Hyo-Suk Lee

p53 mutation in hepatocellular carcinoma after aflatoxin exposure

Mutations of the p53 gene are found in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Specific mutations might reflect exposure to specific carcinogens and we have screened HCC samples from patients in 14 different countries to determine the frequency of a hotspot mutation at codon 249 of the tumour suppressor p53 gene. We detected mutations in 17% of tumours (12/72) from four countries in south Africa and the southeast coast of Asia. There was no codon 249 mutation in 95 specimens of HCC from other geographical locations including North America, Europe, Middle East, and Japan. Worldwide, the presence of the codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus (HBV). Further studies were completed in two groups of HBV-infected patients at different risks of exposure to aflatoxins. 53% of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumour with a codon 249 mutation, in contrast with 8% of patients from Transkei (1/12) who were at low risk. HCC is an endemic disease in Mozambique and accounts for up to two thirds of all tumours in men. A codon 249 mutation of the p53 gene identifies an endemic form of HCC strongly associated with dietary aflatoxin intake.

Aberrant CpG Island Hypermethylation Along Multistep Hepatocarcinogenesis

To determine the methylation profile of multiple tumor-related genes during multistep hepatocarcinogenesis, we investigated the methylation status of CpG islands of 9 genes, using methylation-specific polymerase chain reaction for 60 paired hepatocellular carcinoma (HCC) and non-HCC liver tissue samples, 22 dysplastic nodule (DN), 30 liver cirrhosis (LC), 34 chronic hepatitis (CH) and 20 normal liver samples. The methylation status of 9 genes was correlated to the clinicopathological findings of HCC patients. All HCC samples showed methylation of at least one gene, whereas it was shown in 72.7% of DN and 40% of LC, but was not shown in CH and normal liver samples (P < 0.001). The number of genes methylated showed a stepwise increase with the progression of stages (0 for normal liver and CH, 0.5 for LC, 1.5 for DN, and 3.7 for HCC (P < 0.001)). The genes frequently methylated in HCC were APC (81.7%), GSTP1 (76.7%), RASSF1A (66.7%), p16 (48.3%), COX-2 (35%), and E-cadherin (33.3%). COX-2, p16, RASSF1A, and TIMP-3 were not methylated in LC and CH from patients without concurrent HCC. Chronic liver diseases with concurrent HCC showed higher methylation frequencies of the tested genes, and a higher number of methylated genes than those without concurrent HCC. HCC patients with methylation of E-cadherin or GSTP1 showed poorer survival than those without (P = 0.034 and 0.043, respectively). In conclusion, our results indicated that CpG island methylation of tumor-related genes is an early and frequent event, and accumulates step-by-step during a multistep hepatocarcinogenesis. CpG island methylation of E-cadherin or GSTP1 might serve as a potential biomarker for prognostication of HCC patients.

Raf and MEK Protein Kinases Are Direct Molecular Targets for the Chemopreventive Effect of Quercetin, a Major Flavonol in Red Wine

Considerable attention has focused on the health-promoting effects of red wine and its nonflavonoid polyphenol compound resveratrol. However, the underlying molecular mechanisms and molecular target(s) of red wine or other potentially active ingredients in red wine remain unknown. Here, we report that red wine extract (RWE) or the red wine flavonoid quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells. The activation of activator protein-1 and nuclear factor-kappaB induced by TPA was dose dependently inhibited by RWE or quercetin treatment. Western blot and kinase assay data revealed that RWE or quercetin inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 and Raf1 kinase activities and subsequently attenuated TPA-induced phosphorylation of ERK/p90 ribosomal S6 kinase. Although either RWE or quercetin suppressed Raf1 kinase activity, they were more effective in inhibiting MEK1 activity. Importantly, quercetin exerted stronger inhibitory effects than PD098059, a well-known pharmacologic inhibitor of MEK. Resveratrol did not affect either MEK1 or Raf1 kinase activity. Pull-down assays revealed that RWE or quercetin (but not resveratrol) bound with either MEK1 or Raf1. RWE or quercetin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are involved in the activation of MEK/ERK signaling. Docking data suggested that quercetin, but not resveratrol, formed a hydrogen bond with the backbone amide group of Ser(212), which is the key interaction for stabilizing the inactive conformation of the activation loop of MEK1.

