell-free system, processing of procaspases-9 and -3, and cytochrome c release were observed in the postnuclear fraction of liver obtained from GalN/TNF-α-treated mice, but not in that from control mice. Processing of procaspase-3 was inhibited by a caspase-9 inhibitor, but not by inhibitor for caspase-8 or -2. In a reconstitution assay system, procaspase-9 processing occurred, when both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF-α. Ceramide accumulation was observed only in apoptotic liver and preceded cytochrome c release and caspase activation. Conclusion: Cytochrome c release and caspase-9 activation are required for the activation of executor caspase-3 in TNF-α-induced hepatocyte apoptosis, but caspases-8 and -2 play, if any, a minimal role. Ceramide may be implicated in this apoptotic process. [ABSTRACT FROM AUTHOR]

Editorial. Discusses how hepatocytes are sensitized to apoptosis. Three structurally related cell-surface receptors implicated in the transduction of apoptotic cell death signals; Effect of administration of tumor necrosis factor (TNF) on mice; Comparison of apoptosis mediated with TNF receptor-1 and Fas ligand.

Abstract: Gemcitabine, an antitumor drug, is currently considered to be the standard of care for the treatment of advanced pancreatic cancer, but the clinical outcome is still not satisfactory. Although heat shock protein (HSP) 27 is implicated in the resistance to chemotherapy in several types of cancers, the precise role of phosphorylated HSP27 in cancer cells remains to be clarified. In this study, we investigated the relationship between the effect of gemcitabine and the phosphorylation status of HSP27 in pancreatic cancer cells, Panc1 and KP3. Gemcitabine suppressed pancreatic cancer cell growth and induced apoptosis. Gemcitabine caused activation of p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase 2 (MAPKAPK-2) and subsequently phosphorylation of HSP27 at Ser15, 78 and 82 without affecting total HSP27 levels. The inhibitions of p38 MAPK and MAPKAPK-2 reduced the phosphorylation of HSP27 and apoptosis in gemcitabine-treated cells. To further investigate the role of phosphorylated HSP27, we established Panc1 cell lines which were stably transfected with empty vector (empty cells), wild-type HSP27-encoding vector (WT cells) and 2 mutant HSP27-encoding vectors that mimic non-phosphorylated (3A), and phosphorylated (3D), respectively. In comparison of empty cells with WT cells, there was no difference in cell growth rate and the sensitivity to gemcitabine. Interestingly, cell growth of 3D cells was retarded as compared to that of 3A cells. Taken together, our results strongly suggest that phosphorylation status of HSP27 plays a key role in gemcitabine-induced growth suppression of pancreatic cancer. [Copyright &y& Elsevier]

Abstract: Although gemcitabine is recognized as the standard drug for the treatment of advanced pancreatic cancer, the clinical outcome is not satisfactory. We recently reported that relatively high dose ultraviolet-C (UV-C; 200J) inhibits cell growth by desensitization of epidermal growth factor receptor (EGFR) in human pancreatic cancer cells. In the present study, we investigated the combination effects of low dose UV-C (10J) and gemcitabine on apoptosis and cell growth in these cells. UV-C enhanced gemcitabine-induced suppression of cell viability. In addition, the combination use clearly induced apoptosis, while neither UV-C nor gemcitabine alone did. Concurrently, combination use caused the decrease in the EGFR protein level and reduced EGF-induced activation of Akt pathway, subsequently resulting in accumulation of β-catenin. The order of the treatment with UV-C and gemcitabine did not affect their synergistic effects on apoptosis and cell growth. Interestingly, combination use synergistically induced phosphorylation of 5′ AMP-activated protein kinase (AMPK) alpha at Thr172 and acetyl-CoA carboxylase at Ser79 as a downstream molecular target of AMPK. AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-riboside, induced apoptosis and suppressed cell growth in these cells, thus suggesting that combination effects of UV-C and gemcitabine is due to the activation of AMPK. Together, our findings could provide a new aspect of pancreatic cancer therapy. [Copyright &y& Elsevier]

Abstract: Pancreatic cancer is a serious healthcare problem worldwide because of its high mortality. Gemcitabine, a DNA synthesis inhibitor, is the standard first-line treatment for advanced pancreatic cancer and is also expected as a key drug for the combination therapy of this malignancy. Retinoids, which are derivatives of vitamin A, exert anti-tumor effects in various types of human malignancies, including pancreatic cancer. This study examined whether combination therapy with gemcitabine and acyclic retinoid (ACR), a new synthetic retinoid, had enhanced anti-tumor efficacy in pancreatic cancer. ACR, 9-cis-retinoic acid and gemcitabine preferentially inhibited the growth of human pancreatic cancer cells (Panc-1 and KP-2) in comparison to PE normal human pancreatic epithelial cells. The combination of ACR plus gemcitabine synergistically inhibited the growth of Panc-1 cells. The combined treatment with these two agents also acted synergistically to induce apoptosis and to inhibit Ras activation in these cancer cells. In vivo, the combination therapy augmented tumor growth inhibition through the induction of apoptosis and inhibition of cell proliferation in tumor tissue. These results suggest that the combination of ACR plus gemcitabine may therefore be an effective regimen for the chemotherapy of pancreatic cancer. [Copyright &y& Elsevier]

Background/Aims: Stem cell induction via granulocyte colony-stimulating factor (G-CSF) administration is utilized in the treatment of various diseases. Therefore, we examined the effect of G-CSF administration to a liver fibrosis model induced by dimethylnitrosamine (DMN). Methods: ICR mice were subcutaneously injected with either G-CSF (150μg/kg) or saline at days 0, 3, 7 and 10. Subacute liver injury was established by intraperitoneal injection of DMN (10mg/kg) on three consecutive days of each week. Results: G-CSF administration significantly decreased the survival rate of mice treated with DMN. There was no difference in the degree of liver injury or fibrosis between either group of mice. However, assessment by proliferating cell nuclear antigen (PCNA) revealed that the G-CSF-treated mice experienced a greater degree of inhibition of liver cell proliferation than the control mice. Interleukin-1β (IL-1β) mRNA expression increased in the livers of G-CSF-treated mice. PCNA staining and analysis of cell cycle-related proteins also revealed that passive immunization with anti-IL-1β-neutralizing antibody improved the impaired hepatocellular regeneration and resulted in an improved survival rate of mice treated with G-CSF and DMN. Conclusions: G-CSF administration suppressed liver cell proliferation through the up-regulation of IL-1β expression in DMN-induced liver injury. [Copyright &y& Elsevier]