Cirsium brevicaule A. GRAY leaf inhibits adipogenesis in 3T3-L1 cells and C57BL/6 mice.

BACKGROUND: Various flavonoids obtained from the genus Cirsium have been reported to exhibit beneficial effects on health. The present study evaluated the antiobesity effects of Cirsium brevicaule A. GRAY leaf (CL) by using 3T3-L1 cells and C57BL/6 mice that were fed a high-fat diet (HFD). METHODS: Dried CL powder was serially extracted with solvents of various polarities, and these extracts were tested for antiadipogenic activity using 3T3-L1 adipocytes. Mice were fed experimental HFD supplemented with dried CL powder for 4 wk. Lipid levels and mRNA levels of genes related to lipid metabolism were determined in 3T3-L1 adipocytes and the white adipose tissue (WAT) and liver of mice fed on a HFD. RESULTS: Treatment of 3T3-L1 adipocytes with a hexane extract of CL significantly reduced cellular lipid accumulation and expression of the fatty acid synthase (FASN) gene. Dietary CL reduced the serum levels of non-esterified fatty acids in HFD-fed mice. Significant decreases in subcutaneous WAT weight and associated FASN gene expression were observed in the mice fed the experimental CL diet. Dietary CL also reduced the hepatic lipid and serum levels of a hepatopathic indicator in the HFD-fed mice. A significant reduction in mRNA levels of FASN and HMG-CoA reductase were observed in the livers of the CL-diet group. Dietary CL, on the other hand, increased in the hepatic mRNA levels of genes related to ß-oxidation, namely peroxisome proliferator-activated receptor α, calnitine palmitoyltrasferase 1A, and uncoupling protein 2. Expression of the insulin receptor gene was also significantly increased in the livers of mice-fed the CL diet. CONCLUSIONS: The present study therefore demonstrated that CL suppresses lipid accumulation in the WAT and liver partly through inhibiting mRNA levels of FASN gene and enhancing the lipolysis-related gene expression.

Adult thymus transplantation with allogeneic intra-bone marrow-bone marrow transplantation from same donor induces high thymopoiesis, mild graft-versus-host reaction and strong graft-versus-tumour effects.

Although allogeneic bone marrow transplantation (BMT) plus donor lymphocyte infusion (DLI) is performed for solid tumours to enhance graft-versus-tumour (GVT) effects, a graft-versus-host reaction (GVHR) is also elicited. We carried out intra-bone marrow-bone marrow transplantation (IBM-BMT) plus adult thymus transplantation (ATT) from the same donor to supply alloreactive T cells continually. Normal mice treated with IBM-BMT + ATT survived for a long time with high donor-derived thymopoiesis and mild GVHR. The percentage of CD4(+) FoxP3(+) regulatory T cells in the spleen of the mice treated with IBM-BMT + ATT was lower than in normal B6 mice or mice treated with IBM-BMT alone, but higher than in mice treated with IBM-BMT + DLI; the mice treated with IBM-BMT + DLI showed severe GVHR. In tumour-bearing mice, tumour growth was more strongly inhibited by IBM-BMT + ATT than by IBM-BMT alone. Mice treated with IBM-BMT + a high dose of DLI also showed tumour regression comparable to that of mice treated with IBM-BMT + ATT but died early of GVHD. By contrast, mice treated with IBM-BMT + a low dose of DLI showed longer survival but less tumour regression than the mice treated with IBM-BMT + ATT. Histologically, significant numbers of CD8(+) T cells were found to have infiltrated the tumour in the mice treated with IBM-BMT + ATT. The number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL)-positive apoptotic tumour cells also significantly increased in the mice treated with IBM-BMT + ATT. Allogeneic IBM-BMT + ATT thus can induce high thymopoiesis, preserving strong GVT effects without severe GVHR.

Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes.

UNLABELLED: During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1beta (IL-1beta) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3'-untranslated region (3'UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5-kilobase region 3'-flanking to iNOS gene exon 27 was involved in IL-1beta induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3'UTR. These results together with the data in a yeast RNA-hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The iNOS mRNA colocalized with the AU-rich element-binding protein HuR, a human homolog of embryonic lethal-abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA-binding proteins may mutually interact. CONCLUSION: The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA-RNA interaction may be a new therapeutic target for NO-mediating inflammatory diseases.

