2-Acetylaminofluorene ; Angiogenesis Inhibitors ; pharmacology ; Animals ; Carcinoma, Hepatocellular ; chemically induced ; metabolism ; pathology ; Immunohistochemistry ; Liver ; drug effects ; metabolism ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thalidomide ; pharmacology

BACKGROUND/AIMS: ErbB receptor proteins are transmembrane tyrosine kinase receptors; when they are activated by interaction with ligands, they generate diverse cellular responses, especially during lesion development and progression to cancer. In this study the expression of ErbB receptors and TGF-alpha were investigated using an experimental cirrhosis rat model giving rise to hepatocellular neoplasms, similar to human liver diseases. METHODS: Fifty three male rats received intraperitoneal injection of diethylnitrosamine (DEN, 50 mg/kg), weekly for 18 weeks. Until the eighth week, two rats were sacrificed every two weeks and from the tenth to the eighteenth week, five rats were sacrificed weekly. Grossly, dyschromatic and dysmorphic nodules were counted and categorized into three groups: N1/N2/N3: 3 mm < or = x < 5 mm/5 mm < or = x < 10 mm/x > or = 10 mm in diameter. All nodules were examined, histologically. Antibodies for GSTp, TGF-alpha, EGF-R, ErbB2, ErbB3 and ErbB4 were used for immunohistochemistry. RESULTS: The onset of cirrhoses was noted from the twelfth week. Preneoplastic foci, hepatocellular adenomas (HCA) and hepatocellular carcinomas (HCC) were noted from the second, eleventh and fifteenth week, respectively. The nodules (N1/N2/N3: 397/258/64) included regenerating nodule; RN (N1/N2/N3: 72.3%/15.9%/0%), HCA (N1/N2/N3: 27.2%/82.2%/7.6%) and HCC (N1/N2/N3: 0.5%/ 1.9%/92.4%). EGF-R was expressed in 12.5% of RN, 64.7% HCA and 75.2% HCC. TGF-alpha was expressed in 92.4% of RN, 91.3% HCA and 93.2% HCC. Sixty eight percent of TGF-alpha expressing nodules showed concurrent EGF-R expression. ErbB2 was expressed in 83.6% of RN, 72.9% HCA and 88.7% HCC. ErbB4 was expressed in 95.2% of RN, 86.3% HCA and 62.5% HCC. CONCLUSIONS: Increased expression of EGF-R and decreased expression of ErbB4, might be related with tumor progression during DEN-induced hepatocarcinogenesis.
Adenoma, Liver Cell/chemically induced/metabolism/pathology ; Animals ; Carcinoma, Hepatocellular/chemically induced/*metabolism/pathology ; Diethylnitrosamine ; Glutathione Transferase/metabolism ; Immunohistochemistry ; Liver Neoplasms, Experimental/chemically induced/*metabolism/pathology ; Male ; Rats ; Rats, Wistar ; Receptor, Epidermal Growth Factor/*metabolism ; Receptor, erbB-2/*metabolism ; Transforming Growth Factor alpha/*metabolism

To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.
Actins ; metabolism ; Alanine Transaminase ; blood ; Animals ; Antineoplastic Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Bile ; chemistry ; Bilirubin ; blood ; Body Weight ; drug effects ; Carcinoma, Hepatocellular ; blood ; chemically induced ; pathology ; Diethylnitrosamine ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; pathology ; Liver Neoplasms, Experimental ; blood ; chemically induced ; pathology ; Male ; Powders ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Ursidae

