| Part1:Zileuton, a5-lipoxygenase specific inhibitor prevents against hepatocarcinogenesis induced by diethylnitrosamine in rats and its mechanismBackground and aims:Hepatocellular carcinoma (HCC) is a major health problem worldwide, and its incidence is increasing annually. China is a high-incidence area of HCC. Eicosanoids derived from the arachidonic acid cascade have been considered important in tumor promotion, progression, and metastasis. However, its role in HCC is unknown. In the present study, we examined the expression of5-lipoxygenase in rat HCC, investigated whether its specific inhibitor zileuton prevents against hepatocarcinogenesis, and explored its mechanism.Methods:Male Wistar rats were randomly divided into three groups:normal control (n=5), model (n=8) and zileuton (n=8). Rat HCC was induced by intraperitoneal injections of diethylnitrosamine (DEN) weekly for16weeks followed by a2-week wash-out period. Rats in zileuton group received daily zileuton at a dose of50mg/kg via gastrogavage. Expressions of5-lipoxygenase protein and mRNA were detected using immunochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay was used to detect apoptosis in the liver.Results:Moderately or intensely staining for5-lipoxygenase protein was detected in DEN-induced rat HCC, and mainly located in the cytoplasm. In control rats,5-lipoxygenase mRNA was detected in the liver; but significant elevation of its mRNA was observed in the liver of DEN-induced rats. DEN-induced rats developed numerous nodules on the surface of the livers with the nodule incidence of100%(8/8). Zileuton treatment reduced the nodule incidence (5/8,62.5%), which was not statistically significant compared with model group. The mean number of nodules per nodule-bearing liver (nodule multiplicity) in zileuton group (10.4±3.4) was significantly lower than in DEN group (17.2±3.5). TUNEL assay showed that zileuton treatment induced apoptosis in the liver, and the apoptotic index (AI) was significantly higher in zileuton group (0.70±0.12) than in model group (0.30±0.09).Conclusions:5-lipoxygenase is expressed in rat HCC. Inhibition of5-lipoxygenase by zileuton exerts inhibitory effect on the development of HCC in rats, and the mechanism may be associated with induction of apoptosis. Part2:Effect of zileuton, a5-lipoxygenase specific inhibitor on cell proliferation and apoptosis in human hepatoma cells (HepG2)Background and aims:5-lipoxygenase is a key enzyme in the metabolism of arachidonic acid to leukotrienes. An increase in expression of5-lipoxygenase and their metabolites has been detected in a variety of human cancer cell lines including pancreatic, esophageal, prostate, and colon cancers. However, its expression in HCC cells is unknown. In the present study, we examined the expression of5-lipoxygenase in human hepatoma cell line (HepG2), investigated the effect of its specific inhibitor zileuton on cell proliferation and apoptosis.Methods:Immunochemical staining and RT-PCR were used to detect expressions of5-lipoxygenase protein and mRNA, respectively. Cell proliferation and apoptosis were determined by MTT assay and flow cytometry, respectively.Results:5-lipoxygenase protein was detected in the cytoplasm, with focal nuclear membrane staining. The mRNA of5-lipoxygenase was detected in HepG2cells, and the mean value of its expression (relative quantity to GAPDH) was0.35. Zileuton reduced cell proliferation and induced apoptosis in HepG2cells in a concentration-dependent manner.Conclusions:5-lipoxygenase is expressed in human hepatoma cell line (HepG2). Inhibition of5-lipoxygenase by zileuton reduces cell proliferation and induces apoptosis in a concentration-dependent manner. Part3:Expression of5-lipoxygenase in human hepatocellular carcinoma and its clinical significanceBackground and aims:Overexpression of5-lipoxygenase has been detected in a variety of human cancers including pancreatic, esophageal, prostate, and colon cancers. In colorectal cancer,5-lipoxygenase overexpression is associated with clinical prognosis, and tumor size, depth or vessel invasion. In the present study, we examined the expression of5-lipoxygenase in hepatocellular carcinoma, and investigated its correlation with clinicopathological characteristics.Methods:The expression of5-lipoxygenase protein was detected in human paraffin-embedded samples of cancer and paracancerous normal tissues using immunohistochemistry. Fisher’s exact or x2test was employed to analyze its correlation with clinicopathological characteristics.Results:In human paracancerous normal liver tissues, no or faint immunoreactive staining for5-lipoxygenase was detected. However, moderately staining, with focal strong staining, was observed in the cancer tissues from16/20cases (80%). The staining was present in the cytoplasm of cancer cells or inflammatory cells. However, there was no significant association between their expressions with the patients’ age, gender, tumor size, tumor differentiation, AJCC staging, and AFP levels.Conclusions:5-lipoxygenase is expressed in human hepatocellular carcinoma, but has no significant correlation with clinicopathological characteristics. |
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