A Changing Paradigm for the Treatment of Intermediate-Stage Hepatocellular Carcinoma: Asia-Pacific Primary Liver Cancer Expert Consensus Statements.

The Asia-Pacific Primary Liver Cancer Expert (APPLE) Consensus Statement on the treatment strategy for patients with intermediate-stage hepatocellular carcinoma (HCC) was established on August 31, 2019, in Sapporo, Hokkaido during the 10th Annual APPLE Meeting. This manuscript summarizes the international consensus statements developed at APPLE 2019. Transarterial chemoembolization (TACE) is the only guideline-recommended global standard of care for intermediate-stage HCC. However, not all patients benefit from TACE because intermediate-stage HCC is a heterogeneous disease in terms of tumor burden and liver function. Ten important clinical questions regarding this stage of HCC were raised, and consensus statements were generated based on high-quality evidence. In intermediate-stage HCC, preservation of liver function is as important as achieving a high objective response (OR) because the treatment goal is to prolong overall survival. Superselective conventional TACE (cTACE) is recommended as the first choice of treatment in patients eligible for effective (curative) TACE, whereas in patients who are not eligible, systemic therapy is recommended as the first choice of treatment. TACE is not indicated as the first-line therapy in TACE-unsuitable patients. Another important statement is that TACE should not be continued in patients who develop TACE failure/refractoriness in order to preserve liver function. Targeted therapy is the recommended first-line treatment for TACE-unsuitable patients. Especially, the drug, which can have higher OR rate, is preferred. Immunotherapy, transarterial radioembolization, TACE + targeted therapy or other modalities may be considered alternative options in TACE-unsuitable patients who are not candidates for targeted therapy. Better liver function, such as albumin-bilirubin grade 1, is an important factor for maximizing the therapeutic effect of systemic therapy.

CYP2C19 polymorphism has no influence on rabeprazole-based hybrid therapy for Helicobacter pylori eradication.

AIM: To evaluate the impact of cytochrome P450 2C19 (CYP2C19) and interleukin-1ß (IL-1ß) polymorphisms on the efficacy of Helicobacter pylori (H. pylori) eradication by using rabeprazole-based hybrid therapy. METHODS: A total of 88 H. pylori-infected patients were recruited to receive 14-d of hybrid therapy from March 2013 to May 2014. Three patients were excluded from analysis because of incomplete compliance. Either a follow-up endoscopy or 13C-urea test was performed to determine the results of H. pylori eradication therapy. The genotypes of CYP2C19 and IL-1ß were analyzed to investigate the impact on treatment effect. RESULTS: The total eradication rate of H. pylori was 92.94% (79/85). According to the CYP2C19 genotypes, the rates of H. pylori eradication were 89.19% in extensive metabolizers (EM) and 95.83% in non-EM. The H. pylori eradication rates regarding the IL-1ß genotypes were 92.59% in the normal acid secretion group and 93.10% in the low acid secretion group. After multivariable logistic regression analysis, both the genotypes of CYP2C19 and IL-1ß had no significant influences on the eradication rates of H. pylori. CONCLUSION: The CYP2C19 and IL-1ß polymorphisms are not significantly independent factors of H. pylori eradication using rabeprazole-based hybrid therapy.

Niemann-Pick Type C2 Protein Regulates Free Cholesterol Accumulation and Influences Hepatic Stellate Cell Proliferation and Mitochondrial Respiration Function.