Hepatic steatosis index: A simple screening tool reflecting nonalcoholic fatty liver disease

BACKGROUND/AIMS To optimize management of nonalcoholic fatty liver disease (NAFLD), a simple screening tool is necessary. In this study, we aimed to devise a simple index of NAFLD. STUDY A cross-sectional study with 10,724 health check-up subjects (5362 cases with NAFLD versus age- and sex-matched controls) was conducted. Study subjects were randomly assigned to a derivation cohort or a validation cohort. RESULTS Multivariate analysis indicated that high serum alanine aminotransferase (ALT) to serum aspartate aminotransferase (AST) ratio, high body mass index (BMI), and diabetes mellitus were independent risk factors of NAFLD (all P<0.001). Using these variables, a formula was derived by a logistic regression model: hepatic steatosis index (HSI)= 8 x(ALT/AST ratio)+BMI (+2, if female; +2, if diabetes mellitus). HSI had an area under receiver-operating curve of 0.812 (95% confidence interval, 0.801-0.824). At values of <30.0 or >36.0, HSI ruled out NAFLD with a sensitivity of 93.1%, or detected NAFLD with a specificity of 92.4%, respectively. Of 2692 subjects with HSI <30.0 or >36.0 in the derivation cohort, 2305 (85.6%) were correctly classified. HSI was validated in the subsequent validation cohort. CONCLUSION HSI is a simple, efficient screening tool for NAFLD that may be utilized for selecting individuals for liver ultrasonography and for determining the need for lifestyle modifications.

Transarterial Chemoembolization Can Be Safely Performed in Patients with Hepatocellular Carcinoma Invading the Main Portal Vein and May Improve the Overall Survival

PURPOSE To determine the efficacy and safety of transarterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC) and main portal vein (MPV) invasion. MATERIALS AND METHODS This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. The authors retrospectively assessed the electronic medical records of patients in whom HCC with MPV invasion was newly diagnosed from January 2004 to December 2007 at a single tertiary medical center. Patients with decompensated hepatic function were excluded. Outcomes of patients treated with TACE were compared with those of patients given supportive care according to Child-Pugh class. RESULTS One hundred twenty-five patients (104 men and 21 women; mean age, 55.7 years; age range, 33.4-83.0 years) were included. The median overall survival was 3.7 months (range, 0.2-33.3 months). Eighty-three of the 125 patients (66.4%) were treated with TACE and 42 (33.6%) received supportive care. Repeated TACE showed significant survival benefits compared with supportive care in patients with Child-Pugh class A (median survival, 7.4 months vs 2.6 months, respectively; P < .001) and class B (median survival, 2.8 months vs 1.9 months, respectively; P = .002) disease. Results of multivariate analysis showed that treatment with TACE (hazard ratio, 0.263; 95% confidence interval [CI]: 0.164, 0.424; P < .001) and Child-Pugh class A status (hazard ratio, 0.550; 95% CI: 0.368, 0.822; P = .004) were independent predictive factors of a favorable outcome. There were no procedure-related deaths within 4 weeks after TACE, and patient morbidity was 28.9% (24 of 83 patients). CONCLUSION TACE can be performed safely and may improve the overall survival of patients with HCC and MPV invasion.