The impact of meticulous management for hepatic artery thrombosis on long-term outcome after pediatric living donor liver transplantation.

To analyze the risk factors in the development of hepatic artery thrombosis (HAT) and assess the impact of our perioperative management for HAT on the long-term outcome after pediatric living donor liver transplantation (LDLT), we reviewed 382 patients under 12 yr of age who underwent 403 LDLT from January 1996 to December 2005. One- and 10-yr patient survival rates were 78% and 78% in the patients with HAT (27 patients; 6.7%), and 84% and 76% in the patients without HAT, respectively (p = n.s.). Univariate analysis showed gender (female), body weight (lower), and graft-to-recipient weight ratio (higher) were significant risk factors in the patients with HAT (p < 0.05). Patients with Doppler ultrasound signal loss of the hepatic artery (HA) accompanied by an increase of liver enzymes underwent thrombectomy and reanastomosis (S-group, n = 13), and patients with a weak HA signal underwent anticoagulant therapy (M-group, n = 13). One patient underwent re-LDLT. One- and five-yr patient survival rates were 83% and 83% in the S-group, and 77% and 77% in the M-group (p = n.s.). The incidence of biliary complications in the S-group (58%) was significantly higher than that of the M-group (15%). For a successful long-term outcome, the early detection of HAT and prompt medical and surgical intervention are crucial to minimize the insult of HAT.

Pre-operative patient selection of pancreatic cancer patients by multi-detector row CT.

BACKGROUND/AIMS: Accurate pre-operative staging in patients with pancreatic cancer is crucial for avoiding unnecessary laparotomy and for selecting patients accurately for curative resection. In this study, tumor resectability and residual tumor grading in patients evaluated by MD-CT (Multi-detector row CT) or by SD-CT (single-detector CT) were compared to determine whether more accurate imaging has a significant clinical impact on patient selection and surgical outcomes. METHODOLOGY: One hundred-fifty consecutive patients with pancreatic cancer evaluated from January 2000 to April 2005 were included in this retrospective study. Seventy pancreatic cancer patients underwent pre-operative evaluation using SD-CT and angiography (5-7 mm slice thickness, 1st period 2000-2002), and 80 patients underwent MD-CT (1.25 mm slice thickness, 2nd period 2002-2005). RESULTS: The introduction of MD-CT had a significant impact on the selection of suitable patients, this group showing a lower frequency of surgical intervention in cases of incurable disease (p = 0.0383). Pre-operative evaluation using MD-CT in the resected cases also provided a higher percentage of accurate R0/R1 grading relative to SD-CT evaluations (p = 0.0164). CONCLUSION: MD-CT imaging has a significant impact on preventing unnecessary exploratory surgery and on the selection of appropriate pancreatic cancer patients for surgical resection.

Edaravone prevents Fas-induced fulminant hepatic failure in mice by regulating mitochondrial Bcl-xL and Bax.

Fulminant hepatic failure is a serious disease that has a poor cure rate unless liver transplantation is performed. Edaravone, a free radical scavenger, has been approved for the treatment of acute cerebral infarction, and its mechanism of action involves scavenging free radicals generated in ischemic tissues. We assessed the ability of 3-methyl-1-phenyl-2-pyrazolim-5-one (edaravone) to prevent Fas-induced acute liver failure in mice and examined the mechanisms underlying the observed effects. BALB/c mice were administered 0.25 microg/g (i.v.) body weight of a purified hamster agonist anti-Fas monoclonal antibody (clone Jo2). The mice also received either edaravone or isotonic sodium chloride solution before or after Jo2 treatment. Edaravone improved the survival rate of the mice markedly. Histopathological findings and serum aspartate aminotransferase levels showed that edaravone reduced the degree of liver injury caused by Jo2. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining showed that edaravone reduced the number of apoptotic hepatocytes. Edaravone also prevented cytochrome c release and caspase 3 activity, recognized as markers of apoptosis after mitochondrial disruption. Therefore, we considered that the antiapoptotic activity of edaravone involved blocking signals in the mitochondria-dependent pathway of Fas-induced apoptosis. Mitochondrial Bcl-xL and Bax, which form a channel in the mitochondrial membrane and, by their balance, regulate its permeability, are involved in mitochondrial disruption. Western blotting showed that the Bcl-xL-Bax ratio of the edaravone group was much higher than that of the control group. In conclusion, edaravone might protect hepatocytes from Fas-induced mitochondria-dependent apoptosis by regulating mitochondrial Bcl-xL and Bax.