Liver fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. A high-cholesterol diet is associated with liver fibrosis via the accumulation of free cholesterol in hepatic stellate cells (HSCs). Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular free cholesterol homeostasis via direct binding with free cholesterol. Previously, we reported that NPC2 was downregulated in liver cirrhosis tissues. Loss of NPC2 enhanced the accumulation of free cholesterol in HSCs and made them more susceptible to transforming growth factor (TGF)-ß1. In this study, we showed that knockdown of NPC2 resulted in marked increases in platelet-derived growth factor BB (PDGF-BB)-induced HSC proliferation through enhanced extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK), and protein kinase B (AKT) phosphorylation. In contrast, NPC2 overexpression decreased PDGF-BB-induced cell proliferation by inhibiting p38, JNK, and AKT phosphorylation. Although NPC2 expression did not affect caspase-related apoptosis, the autophagy marker light chain 3ß (LC3B) was decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs. The mitochondrial respiration functions (such as oxygen consumption rate, ATP production, and maximal respiratory capacity) were decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs, while NPC2-overexpressed cells remained normal. In addition, NPC2 expression did not affect the susceptibility of HSCs to lipopolysaccharides (LPS), and U18666A treatment induced free cholesterol accumulation, which enhanced LPS-induced Toll-like receptor 4 (TLR4), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation, interleukin (IL)-1 and IL-6 expression. Our study demonstrated that NPC2-mediated free cholesterol homeostasis controls HSC proliferation and mitochondrial function.

Glycine N-methyltransferase deficiency in female mice impairs insulin signaling and promotes gluconeogenesis by modulating the PI3K/Akt pathway in the liver.

BACKGROUND: Glycine N-methyltransferase (GNMT) is abundantly expressed in the normal liver but is down-regulated in liver cancer tissues. GNMT knockout (Gnmt-/-) mice can spontaneously develop chronic hepatitis, fatty liver, and liver cancer. We previously demonstrated that hepatic GNMT is decreased in high-fat-diet-induced type 2 diabetes mellitus, but its contribution to metabolic syndrome is unclear. Here we show that GNMT modulates key aspects of metabolic syndrome in mice. METHODS: Eleven-week-old Gnmt-/- and wild-type (WT) mice with a C57BL/6 genetic background were used in this study. The metabolic defects of GNMT deficiency were measured by glucose and insulin tolerance tests, lipid homeostasis, gluconeogenesis, and insulin signaling. RESULTS: Gnmt-/- mice, especially females, exhibited glucose intolerance and insulin resistance. However, their body fat and lean mass, food and water intakes, and energy expenditure did not differ from those of WT mice. In addition, glucose-stimulated insulin secretion and insulin-stimulated glucagon secretion were normal in the serum and pancreatic islets of Gnmt-/- mice. Importantly, we found that GNMT deficiency increased lipogenesis and triglycerides in the liver. The elevated triglycerides disrupted the ability of insulin to induce Akt and S6 ribosomal protein phosphorylation, and then triggered insulin resistance and gluconeogenesis in female Gnmt-/- mice. CONCLUSIONS: Our data indicate that hepatic GNMT regulates lipid and glucose homeostasis, and provide insight into the development of insulin resistance through modulating the PI3K/Akt pathway.

Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation.

In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-ß1 (TGF-ß1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-ß1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-ß1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

Hybrid vs sequential therapy for eradication of Helicobacter pylori in Taiwan: A prospective randomized trial.

AIM: To evaluate the efficacy of sequential vs hybrid therapy in patients with Helicobacter pylori (H. pylori) infection. METHODS: From March 2013 to May 2014, one hundred and seventy-five H. pylori infected patients who had not been treated for H. pylori before were randomized to receive either sequential therapy (rabeprazole 20 mg and amoxicillin 1 g twice daily for 5 d, followed by rabeprazole 20 mg, clarithromycin 500 mg and metronidazole 500 mg twice daily for 5 d) or hybrid therapy (rabeprazole 20 mg and amoxicillin 1 g for 7 d, followed by rabeprazole 20 mg, amoxicillin 1 g, clarithromycin 500 mg and metronidazole 500 mg twice daily for 7 d). H. pylori status was confirmed by positive results of both rapid urease test and histology examination or a positive result of culture. Eradication efficacy was assessed by follow-up endoscopy with rapid urease test and histological examination 8 wk after the end of anti-H. pylori therapy, or (13)C-urea breath test at least 4 wk after completion of treatment. The primary outcome was H. pylori eradication by intention-to-treat (ITT) and per-protocol (PP) analyses. RESULTS: One hundred and sixty-seven patients (83 patients in the sequential group and 84 patients in the hybrid group) completed the study. The compliance rates were 97.6% and 97.7% for the two groups, respectively. The eradication rate was 78.2% for the sequential group and 92% for the hybrid group by ITT analysis (P = 0.01). The eradication rate was 81.9% for the sequential group and 96.4% for the hybrid group by PP analysis (P = 0.01). Univariate analysis for the clinical and bacterial factors did not identify any risk factors associated with treatment failure. Severe adverse events were observed in 2.3% of patients in the sequential group and 2.4% of those in the hybrid group. CONCLUSION: Due to a grade A (> 95%) success rate for H. pylori eradication by PP analysis, similar compliance and adverse events, hybrid therapy seems to be an appropriate eradication regimen in Taiwan.

Niemann-Pick type C2 protein regulates liver cancer progression via modulating ERK1/2 pathway: Clinicopathological correlations and therapeutical implications.

Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death. It is important to identify new targets for early diagnosis and treatment of HCC. Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumorigenesis. In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. The patients with NPC2 downregulation expressed much higher α-fetoprotein, multiple tumor type, vascular invasion, later pathological stage and shorter survival rate. Knockdown NPC2 in liver cancer cell lines promote cell proliferation, migration and xenograft tumorigenesis. In contrast, NPC2 overexpression inhibits HuH7 promoted tumor growth. Furthermore, administration of hepatotropic adeno-associated virus 8 (AAV8) delivered NPC2 decreased the inflammatory infiltration, the expression of two early HCC markers-glypican 3 and survivin and suppressed the spontaneous HCC development in mice. To identify the NPC2-dependent mechanism, we emphasized on the status of MAPK/ERK signaling. MEK1/2 inhibitor treatment demonstrated that the expression of NPC2 affected the activation of ERK1/2 but not MEK1/2. In addition, cholesterol trafficking inhibitor treatment did not alter the cell proliferation and the activation of MEK/ERK. In conclusion, our study demonstrates that NPC2 may play an important role in negatively regulate cell proliferation and ERK1/2 activation that were independent of cholesterol accumulation. AAV-NPC2 may thus represent a new treatment strategy for liver cancer.

Characterization of Niemann-Pick Type C2 protein expression in multiple cancers using a novel NPC2 monoclonal antibody.

Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.

Performance of Routine Helicobacter pylori Invasive Tests in Patients with Dyspepsia.

Background. This study was designed to compare the accuracy of three different invasive methods for the detection of Helicobacter pylori (H. pylori) infection in patients with dyspepsia. These tests included culture, histology, and the rapid urease test (CLO test). Methods. H. pylori infection was diagnosed prospectively in 246 untreated dyspeptic patients who underwent upper gastrointestinal endoscopy. The gold standard for H. pylori infection was based on a positive culture or both a positive histological examination and a CLO test. Results. H. pylori was diagnosed in 33.3% of the patients. The sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy were as follows: histology from the antrum (95.12; 95.12; 90.7; 97.5; 95.12%); histology from the antrum and corpus (95.12; 95.12; 90.7; 97.5; 95.12%); histology from the corpus (76.83; 96.95; 92.65; 89.33; 90.24%); culture (91.46; 100; 100; 95.91; 97.15%); a CLO test from the antrum and corpus (85.59; 100; 100; 93.71; 95.52%); a CLO test from the antrum (64.63; 100; 100; 84.97; 88.21%); a CLO test from the corpus (69.51; 100; 100; 96.77; 89.83%), respectively. Conclusions. Antral biopsy histology and culture are the best methods for the diagnosis of H. pylori infection in our cohort of patients with dyspepsia.

Glycine N-methyltransferase deficiency affects Niemann-Pick type C2 protein stability and regulates hepatic cholesterol homeostasis.

Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt(-/-)) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt(-/-) mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with (131)I-labeled 6ß-iodocholesterol demonstrated that Gnmt(-/-) mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 [ABCA1]), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt(-/-) mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81-105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171-295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept.

Deficiency of glycine N-methyltransferase results in deterioration of cellular defense to stress in mouse liver.