A phase II dose‐escalating trial of clevudine in patients with chronic hepatitis B

Current therapies available for the treatment of chronic hepatitis B are limited in their ability to result in a cure. Clevudine is a new pyrimidine analog with potent anti‐hepatitis B virus (HBV) activity in vitro. A multicenter dose‐escalation study evaluated clevudine at 10, 50, 100, and 200 mg once daily for 28 days. Eligible patients had HBV DNA levels of 3 × 106 copies/mL or more, had not undergone nucleoside treatment, and were without human immunodeficiency or hepatitis C virus coinfection. Thirty‐two patients were enrolled (5, 10, 10, and 7 patients in the 10‐, 50‐, 100‐, and 200‐mg dose groups, respectively), 81% were male, 81% Asian, and 88% were hepatitis Be antigen (HBeAg) positive at baseline. Median pretreatment serum HBV DNA levels ranged from 7.3 to 8.8 log10 copies/mL. After 28 days, the median HBV DNA log10 change from baseline was −2.5, −2.7, −3.0, and −2.6 log10. Six months after dosing, median changes from baseline were −1.2, −1.4, −2.7 and −1.7 log10 in the 10‐, 50‐, 100‐, and 200‐mg cohorts, respectively. Six of 27 patients lost HBeAg, and 3 of 27 patients seroconverted to HBe antibody. Clevudine was well tolerated, with no dose‐limiting toxicities. A transient increase in alanine aminotransferase of up to 7.8 times the upper limit of normal (increase ranged from 20 to 186 IU/L) was observed in six patients in the 100‐mg cohort, without signs of liver failure. These increases were associated with improved viral suppression. The pharmacokinetic profile of clevudine was proportional to the dose. In conclusion, these results demonstrate the tolerability and potent activity of clevudine in HBV‐infected patients and support further clinical study. (HEPATOLOGY 2004;40:140–148.)

Liver abscess after transcatheter oily chemoembolization for hepatic tumors: incidence, predisposing factors, and clinical outcome.

PURPOSE To evaluate the incidence of, predisposing factors for, and clinical outcome of liver abscess developing in patients with hepatic tumors after transcatheter oily chemoembolization (TOCE). MATERIALS AND METHODS During the past 6-year period, 2,439 patients with hepatic tumors underwent a total of 6,255 TOCE procedures. With a retrospective review of medical records, the authors evaluated the occurrence of liver abscess, the statistical significance of potential predisposing factors including portal vein obstruction, metastatic tumors, biliary abnormalities (type 1, simple biliary obstruction; type 2, status prone to ascending biliary infection), malignant gastrointestinal mucosal lesions, and additional gelatin sponge particle embolization in liver abscess formation, and the clinical outcome of abscess. RESULTS Fifteen liver abscesses occurred in 14 patients (0.2%). Liver abscesses developed in three of 987 (0.3%) TOCE procedures for portal vein obstruction, three of 114 (2.6%) procedures for metastatic tumors, one of 49 (1.8%) for type 1 biliary abnormality, four of 55 (7.4%) for type 2 biliary abnormality, two of 18 (11.1%) for malignant gastrointestinal mucosal lesion, and nine of 2,108 (0.4%) for additional gelatin sponge particle embolization. Univariate and multivariate statistical analysis showed that type 2 biliary abnormality was a significant predisposing factor. The mortality related to liver abscess occurred in two patients (13.3%). Thirteen liver abscesses were successfully treated with parenteral antibiotics and percutaneous catheter drainage. However, irreversible deterioration of liver function occurred in two patients. Two of nine further TOCE procedures in three patients caused recurrent septicemia and liver abscess. CONCLUSION The biliary abnormality prone to ascending biliary infection was the most important predisposing factor to the development of liver abscess after TOCE. Postembolic liver abscess could be effectively managed with percutaneous catheter drainage.

Twenty‐four‐week clevudine therapy showed potent and sustained antiviral activity in HBeAg‐positive chronic hepatitis B

