Agrocybe aegerita polysaccharide combined with chemotherapy improves tumor necrosis factor‐α and interferon‐γ levels in rat esophageal carcinoma

Natural compounds have generated great interest as alternative treatments of diseases like cancer. Here, we investigated the anti‐tumor mechanism of one such compound, Agrocybe aegerita polysaccharide, by assessing expression of tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) in rat esophageal carcinoma (EC). EC was induced in healthy Wistar rats by methyl‐n‐amyl nitrosamine. Subsequently, rats were administered cancer treatment daily for 4 weeks, as follows: the normal control group (the only group not treated with methyl‐n‐amyl nitrosamine) and model group received only distilled water; the chemotherapy group received tegafur treatment; and the combination group received tegafur combined with A. aegerita polysaccharide. In normal and combination groups, body weight increased gradually after each week of treatment (P < 0.05), while body weights did not change in model and chemotherapy groups. Using enzyme linked immunosorbent assay, we found serum TNF‐α was lower in the combination group (31.56 ± 7.20 pg/L) than either the model (46.24 ± 8.52 pg/L) or chemotherapy (52.39 ± 9.16 pg/L) group, and, while higher, was more similar to the normal controls (25.08 ± 2.93 pg/L; P < 0.05), a finding that was confirmed by the immunohistochemistry of esophageal samples. In contrast, serum IFN‐γ was higher in the combination group (97.20 ± 10.92 pg/L) than in either the model (76.11 ± 11.92 pg/L) or chemotherapy (76.04 ± 9.85 pg/L) group, but lower than in the normal group (117.56 ± 10.88; P < 0.05), also confirmed by immunohistochemistry. Therefore, Agrocybe aegerita polysaccharide, when combined with chemotherapy, can regulate immune function in EC, potentially by modulating cytokine activity, specifically downregulation of TNF‐α and upregulation of IFN‐γ.     

Effect of interferon α, γ, and tumor necrosis factor α on the HLA-A,B, C expression of cell lines derived from human liver

ABSTRACT— Our study was undertaken to determine whether human recombinant interferon α(rIFNα), γ(rIFNγ), and tumor necrosis factor α(rTNFα) exert an effect on the HLA-A, B, C expression of human liver cell lines. The HLA-A, B, C expression was assayed by immunoperoxidase staining and enzyme-linked immunosorbent assay. rIFNα and γ enhanced the HLA-A, B, C expression of the three cell lines tested, Chang cells, SK-Hep-1, and PLC/PRF/5. The activity of rIFNγ proved more than 8000 times more potent than that of rIFNα in Chang cells, 30 times in SK-Hep-1, and 20 times in PLC/PRF/5, respectively. rTNFα also enhanced the HLA-A, B, C expression of the three cell lines. The enhancement of HLA-A, B, C expression by rIFNα and γ reached a peak on day 3, and that by rTNFα on day 5. These findings suggest that IFNα, IFNγ, and TNFα may play similar roles in enhancement of HLA-A, B, C expression of hepatocytes in hepatitis and hepatoma cells.     

Recent topics on α‐fetoprotein

Zinc‐fingers and homeoboxes 2 (ZHX2) and zinc‐finger and BTB domain containing 20 (ZBTB20) repress the postnatal expression of α‐fetoprotein (AFP) by interacting with the AFP gene promoter regions. ZHX2 inhibits the expression of AFP and cyclins A and E. ZBTB20 is negatively regulated by CUX1, which promotes cell‐cycle progression, suggesting that AFP reactivation is closely linked to hepatocyte proliferation. A slight elevation in the serum AFP level often occurs in patients with chronic hepatitis C in the absence of hepatocellular carcinoma (HCC) and is an independent risk factor for HCC development to complement the fibrosis stage. In addition, the sustained elevation of AFP after interferon therapy is a risk factor of HCC development. AFP levels are clinically useful in predicting the outcomes of liver transplantation and sorafenib therapy for HCC patients. A low preoperative AFP level is a predictor of long‐term survival and is associated with a low recurrence rate of HCC after liver transplantation. AFP response (≥20% decrease in AFP during 6–8 weeks of treatment) rather than radiological outcomes is a significant prognostic factor for survival in sorafenib‐treated HCC patients. Highly sensitive Lens culinaris agglutinin‐reactive AFP (AFP‐L3) is 5–10 times more sensitive than conventional AFP‐L3, and useful for early detection of HCC in patients with total AFP below 20 ng/mL.     