PURPOSE: Previously, we reported that glycine N-methyltransferase (GNMT) interacts with benzo[a]pyrene (BaP) and inhibits BaP-DNA adducts formation. In addition, Gnmt knockout (Gnmt(-/-)) mice developed chronic hepatitis and hepatocellular carcinoma (HCC). The aims of this study were to understand the gene expression profile of Gnmt(-/-) mice and to study the interaction between BaP and GNMT deficiency in vivo. EXPERIMENTAL DESIGN: Gene expression profiles of Gnmt(-/-) mice were analyzed by 2-D PAGE and real-time PCR. Both wild-type and Gnmt(-/-) mice were challenged with BaP and sacrificed at the age of 13 months. RESULTS: Compared with the wild-type mice, proteins involved in the anti-oxidation/detoxification response, glycolytic energy metabolism and one-carbon metabolism pathways were down-regulated significantly in Gnmt(-/-) mice. Malondialdehyde assay showed that lipid peroxidation was significantly increased in the Gnmt(-/-) mice liver. H(2)O(2) treatment demonstrated that the survival rate of HuH-7 cells overexpressing GNMT was significantly higher than the controls. BaP challenge experiments showed that 71.4% (5/7) of male and all (7/7) female Gnmt(-/-) mice developed HCC, while only 16.7% (1/6) of male and 20% (1/5) of female wild-type mice had HCC. CONCLUSION AND CLINICAL RELEVANCE: GNMT regulates genes related to detoxification and anti-oxidation pathways. BaP is a liver cancer carcinogen especially during GNMT deficiency.

Neuroprotective activity of Vitis thunbergii var. taiwaniana extracts in vitro and in vivo.

Vitis thunbergii var. taiwaniana (VTT) is a wild grape native to Taiwan, where it has been used as a folk medicine. In this study, we found that the branch and leaf ethanol extracts of VTT significantly inhibited the inducible nitric oxide (NO) synthase protein expression and NO production in BV2 microglia. Using primary neuronal cells, kainic acid (KA) significantly increased hydrogen peroxide production in a dose-dependent manner. All four ethanol extracts of VTT significantly decreased hydrogen peroxide production. However, only root and branch ethanol extracts were able to prevent the neuronal cell death induced by KA in vitro. In the animal study, administration of all four plant part extracts of VTT delayed the onset of seizure and decreased the hippocampus neuronal cell loss, and the neuroprotective activity could be ranked as follows: branch approximately leaf > root > trunk. The results suggest that VTT extracts have a potential to prevent neurodegeneration through the antioxidative activity by their ability to inhibit NO and hydrogen peroxide production.

Serum prohepcidin levels correlate with hepatic iron stores in chronic hepatitis C patients.

BACKGROUND/AIMS: Increased serum iron indices and hepatic iron stores are frequent in patients with chronic hepatitis C (CHC). The antimicrobial peptide hepdicin produced in the liver plays a pivotal role in iron homeostasis. METHODOLOGY: To determine the expression of hepcidin, the serum levels of prohepcidin were measured in 58 CHC patients and 144 healthy controls. The hepatic iron stores were scored by Perls' stain on liver biopsy specimens in 39 CHC patients. The serum prohepcidin levels were correlated with biochemical inflammation markers, histological necroinflammation grades, hemoglobin levels and iron status in CHC patients. RESULTS: The concentrations of serum prohepcidin were significantly higher in CHC patients than in healthy controls (142.07 +/- 67.06 vs. 89.07 +/- 37.32 ng/mL, p < 0.001). The CHC patients with positive hepatic iron stains had significantly higher serum prohepcidin levels than the CHC patients without (221.20 +/- 117.74 vs. 123.81 +/- 60.53 ng/mL, p = 0.037). The serum prohepdicin levels were not significantly correlated with the ages (r = -0.041, p = 0.760), hemoglobin (r = 0.127, p = 0.346), alanine aminotransferase (r = -0.032, p = 0.813), transferrin saturation (r = 0.025, p = 0.862), ferritin levels (r = 0.211, p = 0.133) and hepatic inflammation grades (r = 0.153, p = 0.352) in CHC patients. CONCLUSIONS: The expression of serum prohepcidin is independent of the degree of hepatic inflammation as measured by the histological activity or aminotransferase level. The serum prohepcidin levels are associated with hepatic iron stains and significantly higher in CHC patients than in healthy controls. Our results suggest that CHC may induce the expression of hepcidin possibly by increased hepatic iron stores.