To study the effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl(4)/ethanol. The wild-type mice (Smad4 +/+) and the Smad4 knockout mice (Smad4 +/-) were injected subcutaneously with carbon tetrachloride(CCl(4))/ethanol twice a week for twenty weeks. The expression of Smad4, TGFbeta1, Smad2, Smad3, Smad6, TIMP1, MMP2 and MMP9 was detected by RT-PCR. In the cirrhotic liver, the expression of Smad4 mRNA was significantly higher than that in the normal liver. Comparing with wild-type mice (Smad4 +/+), the TGFbeta1-Smad4 signaling was markedly attenuated in the Smad4 knockout mice (Smad4 +/-). After induction by CCl(4)/ethanol, the hepatic fibrosis in the Smad4 knockout mice (Smad4 +/-) was obviously alleviated compared with the wild-type mice (Smad4 +/+), and the incidence rate of hepatocarcinogenesis of the former was also lower than that of the latter(32.0% vs 41.9%). These results indicate that knocking out Smad4 can delay the progression of liver fibrosis and liver cancer.
Animals ; Carbon Tetrachloride ; administration & dosage ; Disease Models, Animal ; Ethanol ; administration & dosage ; Female ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Mice ; Mice, Knockout ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Smad Proteins ; genetics ; metabolism ; Smad4 Protein ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo screen the differentially expressed proteins especially at the precancerous stage of diethylnitrosamine (DEN) induced hepatocarcinogenesis by comparative proteome research.

METHODSRats were divided into normal and DEN groups and sacrificed periodically. The liver samples were stained with gamma-glutamyl transpeptidase (GGT) and HE to distinguish the preneoplastic lesion (pre-HCC) from the normal and HCC tissues. The two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS/MS) were then applied to analyze the differentially expressed protein between pre-HCC and normal tissues, pre-HCC and HCC, as well as HCC and normal tissues. A few of the candidate proteins such as laminin receptor 1 (67LR) and agmatinase were validated by Western blot and RT-PCR.

RESULTSTotally, there were 82 proteins that differentially expressed two fold or more in one kind of tissues sample than the other, 47 of which occurred in the pre-HCC tissues. Eight proteins including 67LR were consistently up-regulated from normal tissue to pre-HCC and then to HCC tissues, while 22 proteins including agmatinase showed progressively down-regulated in these tissues samples.

CONCLUSIONThe protein expression profiles are different during the process of hepatocarcinogenesis. Further study on the differentially expressed protein, especially these upregulated in the precancerous stage such as 67LR and agmatinase, might contribute to prevention and early diagnosis of human HCC.

Animals ; Blotting, Western ; Carcinoma, Hepatocellular ; chemically induced ; metabolism ; pathology ; Diethylnitrosamine ; Liver ; metabolism ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Neoplasm Proteins ; metabolism ; Precancerous Conditions ; metabolism ; pathology ; Proteins ; metabolism ; Proteome ; Rats ; Rats, Wistar ; Receptors, Laminin ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Ureohydrolases ; metabolism ; gamma-Glutamyltransferase

OBJECTIVETo investigate the changes in transforming growth factor beta 1 (TGF-beta1)/Smads signaling pathway in rats with chemical hepatocarcinogenesis.

METHODSFresh diethylnitrosamine (DENA) solution was administered in SD rats to induce hepatocellular carcinoma (HCC). The protein expressions of TGF-beta1, phosphorylated Smad2, Smad4 and Smad7 were detected in these rats with immunohistochemistry, and the mRNA expression of Smad4 was evaluated with RT-PCR.

RESULTSCirrhotic nodules occurred in the rats 8 weeks after DENA treatment, and HCC nodules were found 16 weeks after the treatment. In the normal liver tissue, very low levels of TGF-beta1 and Smad4 expressions, low Smad7 expression and high phosphorylated Smad2 expression were detected. The development of liver cirrhosis was accompanied by increased expressions of TGF-beta1, Smad4 and Smad7 but at 8 weeks after DENA treatment, the expression of phosphorylated Smad2 was significantly decreased, followed then by gradual increment till nearly the normal level. Twenty-two weeks after DENA treatment, Smad4 expression in liver tissue decreased markedly as compared with the levels at 8 and 16 weeks. The expressions of Smad4 and phosphorylated Smad2 in the HCC tissue was significantly lower than those in normal liver tissue.

CONCLUSIONHepatocarcinogenesis involves very complex mechanisms, can can be related partially to the decreased Smad4 and phosphorylated Smad2 expression and TGFbeta1 and Smad7 overexpression in advanced stage of liver cirrhosis.

Animals ; Diethylnitrosamine ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism