Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling.

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.

BCAA-enriched snack improves nutritional state of cirrhosis.

OBJECTIVE: A late evening snack improves the catabolic state in patients with advanced liver cirrhosis. We tested whether long-term (3 mo) late evening snacking that included a branched-chain amino acid (BCAA)-enriched nutrient mixture produces a better nutritional state and better quality of life than ordinary food in patients with hepatitis C virus-positive liver cirrhosis. METHODS: In a multicenter, randomized study, 48 patients with liver cirrhosis received late-evening supplementation with the BCAA-enriched nutrient mixture or ordinary food, such as a rice ball or bread, for 3 mo. During the study period, each patient was instructed on energy and protein intake. Blood biochemical data, nitrogen balance, respiratory quotient, and health-related quality of life (Short Form 36 questionnaire) were evaluated at baseline and at the end of the study. RESULTS: Total and late-evening energy intakes were similar in the two groups at 3 mo. Serum albumin level, nitrogen balance, and respiratory quotient were significantly improved by the BCAA mixture but not by ordinary food. The parameters of the Short Form 36 did not statistically significantly improve over 3 mo in either group. CONCLUSION: Long-term oral supplementation with a BCAA mixture is better than ordinary food in a late evening snack at improving the serum albumin level and the energy metabolism in patients with cirrhosis.

Overweight and obesity increase the risk for liver cancer in patients with liver cirrhosis and long-term oral supplementation with branched-chain amino acid granules inhibits liver carcinogenesis in heavier patients with liver cirrhosis.

We conducted a multicenter, randomized, controlled trial to investigate the effect of long-term oral supplementation with branched-chain amino acids (BCAA) on the event-free survival in 622 patients with decompensated cirrhosis. In the present study, the development of liver cancer was analyzed as an endpoint in particular. Subjects received either treatment with BCAA at 12g/day or dietary therapy containing the matched daily energy and protein intake. A Cox regression analysis was carried out to estimate the hazard ratios for different background factors stratified by treatment group. Liver cancer was noted in 89 patients. The risk for liver cancer was significantly higher for males, patients with concurrent diabetes mellitus, patients with an alpha-fetoprotein (AFP) level of 20ng/mL or higher, patients with higher body mass index (BMI), and patients with lower serum albumin levels. When the BCAA group and the diet group were compared for factors that interacted with the treatment arms, the risk for liver cancer was significantly reduced in the BCAA group with a BMI of 25 or higher and with an AFP level of 20ng/mL or higher. Oral supplemental treatment with BCAA may reduce the risk of liver cancer in cirrhotic patients with these specific factors.

Fas-associated phosphatase-1 promotes Fas-mediated apoptosis in human colon cancer cells: novel function of FAP-1.

BACKGROUND AND AIM: Fas-associated phosphatase-1 (FAP-1) has been thought as an inhibitor in Fas-mediated apoptosis. Here, we investigated the role of FAP-1 in Fas-mediated apoptosis of human colon cancer cells. METHOD: The viability of four colon cancer cell lines treated with agonistic anti-Fas antibody was determined using WST-1 assay and cell death detection ELISA. pRc/CMV-FAP-1 was transfected to a FAP-1-negative, Fas-resistant colon cancer cell line SW480 by lipofection and the clones expressing FAP-1 protein were selected by limiting dilution. In the clones, expression of 550 genes was analyzed by cDNA microarrays. Protein expression of FAP-1 and molecules related to apoptosis was examined by western blot. RESULTS: We obtained two FAP-1 overexpressed clones which were much more susceptible to Fas-mediated apoptosis than control cells. In the clones, caspase 8 and caspase 3 were fully activated by agonistic anti-Fas antibody treatment. Bcl-2 family proteins were not related to the high susceptibility of these clones, because caspase 9 was not activated. Transfection of FAP-1 did not suppress the survival actions of insulin-like growth factor (IGF-1) which enhanced survival signal through Akt phosphorylation. Upregulation in 21 genes and downregulation in 29 genes was revealed by cDNA arrays. We confirmed protein expression of p21 and phosphorylated p21 were much more enhanced in the clones than in control cells. CONCLUSIONS: Overexpression of FAP-1 enhanced susceptibility to Fas-mediated apoptosis in SW480 and upregulation of p21 may contribute to this phenomenon. Our results indicate a novel function of FAP-1 in Fas-mediated apoptosis of human colon cancer cells.

Effects of oral branched-chain amino acid granules on event-free survival in patients with liver cirrhosis.