Insulin-like growth factor 1 prevents liver injury through the inhibition of TNF-alpha and iNOS induction in D-galactosamine and LPS-treated rats.

Insulin-like growth factor (IGF) 1 has protective effects in liver failure. However, the effect of IGF-1 on inflammatory mediators such as proinflammatory cytokines and NO remains to be clarified. We hypothesized that IGF-1 inhibited the induction of these cytokines and iNOS, resulting in beneficial effect in the liver. Rats were treated with D-galactosamine (400 mg kg(-1)) and LPS (16 microg kg(-1)) (GalN/LPS) to induce acute liver failure. Insulin-like growth factor 1 (3.2 mg kg(-1)) was administered subcutaneously before GalN/LPS injection. Insulin-like growth factor 1 increased the survival rate in GalN/LPS-treated rats and prevented the increases of transaminases and total bilirubin in serum. Histopathological analysis revealed that IGF-1 decreased the incidence of hepatic apoptosis and neutrophil infiltration. Insulin-like growth factor 1 inhibited increases in TNF-alpha, IL-1 beta, and cytokine-induced neutrophil chemoattractant 1 caused by GalN/LPS in serum and liver and enhanced serum IL-10. Insulin-like growth factor 1 reduced the induction of iNOS mRNA and its protein in GalN/LPS-treated liver and resulted in a decrease in NO production. However, IGF-1 had no effect on the activation of nuclear factor-kappa B. Analysis of iNOS antisense-transcript revealed that IGF-1 accelerated the degradation of iNOS mRNA, rather than the inhibition of its synthesis. Insulin-like growth factor 1 may inhibit the induction of proinflammatory cytokines and iNOS through an nuclear factor-kappa B-independent pathway and have a novel therapeutic potential in the prevention of liver injury.

FKBP51 expressed by both normal epithelial cells and adenocarcinoma of colon suppresses proliferation of colorectal adenocarcinoma.

It has been reported, as a result of Western blot analyses, that FKBP51 is expressed in various tissues, but that it is not expressed in the pancreas, lung, colon, stomach, or spleen. In this paper, we show, using Western blot analyses, reverse transcriptase polymerase chain reaction, and immunohistochemical analyses of samples from colon cancer patients, that both normal epithelial cells and adenocarcinoma in the human colon express FKBP51, and that there are no significant differences in the expressions of FKBP51 between them. We also show that FKBP51 suppresses the proliferation of colorectal adenocarcinoma, possibly due to the suppression of functions of the glucocorticoid receptors.

Immunological effect of active hexose correlated compound (AHCC) in healthy volunteers: a double-blind, placebo-controlled trial.

The aim of this study was to evaluate the effects of active hexose correlated compound (AHCC) intake on immune responses by investigating the number and function of circulating dendritic cells (DCs) in healthy volunteers. Twenty-one healthy volunteers were randomized to receive placebo or AHCC at 3.0 g/day for 4 wk. The number of circulating cluster of differentiation (CD)11c(+) DCs (DC1) and CD11c(-) DCs (DC2) were measured. Allogeneic mixed-leukocyte reaction (MLR) was performed. Natural killer (NK) cell activity and the proliferative response of T lymphocytes toward mitogen (phytohemagglutinin [PHA]) were measured. We also measured cytokine production stimulated by lipopolysaccharide [interleukin (IL)-2, IL-4, IL-6, IL-10, interferon gamma-gamma, tumor necrosis factor-alpha). The AHCC group (n = 10) after AHCC intake had a significantly higher number of total DCs compared to that at baseline and values from control subjects (n = 11). The number of DC1s in the AHCC group after intake was significantly higher than at baseline. DC2s in the AHCC group were significantly increased in comparison with controls. The MLR in the AHCC group was significantly increased compared to controls. No significant differences in PHA, NK cell activity, and cytokine production were found between groups. AHCC intake resulted in the increased number of DCs and function of DC1s, which have a role in specific immunity.