Effects of oral administration of yam tuber storage protein, dioscorin, to BALB/c mice for 21-days on immune responses.

Dioscorin, the tuber storage protein of yam, was reported to have immunomodulatory activity in RAW264.7 murine macrophage cell lines ( Food and Chemical Toxicology , 2007 , 45 , 2312 -2318 ). However, the immunomodulatory function of dioscorin after being ingested was not elucidated in vivo. Hence, BALB/c mice were given oral dioscorin (2.5 and 20 mg/kg/day) once a day for 21-days. Lymphocyte subpopulation changes in the peripheral blood and splenocytes, stimulation in phagocytosis of the polymorphonuclear cell (PMN) and monocytes, the natural killer (NK) cell cytotoxicity, the splenocyte proliferation, and cytokine secretions in the presence of PHA were determined. The number changes of Peyer's patches and secreted IgA (sIgA) in the feces were determined. Oral dioscorin for 21-days increased the subpopulation in natural killer cells (CD49(+)) and/or B cells (CD19(+)), elevated the phagocytosis of PMN (p < 0.01) and MON (p < 0.01), and the NK cell cytotoxic activity (p < 0.05), and stimulated splenocyte proliferations in the presence of LPS or PHA (p < 0.05) in comparison with those of the control. Cytokines of INF-gamma, IL-4, and IL-10 secretions, the numbers of Peyer's patches, and sIgA in the feces showed higher levels in oral dioscorin and significant difference to those of the control (p < 0.001). These results suggested that dioscorin exhibited systemic and mucosal immunomodulatory activities after being ingested in vivo.

Osthole, a potential antidiabetic agent, alleviates hyperglycemia in db/db mice.

Osthole is an agent isolated from Cnidium monnieri (L.) Cusson and Angelica pubescens and has been used to treat several diseases, including metabolic syndromes. To investigate the hypoglycemic effects of osthole in diabetic db/db mice and the underlying mechanisms of these effects by in vitro assay, diabetic db/db mice and cell experiments were utilized to understand its possible effects. Osthole significantly activated both PPARalpha and PPARgamma in a dose-dependent manner based on the results of the transition transfection assay. The activation of PPARalpha and PPARgamma by osthole also resulted in an increase in the expression of PPAR target genes such as PPAR itself, adipose fatty acid-binding protein 2, acyl-CoA synthetases, and carnitine palmitoyltransferase-1A. In vitro results suggested that osthole might be a dual PPARalpha/gamma activator, but its chemical structure differed from that of the thiazolidinedione class of antidiabetic drugs. In addition, osthole markedly activated the AMP-activated protein kinase and its downstream acetyl CoA carboxylase molecules by increasing their phosphorylation levels. Finally, obese diabetic db/db mice were treated with osthole by different administered routes, and osthole was found to markedly reduce blood glucose level. Interestingly, osthole did not reduce the blood insulin or lipid levels, two phenomena that did occur in animals treated with insulin sensitizers like PPAR agonists. These results suggest that osthole can alleviate hyperglycemia and could be potentially developed into a novel drug for treatment of diabetes mellitus.

Immunomodulatory activity of dioscorin, the storage protein of yam (Dioscorea alata cv. Tainong No. 1) tuber.

The purified dioscorin from yam (Dioscorea alata L. cv. Tainong 1) tuber was previously reported (Hsu et al., 2002. J. Agric. Food Chem., 50, 6109-6113). In this report, we evaluated its immunomodulatory ability in vitro in the presence of polymyxin B (50 microg/ml) to eliminate lipopolysaccharide (LPS) contamination. Dioscorin (5-100 microg/ml) was able to stimulate nitric oxide production (expressed as nitrite concentrations) in RAW264.7 cells. The stimulation index on the phagocytosis of RAW264.7 cells against E. coli and the oxidative burst (determined by the intensity of rhodamine fluorescence) of RAW264.7 cells were both enhanced by different concentrations of dioscorin (5-100 microg/ml). The cytokine production, including IL-6, TNF-alpha, and IL-1beta in dioscorin-treated RAW264.7 cells or human monocytes, was measured in the cultured medium. Dioscorin (5-100 microg/ml) was found able to induce IL-6, TNF-alpha, and IL-1beta production in RAW264.7 cells and human monocytes. To evaluate the effects of dioscorin on the proliferation of spleen cells from BALB/c mice, phytohemagglutinin (PHA, 2 microg/ml) alone or PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml) was used to treat spleen cells for 24h. The stimulated proliferation index of splenic cells ranged from 1.38- to 1.48-fold of PHA alone for PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml). We suggest that the tuber storage protein of yam dioscorin functions as an immunomodulatory substance.