BACKGROUND & AIMS: Nutritional intervention with branched-chain amino acid (BCAA) is reported to increase serum albumin concentration in patients with decompensated cirrhosis. However, a definite conclusion on whether it can improve patients' survival has not yet been reached. The present study aimed to test possibilities of improving survival of patients with decompensated cirrhosis by using a BCAA preparation that is suitable for long-term oral administration. METHODS: A multicenter, randomized, and nutrient intake-controlled trial on the comparative effects of BCAA orally administered at 12 g/day for 2 years versus diet therapy with defined daily food intake (1.0-1.4 g protein kg(-1) day(-1) including BCAA preparation and 25-35 kcal kg(-1) day(-1)) was conducted in 646 patients with decompensated cirrhosis. The primary end point was a composite of death by any cause, development of liver cancer, rupture of esophageal varices, or progress of hepatic failure (event-free survival). The secondary end points were serum albumin concentration and health-related quality of life (QOL) measured by Short Form-36 questionnaire. RESULTS: The incidence of events comprising the primary end point significantly decreased in the BCAA group as compared with the diet group (hazard ratio, 0.67; 95% confidence interval, 0.49-0.93; P = .015; median observation period, 445 days). Serum albumin concentration increased significantly in the BCAA group as compared with the diet group (P = .018). The "general health perception" domain in Short Form-36 measures was also improved (P = .003). Patients' adherence to the prescription was favorable. CONCLUSIONS: Oral supplementation with a BCAA preparation that can be administered for a long period improves event-free survival, serum albumin concentration, and QOL in patients with decompensated cirrhosis with an adequate daily food intake.

CTLA-4Ig suppresses liver injury by inhibiting acquired immune responses in a mouse model of fulminant hepatitis.

Expression of costimulatory molecules is significantly upregulated in various organs in an animal model of severe hepatitis induced by injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). In the present study, we examined whether blockade of costimulatory signals by CTLA-4Ig can suppress the liver injury in this model. We injected an adenovirus encoding CTLA-4Ig (AdCTLA-4Ig) into mice 7 days before, on the same day, or 3 days after P. acnes priming. The virus was found to infect the liver preferentially, and CTLA-4Ig was detected in the serum as early as 2 days after viral injection. After injection of LPS, liver injury and survival rates were examined. Most of the mice not injected with AdCTLA-4Ig died within 12 hours after injection of LPS. In contrast, all the AdCTLA-4Ig-injected mice survived when the virus was injected 7 days before or on the same day as P. acnes priming. Importantly, hemorrhagic liver injury and serum alanine aminotransferase levels were significantly reduced after LPS injection even when AdCTLA-4Ig was injected 3 days after P. acnes priming. Immunological analyses showed that CTLA-4Ig inhibited the activation and expansion of P. acnes-specific CD4+ T cells in the hepatic lymph nodes, leading to a reduction in the recruitment of the cells to the liver. The total amounts of interferon-gamma, interleukin-12, and various chemokines in the liver were then decreased, resulting in inhibition of the secondary recruitment of not only T cells but also macrophages. In conclusion, CTLA-4Ig could be useful for treatment of severe liver injury.

Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model.

Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34(+/-) cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34(+), or CD34(-) cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of alpha-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34(+) and CD34(-) cells were not observed in human hepatocyte-specific expression.

Functional analysis of transactivation by mutants of hepatitis B virus X gene in human hepatocellular carcinoma.

The X protein of the hepatitis B virus transactivates various cellular and viral promoters and enhancers. In this study, wild-type and mutants of the X gene, including the point mutations at codons 130 (AAG-->ATG, lysin-->methionine) and 131 (GTC-->ATC, valine-->isoleucine), commonly found in patients with human hepatocellular carcinoma (HCC), or deletions encompassing the same region, were cloned and inserted into expression vectors. Functional analysis of the mutants of the X gene was performed on the long terminal repeat of the Rous sarcoma virus in a transient transfection assay. A transactivating function was observed in the vector containing point mutations at codons 130 and 131 at the same level as that of wild-type X gene. Two constructs, each containing a different type of 8-nucleotide deletion mutant (codons 128-130 or 130-132,) dramatically lost their transactivating function. These findings suggest that the transactivating function is not necessarily associated with the development of HCC, and that not only transactivation by the X gene but also the mutation-enhanced oncogenic potential of the gene products could contribute to hepatocarcinogenesis.