Impact of fresh frozen plasma on hepatectomy for hepatocellular carcinoma.

BACKGROUND: Little is known about the effect of transfusing fresh frozen plasma on the outcome after hepatectomy for hepatocellular carcinoma. PATIENTS AND METHODS: Among 410 patients who underwent curative resection between 1992 and 2005, 180 patients had perioperative transfusion with whole blood or packed red blood cells and fresh frozen plasma (group A), while 46 patients were only transfused with packed red cells (group B), 43 patients were only transfused with fresh frozen plasma (group C) and 141 patients had no transfusion (group D). RESULTS: Group C had significantly fewer postoperative complications and a shorter hospital stay than group A. Preoperative coagulation was significantly worse in group C. Survival was significantly better in groups C and D than in group A. CONCLUSION: Perioperative transfusion of fresh frozen plasma improves clotting factors without an adverse influence on the survival of patients with liver dysfunction undergoing resection of hepatocellular carcinoma.

Risk factors for early death due to recurrence after resection of large hepatocellular carcinomas.

BACKGROUND/AIM: Long-term survival after resection of hepatocellular carcinomas larger than 5 cm in diameter is worse than after resection of smaller tumors. The risk factors for early death due to recurrence after resection of large tumors have not been clearly elucidated. METHODOLOGY: Among 377 patients who underwent curative resection of hepatocellular carcinoma between 1992 and 2004, 115 patients with tumors larger than 5 cm in diameter were enrolled. They were divided into two groups, i.e., 35 patients who died of recurrent cancer within 2 years after surgery and 80 patients who survived for more than 2 years. RESULTS: The preoperative serum alpha-fetoprotein level, positive surgical margins, number of tumors, the serum levels of albumin and alpha-fetoprotein at 1 and 3 months after surgery, as well as the prothrombin time, cholinesterase and protein induced by vitamin K antagonism-II levels at 3 months, respectively, were significant determinants of survival by univariate analysis. Multivariate analysis showed that an albumin level <3.5 g/dl at 1 month and a cholinesterase level <100 U/L at 3 months after surgery were associated with an increased risk of early death due to recurrence. CONCLUSIONS: The hepatic functional reserve in the early postoperative period significantly influences early recurrence and early death after resection of large hepatocellular carcinomas. It is important to maintain a good perioperative nutritional status and early postoperative adjuvant therapy is required for patients with large tumors.

Overproduction of PGE2 in peripheral blood monocytes of gastrointestinal cancer patients with mucins in their bloodstream.

When monocytes from healthy donors were cultured in the presence of sera from patients with gastrointestinal cancer, PGE2 production from the monocytes was elevated. Serum proteins were fractionated on Sepharose 4B and the inducing activity was found in the excluded fractions. By excluding some mucins from the serum, the inducing activity was reduced effectively. The activity was also reduced by adding binding inhibitors to the scavenger receptor. These results suggest that peripheral blood monocytes in epithelial cancer patients may be continuously stimulated by mucins in the bloodstream through the scavenger receptor, resulting in overproduction of PGE2.

Clinicopathologic study of small hepatocellular carcinoma with microscopic satellite nodules to determine the extent of tumor ablation by local therapy.

To determine the optimal treatment margin of local ablation therapy for small hepatocellular carcinoma (HCC), we investigated characteristics of microscopic satellite HCC nodules (msn) using resected livers, and the incidence of local recurrences in patients who underwent percutaneous microwave coagulation therapy (PMCT) according to whether or not an adequate treatment margin was achieved. We reviewed 117 single small HCCs (tumor size < or =3 cm ) resected with a > or =1-cm surgical margin. Among the surgically resected tumors, none of the msn were detected by preoperative imaging. When an msn was observed in the resected specimen, the maximum distance from the edge of the tumor to the msn was measured. Among the tumors sized < or =2 cm (n=66), the number having msn and the distance (mm) from the main tumor according to the degree of tumor differentiation were as follows: well (n=16), 1 (6.2%) and 1.4 mm; moderate (n=46), 5 (19.2%) and 5.8+/-1.2 mm; and poor (n=4), 1 (25.0%) and 4.8 mm. Among the tumors sized 2 to 3 cm (n=51), the corresponding results were: well (n=11), 3 (27.2%) and 3.5+/-2.2 mm; moderate (n=36), 6 (16.7%) and 5.4+/-1.4 mm; and poor (n=4), 1 (25.0%) and 4.9 mm. Of the 112 vascular tumors, 17 (15%) had msn. The 5 avascular tumors had no msn. Among the patients who underwent PMCT with a treatment margin measuring <5 mm, local recurrence occurred in 2 of 25 patients whose original tumor was < or =2 cm, and 3 of 25 (12%) patients of those with tumors of 2 to 3 cm. No patient with a tumor < or =3 cm and a > or =5-mm treatment margin suffered a local recurrence. The results of this study suggest that the incidence of local recurrence may be reduced by achieving a treatment margin of any width for avascular tumors and a margin of 1 cm for vascular tumors during local ablation therapy for small HCCs of < or =3 cm.