Effect of peroxisome proliferator‐activated receptors‐γ and co‐activator‐1α genetic polymorphisms on plasma adiponectin levels and susceptibility of non‐alcoholic fatty liver disease in Chinese people

Background/Aims: Peroxisome proliferator‐activated receptors‐γ (PPAR‐γ) and its co‐activator‐1α (PGC‐1α) are involved in the regulation of lipid and glucose metabolisms. This study aimed to investigate the genetic polymorphisms of PPAR‐γ and PGC‐1α in Chinese people and their influence on plasma adiponectin levels and non‐alcoholic fatty liver disease (NAFLD) susceptibility. Methods: Ninety‐six patients with NAFLD and 96 healthy controls were included. The single nucleotide polymorphisms (SNPs) of C161T PPAR‐γand Gly482Ser PGC‐1α genes were analysed by polymerase chain reaction and restriction fragment length polymorphism. Result: The CC, CT and TT genotypic distributions of the NAFLD group were significantly different from those of controls (55.2, 39.6, 5.2 vs. 74.0, 25.0, 1.0%; P=0.015). The allelic frequencies of C and T were also different between the two groups (P=0.004). As for the PGC‐1α gene, there was no difference of the genotypic and allelic frequencies between the two groups (P>0.05). In NAFLD patients, the plasma adiponectin concentrations were lower in the PPAR‐γ CT/TT genotypes compared with those in the CC genotype group (3.0±0.6 vs. 4.3±0.9, P=0.02). Multivariate logistic regression analysis showed that CT/TT genotypes of PPAR‐γ, TG, waist hip ratio, hypoadiponectinaemia and homoeostasis model assessment (HOMA)‐IR were the risk factors for NAFLD. Conclusion: SNPs in the PPAR‐γ, but not PGC‐1α, gene are associated with NAFLD susceptibility possibly through the adiponectin pathway.     

Effect of mutant β‐catenin on liver growth homeostasis and hepatocarcinogenesis in transgenic mice

Background: Mutations in the Wnt signalling pathway molecule β‐catenin are associated with liver cancer. Aims: Our aim was to confirm the effects of stabilized β‐catenin on liver growth, identify whether those effects were reversible and cell autonomous or non‐cell autonomous and to model β‐catenin‐induced liver cancer in mice. Methods: Using a liver‐specific inducible promoter, we generated transgenic mice in which the expression of mutant β‐catenin can be induced or repressed within hepatocytes in mice of different ages. Results: Similar to other models, the hepatic expression of mutant β‐catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two‐fold liver enlargement and development of disease with a latency of 1–5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β‐catenin could not increase the growth of transgene‐expressing foci in either growth‐permissive or ‐restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. Conclusions: Our findings indicate that hepatocyte gene expression is directly affected by mutant β‐catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte‐specific expression of mutant β‐catenin is secondary to liver functional alteration or non‐cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene‐expressing cells progressed to carcinoma, confirming the association of mutant β‐catenin with liver cancer.     

Effects of a late evening snack combined with α‐glucosidase inhibitor on liver cirrhosis

Aim: A late evening snack (LES) is recommended for protein‐energy malnutrition in patients with liver cirrhosis. However, many cases of liver cirrhosis have accompanying impaired glucose tolerance and there are concerns that LESs might aggravate glucose intolerance. In this study, we concomitantly used an α‐glucosidase inhibitor with a LES and examined the effects on glucose tolerance. In addition, we examined whether or not there was an improvement in energy metabolism by slowing glucose absorption with the concomitant use of the α‐glucosidase inhibitor. Methods: The subjects were 11 patients with liver cirrhosis. From before the study, all the patients had been taking a LES supplementation with a branched‐chain amino acid (BCAA)‐enriched nutrient mixture. The patients were started on the concomitant use of α‐glucosidase inhibitor (0.2 mg) taken just prior to the LES. The change of glucose tolerance and energy metabolism were examined using a 75‐g oral glucose tolerance test and indirect calorimetry. Results: One week and three months after the start of the concomitant use of the α‐glucosidase inhibitor, the area under the concentration curve for plasma glucose was significantly decreased. Three months after the concomitant use, the non‐protein respiratory quotient was significantly improved. There were no serious side effects during the follow‐ups. Conclusion: The concomitant use of the α‐glucosidase inhibitor use with LES showed the possibility of improving glucose tolerance and energy metabolism. In patients with impaired glucose tolerance, the concomitant use of an α‐glucosidase inhibitor with LES might be a useful measure for nutritional management.     

Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon‐γ and tumour necrosis factor‐α

Background: Interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were thought to be important immune mediators in host defence against hepatitis B virus (HBV) infection. Aims: To examine the synergistic effect of IFN‐γ and TNF‐α on HBV‐expressing HepG2.2.15 cells and its potential mechanisms. Methods: Cell viability was quantitatively measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay. Cell morphology was captured using light microscopy. The typical DNA ladder test was performed using agarose gel electrophoresis. HBsAg and HBeAg titre changes were quantified by the enzyme‐linked immunosorbent assay method. Gene expression was analysed using cDNA macroarrays. Results: Interferon‐γ (1000 U/ml) alone or combined with TNF‐α (5 ng/ml) treatment resulted in apoptosis in HepG2.2.15 cells, but no significant apoptosis in the parent non‐virus expressing HepG2 cells. IFN‐γ‐ and TNF‐α‐mediated apoptosis was reduced by lamivudine treatment in HepG2.2.15 cells. IFN‐γ combined with TNF‐α reduced the titre of hepatitis B surface antigen and hepatitis B e antigen in the HepG2.2.15 cell line. For apoptosis‐related gene changes, IFN regulatory factor 1 (IRF‐1) (12.2‐fold), c‐myc (V00568 4.7‐fold, L00058 2.4‐fold) and caspase 7 (2.3‐fold) genes were upregulated in the combination treatment group. Conclusion: Interferon‐γ and TNF‐α play a role in the cell death of HBV‐expressing HepG2.2.15 cells. Expression of HBV leads to IFN‐γ‐ and TNF‐α‐mediated apoptosis in the cells. Increased IRF‐1, c‐myc and caspase 7 gene expression may be responsible for the synergistic induction of apoptosis by IFN‐γ and TNF‐α.     

Antisense integrin αV and β3 gene therapy suppresses subcutaneously implanted hepatocellular carcinomas

BACKGROUND: Integrin alphaVbeta3 plays a critical role in tumour angiogenesis and metastasis formation, and is recognized as a key therapeutic target in the treatment of cancer. AIM: To investigate whether antisense alphaV and beta3 gene therapy has utility in the treatment of hepatocellular carcinomas. METHODS: Antisense expression plasmids targeting integrin alphaV or beta3 were constructed, and examined by immunohistochemistry and Western blot analyses for their ability to inhibit alphaV and beta3 expression. The antisense alphaV and beta3 expression vectors, either alone or in combination, were injected into HepG2 hepatomas established subcutaneously in nude mice and tumour growth, angiogenesis and apoptosis were monitored. RESULTS: Antisense alphaV and beta3 downregulated the alphaV and beta3 subunits expressed by human umbilical vein endothelial cells, and the alphaV subunit expressed by HepG2 cells. Gene transfer of antisense alphaV and beta3 expression vectors downregulated alphaV and beta3 in HepG2 tumours established in nude mice, inhibited tumour vascularization and growth, and enhanced tumour cell apoptosis. Antisense alphaV suppressed tumour growth more strongly than antisense beta3; however antisense therapy that simultaneously targeted both integrin subunits was more effective than the respective monotherapies. Antisense alphaV and beta3 inhibited tumour angiogenesis to similar extents, by a process that is independent of vascular endothelial growth factor. CONCLUSIONS: Antisense gene therapy targeting alphaV integrins warrants consideration as an approach to treat hepatocellular carcinomas.     

Characterization of elevated alanine aminotransferase levels during pegylated‐interferon α‐2b plus ribavirin treatment for chronic hepatitis C

Aim: Elevation of alanine aminotransferase (ALT) levels during pegylated‐interferon (peg‐IFN) plus ribavirin therapy in patients with chronic hepatitis C [CHC] is a problem that cannot be disregarded. The aim of this study is to assess the frequency and to characterize clinical parameters of this phenomenon. Methods: Two hundred and thirty‐five (235) CHC patients with genotype 1b receiving peg‐IFN α‐2b plus ribavirin therapy were analyzed. Clinical parameters that may be associated with abnormal ALT values during treatment and therapy outcomes were evaluated statistically. One hundred and sixteen (116) patients treated with peg‐IFN α‐2a plus ribavirin were also included for partial analysis. Results: Abnormal ALT values during treatment were observed in 23.0% of patients. It was observed in 14.5% of those with sustained virological response (SVR) and 17.8% of those with relapse, in whom viral clearance was observed during therapy. Multivariate logistic regression analysis revealed that pretreatment ALT values, therapy outcome, and body mass index (BMI) were significant factors related to abnormal ALT values during treatment. Abnormal ALT values during treatment became normal in SVR patients at 6 months after the completion of treatment, but not in NR (non‐response) patients. Mean ALT values were significantly higher at some time points during treatment in patients treated with α‐2a when compared to those treated with α‐2b. Conclusion: Abnormal ALT values during peg‐IFN plus ribavirin treatment are observed relatively frequently, even in patients without detectable HCV RNA. Direct or indirect involvement of drugs is considered as one possible cause.     

