DEN-induced Hepatocarcinogenesis In Rats Research Articles

Lactobacillus acidophilus ATCC 4356 Exopolysaccharides Suppresses Mediators of Inflammation through the Inhibition of TLR2/STAT-3/P38-MAPK Pathway in DEN-Induced Hepatocarcinogenesis in Rats

Probiotics have been suggested as a safe and cost-effective approach to prevent or treat hepatocellular carcinoma (HCC). Some of the exopolysaccharides (EPSs) produced by lactic acid bacteria confer health benefits such as immunomodulatory and antitumor activities. The present study was therefore aimed to investigate the immunomodulatory effect of Lactobacillus acidophilus ATCC 4356 EPSs against diethylnitrosamine (DEN) and gamma radiation (IR) induced HCC either as prevention or treatment in male rats’ model. Biochemical results revealed a significant increase in serum ALT and γ-GT activities as well as MDA, IL-17, TGF-β1, signal transducer and activator of transcription-3 protein (STAT3), mitogen-activated protein kinase p38 (p38MAPK) levels in the liver tissue. The gene expression level of the liver toll-like receptor 2 (TLR-2) gene was also increased. However, prevention and treatment with EPSs ameliorated most of the investigated parameters. The histopathological observations of liver tissues were in agreement with restored biochemical results. In conclusion, Lactobacillus acidophilus ATCC 4356 EPSs are efficacious control against HCC throughout the regulation of TLR2/STAT-3/P38-MAPK Pathway associated with inflammation. Therefore, our novel EPSs ATCC 4356 could be used as a good, safe and effective probiotic to prevent hepatocarcinogenesis in suspected patients.

Protective Effects of Defatted Sticky Rice Bran Extracts on the Early Stages of Hepatocarcinogenesis in Rats.

Use of natural products is one strategy to lessen cancer incidence. Rice bran, especially from colored rice, contains high antioxidant activity. Cancer chemopreventive effects of hydrophilic purple rice bran extract (PRBE) and white rice bran extract (WRBE) on carcinogen-induced preneoplastic lesion formation in livers of rats were investigated. A 15-week administration of PRBE and WRBE did not induce hepatic glutathione S-transferase placental form (GST-P) positive foci formation as the biomarker of rat hepatocarcinogenesis. PRBE and WRBE at 500 mg/kg body weight significantly decreased number and size of GST-P positive foci in diethylnitrosamine (DEN)-initiated rats. The number of proliferating nuclear antigen positive hepatocytes were also reduced in preneoplastic lesions in both PRBE and WRBE fed DEN-treated rats. Notably, the inhibitory effect on GST-P positive foci formation induced by DEN during the initiation stage was found only in rats treated by PRBE for five weeks. Furthermore, PRBE attenuated the expression of proinflammatory cytokines involving genes including TNF-α, iNOS, and NF-κB. PBRE contained a higher number of anthocyanins and other phenolic compounds and vitamin E. PRBE might protect DEN-induced hepatocarcinogenesis in rats via attenuation of cellular inflammation and cell proliferation. Anthocyanins and other phenolic compounds, as well as vitamin E, might play a role in cancer chemopreventive activity in rice bran extract.

Olive fruit (Olea europaea L.): Chemopreventive effect in the rat model of hepatocellular carcinoma

Primary liver cancer or hepatocellular carcinoma (HCC) is an aggressive tumor with poor prognosis. Olea europaea L. commonly called olive contains diverse phytochemical constituents that have preventive effects against different diseases. The purpose of this investigation was to study the chemopreventive effect of aqueous extract of Olea europaea L. (AOE) against diethylnitrosamine (DEN)-induced HCC in rats. Olive-treated rats were pretreated with AOE intragastrically at three different doses two weeks prior to DEN injection that continued until 8 weeks. At three different time points, blood samples were collected for biochemical analysis. In the present investigation, a significant decrease in serum biomarkers of liver damage and cancer, including alfa-fetoprotein (AFP), gamma glutamyl transpeptidase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) were observed in AOE supplemented animals when compared to DEN-treated rats. In addition, AOE counteracted DEN-induced hepatic oxidative stress in rats illustrated by the restoration of reduced glutathione (GSH) and the reduction of lipid peroxidation in HCC rats. Furthermore, AOE prevented increase of relative liver weight as a prognostic marker in HCC. Finally, histopathological investigation in the liver sections confirmed the preventive effect of AOE against DEN-induced HCC. To conclude, olive has a preventive effect against DEN-induced hepatocarcinogenesis in rats.

A novel curcumin analogue is a potent chemotherapy candidate for human hepatocellular carcinoma.