Immunotherapy for malignant tumors using combination of allogeneic intra-bone marrow-bone marrow transplantation, donor lymphocyte infusion and dendritic cells.

We have previously shown that the combination of allogeneic intra-bone marrow-bone marrow transplantation (IBM-BMT) and donor lymphocyte infusion (DLI) using CD4+ cell-depleted spleen cells is effective in suppressing tumor growth, but that this does not induce graft-versus-host disease (GVHD) in mice. In this report, we show that formalin-fixed tumor cell-pulsed dendritic cells (FFTCP DCs) have an additive effect with IBM-BMT plus DLI on the suppression of tumor growth, but that the DCs do not augment GVHD. BALB/c mice, which had been subcutaneously inoculated with Meth A (BALB/c-derived fibrosarcoma), were irradiated at a low dose (5 Gy) and were transplanted with bone marrow cells (BMCs) from C57BL/6 (B6) mice into the bone marrow cavity (IBM-BMT). Simultaneously, the mice were intravenously injected with spleen cells from B6 mice, and subcutaneously injected with FFTCP DCs derived from the bone marrow (BM) of B6 mice. At the point of the induction of DCs from BMCs, formalin-fixed Meth A cells were added into the culture. The mice treated with the combination of FFTCP DCs, IBM-BMT and DLI using CD4+ cell-depleted spleen cells showed smaller tumor sizes and longer survival than the mice treated with IBM-BMT plus FFTCP DCs or IBM-BMT plus DLI using CD4+ cell-depleted spleen cells. These results suggest that the combination of FFTCP DCs, IBM-BMT plus DLI using CD4+ cell-depleted spleen cells has potent anti-tumor effects without showing GVHD.

Postoperative outcomes in patients with hepatocellular carcinomas resected with exposure of the tumor surface: clinical role of the no-margin resection.

HYPOTHESIS: We hypothesized that no-margin resections for hepatocellular carcinoma do not negatively affect patient outcomes. DESIGN: Inception cohort study. SETTING: Department of surgery at a university hospital. PATIENTS: From January 1992 to December 2005 at our institute, 465 consecutive patients with a preoperative diagnosis of hepatocellular carcinoma with curative potential were evaluated. INTERVENTION: Liver resection performed with or without surgical margins. MAIN OUTCOME MEASURES: Overall survival and no-recurrence survival. RESULTS: Of the 465 patients, 62 underwent resections with exposure of the tumor surface at the cut stump (the cut surface of the remnant liver) with no surgical margins (exposure group), because the tumor adhered to the major hepatic vascular structures. The remaining 365 patients underwent resections without exposure of the tumor surface (nonexposure group). There were no significant differences between the 2 groups regarding the recurrence and overall survival rates. There were also no significant differences between the 2 groups with respect to the recurrence rate at the cut stump or the number and the location of intrahepatic recurrences, despite the less favorable clinical histories in the exposure group. CONCLUSIONS: Limited resection with no margin seems to be the best procedure for patients with tumors close to the major hepatic vessels and with hepatic functions that do not permit wide-margin resections.

Risk factors for different patterns of recurrence after resection of hepatocellular carcinoma.