Systemic mastocytosis—a case treated with interferon α and radiotherapy

Summary. Systemic mastocytosis is uncommon. Symptoms result from local infiltration and degranulation of mast cells. Reports in the literature describe successful use of interferon α and radiotherapy to produce reduction in symptoms and bulk of disease. We report a patient who responded to radiotherapy but not interferon α.     

α‐thalassaemia masked by β gene defects and a new polyadenylation site mutation on the α2‐globin gene

We report three examples of chronic anaemia involving complex combinations of α‐ and β‐globin gene defects. The first case had a potential Hb H disease caused by the classic SEA/RW deletions masked by Hb E [β26(B8)Glu→Lys] in the homozygous state. The second had an unusual Hb H disease caused by compound heterozygosity for two different α2 polyadenylation site mutations masked by a β‐thalassaemia heterozygosity. The third had an intermediate α‐thalassaemia with considerable anaemia caused by an as yet unknown polyadenylation site (AATAAA>AATAAC) mutation in combination with a common RW deletion masked by a common Hb C [β6(A3)Glu→Lys] heterozygosity. Diagnostic methods, genotype/phenotype correlations and the chance of overlooking these combinations during risk assessment in a multiethnic society are discussed.     

Postoperative surveillance with monthly serum tumor markers after living‐donor liver transplantation for hepatocellular carcinoma

Aim: Recurrence of hepatocellular carcinoma (HCC) after liver transplantation decreases patient survival. The usefulness of post‐transplant surveillance with tumor markers, however, is not clear. We evaluated our cumulative experience with recurrent HCC detected during post‐transplant surveillance. Methods: We analyzed 100 patients with HCC detected in the explanted liver. Monthly to bimonthly measurement of tumor markers and yearly computed tomography were scheduled postoperatively. Results: Preoperatively, 82 met the Milan criteria. The histological findings indicated that 61 fulfilled the Milan criteria. In nine patients, HCC recurred 10 months (2–29) after liver transplantation in the graft (n = 1), lung (n = 2), bone (n = 3) and multiple organs (n = 3). In all nine recipients, HCC was first suspected based on an increase in tumor marker levels. Recurrent HCC was confirmed by computed tomography (n = 7) or magnetic resonance imaging (n = 2) within 4 months (0–6) after first identifying an increase in the tumor marker levels. Six cases were treated surgically, two of which achieved prolonged survival of 16 and 38 months. Conclusion: Frequent measurement of α‐fetoprotein and des‐γ carboxy prothrombin was useful for detecting recurrent HCC and may be useful long‐term follow‐up markers for post‐transplant surveillance.     

α‐Galactosylceramide activates antitumor immunity against liver tumor

Aim: α‐Galactosylceramide (α‐GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. We investigated the detailed process of activating liver dendritic cells (DC) and immune cells after α‐GalCer treatment in the mouse liver tumor model. Methods: BALB/c mice bearing CMS4 liver tumor (p53 peptide‐expressing tumor) were treated by α‐GalCer. We evaluated the activation of liver DC and immune cells after α‐GalCer treatment. Interferon (IFN)‐γ enzyme‐linked immunosorbent spot (ELISPOT) assay was performed to detect p53 peptide‐specific cytotoxic T lymphocytes (CTL). To assess the impact of systemic acquired immunity by α‐GalCer treatment, 28 days after liver tumor treatment, CMS4 cells or Colon26 cells were re‐challenged s.c. Results: The liver weights of α‐GalCer‐treated mice were significantly lighter than those of vehicle‐treated mice. Depletion experiments revealed that natural killer (NK) cells were essential for the antitumor effect of α‐GalCer. α‐GalCer treatment significantly increased the population of DC and NK cells in the liver. The expressions of co‐stimulatory molecules on liver DC significantly increased with the peak at 1 day after α‐GalCer administration. IFN‐γ ELISPOT assay demonstrated that p53 peptide‐specific CTL was generated efficiently in α‐GalCer‐treated mice. ⁵¹Cr‐release assay revealed that CD8⁺, not CD4⁺, CTL against CMS4 cells were generated in α‐GalCer‐treated mice. The mice that had been protected from CMS4 liver tumor by α‐GalCer injection became resistant against s.c. CMS4 re‐challenge, but not against Colon26 re‐challenge. Conclusion: These results demonstrated the therapeutic potential of α‐GalCer against liver cancer through activating liver DC and immune cells in the liver.