Clevudine is a pyrimidine analogue with potent and sustained antiviral activity against HBV. The present study evaluated the safety and efficacy of 30 mg clevudine once daily for 24 weeks and assessed the durable antiviral response for 24 weeks after cessation of dosing. A total of 243 hepatitis B e antigen (HBeAg)‐positive chronic hepatitis B patients were randomized (3:1) to receive clevudine 30 mg once daily (n = 182) or placebo (n = 61) for 24 weeks. Patients were followed for a further 24 weeks off therapy. Median serum HBV DNA reductions from baseline at week 24 were 5.10 and 0.27 log10 copies/mL in the clevudine and placebo groups, respectively (P < 0.0001). Viral suppression in the clevudine group was sustained off therapy, with 3.73 log10 reduction at week 34 and 2.02 log10 reduction at week 48. At week 24, 59.0% of patients in the clevudine group had undetectable serum HBV DNA levels by Amplicor PCR assay (less than 300 copies/mL). The proportion of patients who achieved normalization of alanine aminotransferase (ALT) levels was 68.2% in the clevudine group and 17.5% in the placebo group at week 24 (P < 0.0001). ALT normalization in the clevudine group was well maintained during post‐treatment follow‐up period. The incidence of adverse events (AEs) was similar between the clevudine group and the placebo group. No resistance to clevudine was detected with 24 weeks of administration of drug. Conclusion: A 24‐week clevudine therapy was well tolerated and showed potent and sustained antiviral effect without evidence of viral resistance during treatment period in HBeAg‐positive chronic hepatitis B. (HEPATOLOGY 2007;45:1172–1178.)

Visceral Obesity and Insulin Resistance as Risk Factors for Colorectal Adenoma: A Cross-Sectional, Case–Control Study

OBJECTIVES:Colorectal adenoma is known to be associated with obesity, but the association between colorectal adenoma and visceral adipose tissue (VAT) area measured by abdominal computed tomography (CT) has not been documented clearly. In addition, the relationship between insulin resistance and colorectal adenomas, which underlies the mechanism that links obesity and colorectal adenoma, has not been studied extensively. The aim of this study was to examine VAT area and insulin resistance as risk factors of colorectal adenoma.METHODS:A cross-sectional, case–control study was conducted in Koreans that presented for health check-ups. Subjects underwent various laboratory tests, abdominal CT, and colonoscopy. VAT, subcutaneous adipose tissue (SAT), and homeostatic metabolic assessment (HOMA) index were evaluated as potential risk factors of colorectal adenoma in 2,244 age- and sex-matched subjects.RESULTS:According to univariate analysis, the prevalences of smoking, hypertension, metabolic syndrome, and family history of colorectal cancer were higher in the adenoma group than in the normal control group. In addition, body mass index, waist circumference, triglyceride, high-density lipoprotein cholesterol, and VAT and SAT areas were significantly different in the two groups. According to the multivariate analysis adjusted for multiple confounders, VAT area was independently associated with the risk of colorectal adenoma (odds ratio (OR)=3.09, 95% confidence interval (CI): 2.19–4.36, highest quintile vs. lowest quintile). Mean HOMA index was higher in the adenoma group than in the control group (OR=1.99, 95% CI: 1.35–2.92, highest vs. lowest quintile).CONCLUSIONS:Visceral obesity was found to be an independent risk factor of colorectal adenoma, and insulin resistance was associated with the presence of colorectal adenoma.

Myricetin Suppresses UVB-Induced Skin Cancer by Targeting Fyn

Skin cancer is currently the most common type of human cancer in Americans. Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods, including red wine. Here, we show that myricetin suppresses UVB-induced cyclooxygenase-2 (COX-2) expression in mouse skin epidermal JB6 P+ cells. The activation of activator protein-1 and nuclear factor-kappaB induced by UVB was dose-dependently inhibited by myricetin treatment. Western blot and kinase assay data revealed that myricetin inhibited Fyn kinase activity and subsequently attenuated UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays revealed that myricetin competitively bound with ATP to suppress Fyn kinase activity. Importantly, myricetin exerted similar inhibitory effects compared with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a well-known pharmacologic inhibitor of Fyn. In vivo mouse skin data also revealed that myricetin inhibited Fyn kinase activity directly and subsequently attenuated UVB-induced COX-2 expression. Mouse skin tumorigenesis data clearly showed that pretreatment with myricetin significantly suppressed UVB-induced skin tumor incidence in a dose-dependent manner. Docking data suggest that myricetin is easily docked to the ATP-binding site of Fyn, which is located between the N and C lobes of the kinase domain. Overall, these results indicated that myricetin exerts potent chemopreventive activity mainly by targeting Fyn in skin carcinogenesis.