Involvement of fatty acid-CoA ligase 4 in hepatocellular carcinoma growth: roles of cyclic AMP and p38 mitogen-activated protein kinase.

AIM: Fatty acid-CoA ligase 4 (FACL4) is an arachidonate-preferring enzyme which has been shown to be up-regulated in human colon cancer tissues and implicated in the colon tumorigenesis. The purpose of this study was to investigate the role of FACL4 in the human hepatocellular carcinoma (HCC) tumorigenesis and the specific signal pathways involved in this process. METHODS: We investigated the expression and regulation of FACL4 in HCC, adjacent non-tumorous liver tissues, and cell lines. RESULTS: In HCC patients, we demonstrated that FACL4 gene expression was markedly elevated in the cancerous tissues than in the adjacent non-cancerous liver tissues. In addition, several human hepatoma cell lines, including Hep3B and HepG2, expressed high levels of FACL4. Stable overexpression of FACL4 knockdown plasmids (small interfering RNA, siRNA) to Hep3B cells significantly decreased FACL4 expression and subsequently limited the cell proliferation. Treatment of Hep3B cells with 8-bromo-cAMP and SB203508 (p38 MAPK inhibitor) significantly suppressed the FACL4 expression. CONCLUSION: FACL4 is involved in the HCC tumorigenesis and both cAMP and p38 MAPK pathways are associated with the regulation of FACL4 in HCC.

Cytochrome C release induces apoptosis of nasopharyngeal carcinoma (NPC) by antitumor glyfoline.

The mechanism of action, leakage of cytochrome c from mitochondria into cytosol, for the antineoplastic compound glyfoline was examined. Additionally, our current studies revealed that glyfoline induced apoptotic changes and arrested cell cycle procession at the G2/M phase in nasopharyngeal carcinoma (NPC). A reverse transcriptase polymerase chain reaction (RT-PCR) showed no specific changes of apoptosis-related gene expression (i. e., bax, ICE-alpha,beta, bcl-2, and c-myc). However, no similar changes were detected in fibroblasts and peripheral lymphocytes after glyfoline treatment suggesting that glyfoline has a higher affinity for tumor cells than for normal cells.

MDM2 expression in EBV-infected nasopharyngeal carcinoma cells.

To understand whether the p53-regulated mdm2 gene expression was altered by the Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), the NPC-TW01 cell line was infected by EBV through IgA receptor-mediated endocytosis. The mdm2 gene was expressed only in a small fraction of the NPC cell population and could be enhanced in the EBV-infected (EBV+) cells. In the animals bearing EBV+ and EBV- NPC xenografts, the MDM2+ cells only appeared in clusters in both EBV+ and EBV- tumors with stronger expression in EBV+ cells. Cotransfection of pmdm2-Luc plus pSV40-p53 plus pCMV-LMP1 in the NPC-TW06 line that had p53 heterozygous point mutation showed stronger mdm2 promoter activity than cells cotransfected with pmdm2-Luc plus pSV40-p53, but no mdm2 promoter activity was seen in cells cotransfected with pmdm2-Luc plus pCMV-LMP1. Only the EBV-LMP1 but not the EBV-LMP2A gene could enhance p53 to upregulated mdm2 expression. Tumor cells in NPC biopsy specimens revealed similar mdm2 expression as in the animal model. It is concluded that although EBV can indirectly enhance mdm2 gene expression in tumor cells that express this gene, it cannot turn on or directly regulate mdm2 expression in cells that do not express this gene. In other words, EBV plays a role as an enhancer in NPC tumorigenesis.