Suppression of invasion of a hamster pancreatic cancer cell line by antisense oligonucleotides mutation-matched to K-ras gene.

The anti-invasive activity of antisense oligonucleotides (ASO) specific to the K-ras gene in hamster pancreatic cancer was investigated. HaP-T1, a cell culture derived from BHP-induced hamster pancreatic cancer, was used. After liposome-mediated transfection with mutation-matched and mutation-mismatched ASO in different concentrations, cell proliferation was studied by MTT and MTT-agarose methods. In vitro chemoinvasion assay with the reconstitution of a matrix of a basement membrane onto a filter in a Boyden chamber was performed. Mutation-matched ASO inhibited the tumor growth and invasiveness of HaP-T1 in a dose-dependent manner, while mutation-mismatched ASO were not effective in inhibiting invasion. The present study suggests that antisense oligonucleotides mutation-matched to the K-ras gene may be a new anticancer strategy for pancreatic cancer since they inhibited not only tumor growth but also invasiveness in vitro.

Different pattern of metastasis in liver implanted pancreatic cancer between young and old Syrian golden hamsters.

We have previously reported on the "return trip" metastases from the liver to the pancreas in a hamster experimental pancreatic cancer model. Because the pancreas is the main metastatic site of liver-implanted pancreatic tumors, our aim was to clarify whether the metastatic sites differ in young and old tumor-bearing animals. HaP-T1, a continuous tissue-cultured cell line, derived from BHP-induced pancreatic adenocarcinoma, was implanted into the liver. The animals were divided into two groups: A) younger than 26 weeks and B) older than 26 weeks. Three animals from each group were sacrificed on Days 35, 42, 49, 56, 63, 70, 77, 84, 91 and 98, to study the metastatic sites. Survival was also studied. After death, necropsy was performed. Resected and necropsied specimens were analyzed histopathologically and by PCR/RFLP analysis to confirm the presence of K-ras point mutation. The success rate of implantation was 100%. Survival was 102.3+/-2.5 days in group A and 95.3+/-1.5 days in group B. Animals of group A, sacrificed weekly until Day 70, showed metastases only to the pancreas ("return trip"), while this phenomenon happened only in animals sacrificed on Day 35 in group B. In group A, on Days 77, 84, 91 and 98, metastases were also found in the kidneys, lymph nodes, ovary and testis. In hamsters of group B, metastases were found in multiple sites such as the pancreas, vas deferens, ovary and testis ("multiple journeys"). All intra-hepatically-implanted tumor and metastatic sites showed the K-ras point mutation. This homologous implantation model may be helpful for further research into the process of metastasis and its relationship with the immunological response.

Clinical comparison of branched-chain amino acid (l-Leucine, l-Isoleucine, l-Valine) granules and oral nutrition for hepatic insufficiency in patients with decompensated liver cirrhosis (LIV-EN study).

This multicenter study compared the effects of branched-chain amino acid granules (Livact((R)) Granules, LIV) and an enteral nutrient for chronic hepatic failure (Aminoleban((R)) EN, EN) on serum albumin in patients with decompensated liver cirrhosis. This study enrolled "patients with decompensated liver cirrhosis associated with hepatic encephalopathy who were suffering from hypoalbuminemia in spite of adequate food intake," a condition for which both drugs are indicated. Enrolled patients were randomized to the two groups according to the central registration method. This study continued for 24 weeks. Selected foods were supplied to each patient in principle so that caloric and protein intakes were standardized between the two groups. A total of 281 patients were enrolled. LIV was not inferior to EN concerning the primary efficacy endpoint changes in serum albumin.

Development of quantitative neuropsychological tests for diagnosis of subclinical hepatic encephalopathy in liver cirrhosis patients and establishment of diagnostic criteria-multicenter collaborative study in Japanese.

At present, there are no generally accepted diagnostic criteria or methods for subclinical hepatic encephalopathy (SHE) associated with liver cirrhosis. We therefore developed an easily conducted computer-aided quantitative neuropsychiatric function test system for use in routine medical practice. We established normal values in healthy Japanese subjects and determined differences between healthy persons and liver cirrhosis patients without clinical encephalopathy in a multi-center clinical trial. The test system consists of eight tests: number connection tests A and B, a figure position test, a digit symbol test, a block design test, and reaction time tests A, B and C. The test results were affected by age, but not by gender or facility. No learning effect was noted. The results were therefore reported by 5-year quartile ranges and differences were evaluated between 542 healthy subjects and 292 cirrhotic patients. When the cut-off value was set at the 10th/90th percentile of the results in healthy subjects, the results of each of the 8 tests were abnormal in about 25% of cirrhotic patients, and at least 1 of the 8 tests gave values greater than the 10th/90th percentile cut-off value in 58.2% of the 292 liver cirrhosis patients. SHE patients were thought to be included in these 58.2% of patients. The developed test makes it possible to quantitatively assess neuropsychiatric function, and the results obtained can be used as a basis for the diagnosis of SHE.