BACKGROUND: Long-term survival of patients with hepatocellular carcinoma after hepatectomy is unsatisfactory because of the high recurrence rate. PATIENTS AND METHODS: Among 396 patients who underwent curative resection of hepatocellular carcinoma, there were 228 patients with clinical recurrence: 85 with solitary intrahepatic recurrence (group A), 109 with two or more intrahepatic recurrences (group B), and 34 who had extrahepatic recurrence (group C). The clinical and pathological factors for each group were investigated for association with long-term survival of each group. RESULTS: The survival rate of group C was significantly lower than that of the other groups. Patients in group C were significantly younger than those in groups A or B, with higher levels of protein induced by absence/antagonism of vitamin K-II, larger tumors, more poorly differentiated tumors, intravascular invasion and a lower incidence of cirrhosis than the other groups. Group A showed a significantly longer period until recurrence and maintained good liver function at recurrence. In group B, the survival rate of Child-Pugh class A patients was significantly higher than that of class B and C patients. Class A patients received significantly more treatments for recurrence than patients in the other classes. CONCLUSION: Postoperative adjuvant chemotherapy should be performed as early as possible after hepatectomy if the patient is younger, has a large tumor and/or has a high level of protein induced by absence/antagonism of vitamin K-II. Patients with solitary intrahepatic recurrence and adequate liver function should receive further curative therapy. It is important to maintain the postoperative nutritional status of patients with multiple intrahepatic recurrences in order to allow repeated and aggressive therapy to be performed.

Effect of thiol-containing molecule cysteamine on the induction of inducible nitric oxide synthase in hepatocytes.

BACKGROUND: Cysteamine, which is a known antioxidant and anti-inflammatory agent, is believed to be a key regulator of essential metabolic pathways in organisms. Cysteamine has beneficial effects in liver damaged by a variety of insults. During liver injury, inducible nitric oxide synthase (iNOS) is induced by lipopolysaccharide or proinflammatory cytokines, leading to excessive nitric oxide (NO) production. Accumulated evidence indicates that NO is an important factor associated with hepatic dysfunction. We examined whether cysteamine influences the induction of iNOS in hepatocytes. METHODS: Primary cultured rat hepatocytes were treated with interleukin (IL)-1beta in the presence and absence of cysteamine. NO production, iNOS induction, and iNOS signal were analyzed. RESULTS: IL-1beta stimulated the inhibitory protein kappaB (IkappaB)/nuclear factor kappaB (NFkappaB) pathway, resulting in the activation of NFkappaB (nuclear translocation and DNA binding), which was followed by the induction of iNOS and NO production. The addition of IL-1beta and cysteamine (1-4 mmol/L) markedly inhibited NO production, with a maximal effect at 4 mmol/L (80%-90% inhibition). Cysteamine also decreased the levels of iNOS protein and mRNA. Transfection experiments revealed that cysteamine decreased the transactivation activity of the iNOS promoter. An electrophoretic mobility shift assay demonstrated that cysteamine inhibited the activation of NFkappaB. Furthermore, cysteamine decreased the mRNA levels of the NFkappaB subunit p65 but increased those of the inhibitory protein IkappaB. CONCLUSIONS: These findings suggest that cysteamine inhibits iNOS induction at the step of NFkappaB activation. Further study is necessary to define the molecular basis of this effect of cysteamine on the regulation of NFkappaB and its pharmacologic implications.

Effect of active hexose correlated compound on the production of nitric oxide in hepatocytes.

BACKGROUND: Active hexose correlated compound (AHCC) is a "complex compound" containing polysaccharides. AHCC has been reported to improve the prognosis of postoperative hepatocellular carcinoma patients. However, the molecular mechanism of this improvement is not fully understood. In the diseased liver, nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) is considered to be a causal factor for various hepatopathies. In this study, the possibility of AHCC regulation of NO production by iNOS was pursued as a potential liver-protecting mechanism. METHODS: Primary cultured rat hepatocytes were treated with interleukin-1beta (IL-1beta) in the presence or absence of AHCC. NO production, iNOS induction, and iNOS signal were analyzed. RESULTS: IL-1beta stimulated iNOS induction through the activation of nuclear factor kappaB (NFkappaB), leading to NO production. The addition of AHCC inhibited NO production, showing >80% inhibition at 8 mg/mL. AHCC also decreased the levels of iNOS protein and mRNA. However, AHCC influenced neither the degradation of inhibitory protein kappaB (IkappaB) nor the activation of NFkappaB stimulated by IL-1beta. Transfection experiments with an iNOS promoter-luciferase construct (iNOS-Luc) revealed that AHCC had no effect on the transactivation activity of the iNOS promoter. By contrast, AHCC inhibited the activity of iNOS-Luc containing a 3'untranslated region (UTR) with adenosine and uridine (AU)-rich elements, which shows the stabilizing activity of iNOS mRNA. CONCLUSIONS: Results indicated that AHCC inhibits the induction of iNOS at the level of transcription, causing a decrease in NO production in hepatocytes. AHCC seems to decrease the levels of iNOS mRNA by reducing mRNA stabilization rather than inhibiting its synthesis.

Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH(2)-terminal kinase during human colorectal carcinogenesis.

Conversion of normal epithelial cells to tumors is associated with a shift in transforming growth factor-beta (TGF-beta) function: reduction of tumor suppressor activity and increase of oncogenic activity. However, specific mechanisms of this functional alteration during human colorectal carcinogenesis remain to be elucidated. TGF-beta signaling involves Smad2/3 phosphorylated at linker regions (pSmad2/3L) and COOH-terminal regions (pSmad2/3C). Using antibodies specific to each phosphorylation site, we herein showed that Smad2 and Smad3 were phosphorylated at COOH-terminal regions but not at linker regions in normal colorectal epithelial cells and that pSmad2/3C were located predominantly in their nuclei. However, the linker regions of Smad2 and Smad3 were phosphorylated in 31 sporadic colorectal adenocarcinomas. In particular, late-stage invasive and metastatic cancers typically showed a high degree of phosphorylation of Smad2/3L. Their extent of phosphorylation in 11 adenomas was intermediate between those in normal epithelial cells and adenocarcinomas. Whereas pSmad2L remained in the cytoplasm, pSmad3L was located exclusively in the nuclei of Ki-67-immunoreactive adenocarcinomas. In contrast, pSmad3C gradually decreased as the tumor stage progressed. Activated c-Jun NH(2)-terminal kinase in cancers could directly phosphorylate Smad2/3L. Although Mad homology 2 region sequencing in the Smad4 gene revealed a G/A substitution at codon 361 in one adenocarcinoma, the mutation did not correlate with phosphorylation. No mutations in the type II TGF-beta receptor and Smad2 genes were observed in the tumors. In conclusion, pSmad3C, which favors tumor suppressor activity of TGF-beta, was found to decrease, whereas c-Jun NH(2)-terminal kinase tended to induce the phosphorylation of Smad2/3L in human colorectal adenoma-carcinoma sequence.

Fibronectin prevents D-galactosamine/lipopolysaccharide-induced lethal hepatic failure in mice.

Plasma fibronectin (FN) has a broad range of biological functions involved in cellular adhesion, motility, differentiation, apoptosis, hemostasis, wound healing, reticuloendothelial system function, and ischemic injury. In this study, we examined the effects of FN on D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant liver failure in mice. Female Balb/c mice received intraperitoneal injection of 50 mug/kg of LPS and 400 mg/kg of GalN simultaneously. Thirty minutes before GalN/LPS administration, human plasma FN (FN group) or the same dose of human serum albumin (control group) was given intravenously. GalN/LPS induced a marked decrease in plasma FN, which was reversed by FN pretreatment. The survival rate of the FN group was markedly improved in a dose-dependent manner compared with that of the control group (survival rate 0%). FN prevented increases in the concentrations of serum enzymes and total bilirubin related to liver injury. FN pretreatment significantly suppressed tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6 levels, and enhanced IL-10 levels in serum and liver tissue compared with the control group. Moreover, TUNEL staining, caspase 3 and 8 activities, and necrosis in the remnant liver were significantly decreased in the FN-treated rats compared with the controls. Furthermore, FN pretreatment inhibited the activation of nuclear factor (NF)-kappaB and increased the expression of Bcl-xL protein in liver tissue. These results suggest that FN protected against GalN/LPS-induced liver failure by a mechanism involving inhibition of NF-kappaB activation, which caused down-regulation of TNF-alpha and involved up-regulation of IL-10, and elevation of Bcl-xL induced a blockage of apoptotic signals, by which apoptosis of hepatocytes caused by GalN/LPS was suppressed.