Antisense therapy specific to mutated K-ras gene in hamster pancreatic cancer model. Can it inhibit the growth of 5-FU and MMC-resistant metastatic and remetastatic cell lines?

UNLABELLED: K-ras point mutation at codon 12 has a relationship greater than 90% with pancreatic cancer. Cancer therapy should also include the treatment of metastatic disease because it is known that the properties of metastatic cells may vary considerably from those of the primary tumor. AIM: To clarify if the same drugs, which can inhibit the tumor growth in the parental cell line, can inhibit the pancreatic metastatic and remetastatic cell lines at the same concentrations and to compare the inhibition with antisense oligonucleotides mismatched to K-ras gene, in Syrian golden hamsters. MATERIALS AND METHODS: HaP-T1, a BHP-induced hamster pancreatic cancer cell line, MS-PaS-1 (a metastatic cell line established from "return trip" metastases from the liver to the pancreas) and MS-PaS-2 named as a "remetastatic cell line", i.e., metastases from MS-PaS-1 were used. MTT and MTT-agarose assays were performed, using 5-Fluorouracil (5-FU), Mitomycin C (MMC) and antisense oligonucleotide specific to K-ras oncogene. RESULTS: The inhibitory concentration (IC50) of 5-FU, which inhibited HaP-T1, had to be increased by 50-fold to inhibit MS-PaS-1 and 100-fold to inhibit MS-PaS-2. MMC had to be increased by 10-fold to inhibit MS-PaS-1 and 50-fold to inhibit MS-PaS-2. However, IC50 was the same when antisense oligonucleotide was tried in these 3 cell lines. CONCLUSION: Antisense oligonucleotide-targeted K-ras gene may be a good choice for therapy because it could inhibit the growth in metastatic and remetastatic cells as well as in primary tumor cells.

Problems in serum albumin measurement and clinical significance of albumin microheterogeneity in cirrhotics.

OBJECTIVES: To clarify problems with the determination of serum albumin levels, the definition of hypoalbuminemia, and the implications of microheterogeneity of albumin, serum albumin was measured by using dye-binding methods and the authentic method (immunoassay) in patients with liver cirrhosis and healthy subjects. METHODS: We enrolled 103 patients with liver cirrhosis and 36 healthy subjects. Serum albumin levels were analyzed by immunoassay and the bromcresol green and bromcresol purple methods. Oxidized albumin and glycoalbumin were determined by high-performance liquid chromatography. RESULTS: In cirrhotic patients, serum albumin levels measured by the bromcresol green method was about 0.2 g/dL higher than that by immunoassay. Serum albumin levels measured by the bromcresol purple method also was higher in cirrhotic patients than those measured by immunoassay and varied widely. In addition, extensive variation was found across serum albumin levels determined by the bromcresol green method at individual institutions (five university hospitals) and those determined by immunoassay at a contract laboratory. The percentages of oxidized albumin and glycoalbumin within total serum albumin increased with progression of liver disease. Further, an increase in oxidized albumin led to an increase in the albumin level as measured by the bromcresol purple method. CONCLUSION: These results show that adequate assessment of the pathophysiology and prognosis of patients with liver cirrhosis and the efficacy of treatment is not possible with dye-binding methods for determination of serum albumin. Further, the conventional definition of hypoalbuminemia as a serum albumin level of 3.5 g/dL or lower should be reconsidered, and the clinical implications of qualitative changes in albumin should be investigated in consideration of the microheterogeneity of albumin, such as oxidized albumin and glycoalbumin.

Cotreatment with interferon-alpha and -gamma reduces liver fibrosis in a rat model.

Background/Aims: Interferon-alpha is used widely to treat viral hepatitis. Interferon-gamma modulates a system attacking infected cells and also has an anti-fibrotic effect. A treatment with interferon-alpha and -gamma has undergone trials in eliminating hepatitis C virus. We investigated effects of cotreatment in a liver fibrosis model to explore anti-fibrotic effects. Methods: Rats were assigned to groups including normal controls (NC), CCl(4) controls, rat interferon-alpha treatment, rat interferon-gamma treatment, and cotreatment. All groups except normal controls received CCl(4) orally for 8 weeks. At the beginning of the third week of exposure, 6 weeks of treatment were initiated according to interferon group. Digitally analyzed immunohistochemistry, biochemical assays, and Northern analysis were performed. Results: Pixels (x10(5)) per field containing immunoreactive type III collagen (fibrotic density) in CCl(4) controls, interferon-alpha, interferon-gamma, and cotreatment groups respectively were [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text]. Liver hydroxyproline content correlated with fibrotic density, and was significantly low in the cotreatment group. Plasma hyaluronate and transaminase were significantly low in cotreatment and interferon-alpha groups. Northern blotting showed lowest mRNA expression for type I collagen, desmin, transforming growth factor (TGF)-beta1, and matrix metalloproteinase-2 mRNA in the cotreatment group; tissue inhibitor of metalloproteinase-1 and -2 mRNAs were significantly low in the interferon-gamma group. Conclusions: Cotreatment can suppress collagen and transforming growth factor-beta1 and has an overall anti-fibrotic effect without exacerbating inflammation.

Comparison of metastatic behavior of parental and metastatic pancreatic cancer cell lines in Syrian golden hamsters.

The major cause of death in patients with pancreatic cancer is metastatic disease. In fact, at the time of diagnosis, patients usually have locally advanced or metastatic disease involving lymph nodes, liver, lungs or peritoneum. Therefore, for a better understanding of the tumoral behavior to design prevention or treatment strategies, in vivo models are important. We report here the results of the metastatic behavior of parental and metastatic cell lines in a hamster pancreatic cancer model when implanted orthotopically (OIH,OIM) or into the liver (LIH,LIM). Metastatic sites and survival were studied. Survival ranged from 72 to 105 days. The OIH group showed spontaneous metastases to lymph nodes. The second target organ was the lung. Liver metastases appeared earlier in the OIM group than OIH. LIH showed only metastases to the pancreas while the LIM group showed metastases to pancreas, vas deferens and testis. This study suggests that the metastatic behavior of parental and metastatic cell lines is different. Thus, this should be considered in the planning of clinical or surgical treatment against pancreatic cancer.

Sodium butyrate enhances Fas-mediated apoptosis of human hepatoma cells.

BACKGROUND/AIMS: Human hepatoma cells have been reported to be resistant to Fas-mediated apoptosis. Sodium butyrate (SB) induced apoptosis of several cancer cells. We investigated the effects of SB on Fas-mediated apoptosis of hepatoma cells. METHODS: In hepatoma cells (HuH-6, HuH-7, Hep-G2, and PLC/PRF/5), susceptibility to Fas-mediated apoptosis and Fas expression were assessed. Caspase-3 activation and cell cycle progression were evaluated in HuH-6. A cDNA microarray assay was performed to screen the changes in the expression of mRNAs. RESULTS: Pretreatment with SB caused an enhancement of the sensitivity to anti-Fas-mediated cytotoxicity, though it did not increase the expression of Fas. The cDNA microarray assay revealed up-regulation of pro-apoptotic Bik, Bak, Bid and c-Jun N-terminal protein kinase-1, and down-regulation of anti-apoptotic Bag-1 and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitor protein. In some molecules, expression of the proteins was confirmed by Western blotting. An increase in truncated-Bid accompanying the reduction in Bid was also observed. CONCLUSIONS: SB enhances the susceptibility of hepatoma cells to anti-Fas-mediated cytotoxicity by altering the mRNA and protein expression and/or the activation status of proteins that could be involved in the Fas signaling pathway. SB may have an important role in the elimination of hepatoma cells.

A case of small cell gastric carcinoma with an adenocarcinoma component operated curatively.

We present a case of a primary advanced gastric tumor that was composed of 2 different pathological components: small cell carcinoma and moderately-differentiated adenocarcinoma. The patient was still alive four years after the surgery was performed, without recurrence. A large part of the tumor consisted of a diffuse sheet of small cell carcinoma, which transitioned into another small portion consisting of moderately-differentiated tubular adenocarcinoma components. Therefore, this case raised the possibility that small cell gastric carcinoma may originate from totipotential stem cells of the stomach. Although small cell carcinoma progresses aggressively, and patients with it have an extremely poor prognosis, this patient recovered uneventfully after the surgical resection, and has remained in good health, without any recurrences.