Curcumin (CUR) has been demonstrated to protect against carcinogenesis and to prevent tumor development in cancer; however, the clinical application of CUR is limited by its instability and poor metabolic properties. The present study offers an strategy for a novel CUR analogue, (1E,4E)-1,5-bis(2-bromophenyl)penta-1,4-dien-3-one (GL63), to be used as a potential therapeutic agent for hepatocellular carcinoma (HCC) in vitro and in vivo. The current study demonstrated that GL63 exhibited more potent inhibition of proliferation of HCC cells than CUR. GL63 induced G0/G1 phase cell cycle arrest and apoptosis in SK-HEP-1 cells in a dose-dependent manner, and was more potent than CUR, according to the flow cytometry data. The present study demonstrated for the first time that the inhibition of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway by GL63 resulted in a protective effect against HCC cell growth. GL63 was more effective than CUR in regulating STAT3 downstream targets, which contributed to the suppression of cell proliferation and the induction of cell apoptosis. In addition, the effects of GL63 were tested in a model of N-nitrosodiethylamine (DEN)-induced HCC in Wistar rats. Although macroscopic and microscopic features suggested that both GL63 and CUR were effective in inhibiting DEN-induced hepatocarcinogenesis, GL63 exerted a stronger effect than CUR. Immunohistochemical analysis for proliferating cell nuclear antigen demonstrated significant differences among the DEN-bearing non-treated, DEN-bearing GL63-treated and DEN-bearing, CUR-treated groups (P=0.039). It was concluded that GL63 was a potent agent able to suppress the proliferation of HCC cells by inhibition of the JAK2/STAT3 signaling pathway, with more favorable pharmacological activity than CUR, and may be a more potent compound for the prevention of DEN-induced hepatocarcinogenesis in rats than CUR.

Preventive Effect of Hydrazinocurcumin on Carcinogenesis of Diethylnitrosamine-induced Hepatocarcinoma in Male SD Rats

The purpose of the present study was to evaluate the preventive effects of hydrazinocurcumin (HZC) on diethylnitrosamine (DEN)-induced hepatocarcinogenesis in a male Sprague Dawley (SD) rat model. One hundred and twenty male SD rats used in this study were divided into six groups. Those receiving DEN with curcumin (CUR) or HZC were studied compared with the DEN-alone group. The study demonstrated that DEN induced severe histological and immunohistochemical changes in liver tissues, significantly increasing the levels of liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and total bilirubin level (TBL)). The hepatocarcinoma incidences were 100.0%, 36.7% and 20.0% in the DEN-alone, DEN-CUR and DEN-HZC groups, respectively. Although macroscopic and microscopic features suggested that both CUR and HZC were effective in inhibiting DEN- induced hepatocarcinogenesis, HZC was exerted a stronger influence. Immunohistochemical analysis with PCNA demonstrated significantly differences among the groups (all P < 0.05). Taken together, the results suggested application of CUR and HZC could prevent the occurrence of carcinogenesis and HZC may be a more potent compound for prevention of DEN-induced hepatocarcinogenesis in rats than CUR.

Anticarcinogenic activity of nanoencapsulated quercetin in combating diethylnitrosamine-induced hepatocarcinoma in rats

Hepatocellular carcinoma is the most common primary hepatic malignancy worldwide. N-Nitroso compounds act as strong carcinogens in various animals, including primates. Diethylnitrosamine (DEN) is a well known carcinogenic substance, which induces hepatic carcinoma. The theme of the study was to evaluate the therapeutic efficacy of nanoencapsulated flavonoidal quercetin (3,5,7,3',4'-pentahydroxy flavone, QC) in combating DEN-induced hepatocarcinogenesis in rats. DEN induced a substantial increase in relative liver weights with proliferation and development of hyperplastic nodules. A significant increase in hepatocellular and nephrotoxicity indicated by serum alkaline phosphatase, aspartate transaminase, alanine transaminase, urea, and creatinine was observed in DEN-treated animals. Maximum protection from such toxicity was provided by nanoparticulated QC. Elevated levels of conjugated diene in DEN-treated rats were lowered significantly by nanoparticulated QC. Antioxidant levels in hepatic cells were reduced significantly by the induction of DEN. Nanoparticulated QC was found most potent for complete prevention of DEN-induced reduction in antioxidant levels in the liver. Upregulation of glutathione-S-transferase activity by DEN induction was reduced maximally by nanoencapsulated QC. Nanoencapsulated QC completely protected the mitochondrial membrane of the liver from carcinoma mediated by DEN injection. A significant correlation could be drawn between DEN-induced tissue reactive oxygen species generation and cytochrome C expression in the liver. Nanoencapsulated QC completely prevented the DEN-induced cytochrome C expression in the liver significantly.

Chemopreventive effect of saikosaponin-d on diethylinitrosamine-induced hepatocarcinogenesis: Involvement of CCAAT/enhancer binding protein�β and cyclooxygenase-2

Cyclooxygenase-2 (COX-2) and CCAAT/enhancer binding protein β (C/EBPβ) have been shown to be involved in inflammation and carcinogenesis, and our previous study revealed that they were co-overexpressed in human hepatocellular carcinoma (HCC) tissue and a positive correlation was found. Saikosaponin-d (SSD), a triterpene saponin extracted from Bupleurum falcatum L. (Umbelliferae), is known to exert inhibitory effects on COX-2 expression, together with inflammation and hepatic fibrosis. These findings prompted us to investigate the chemopreventive potential of SSD against hepatocarcinogenesis and its possible molecular mechanism in vivo. An experimental model with diethylinitrosamine (DEN)-treated Sprague Dawley rats was used in the present study. DEN (50 mg/kg body weight) and SSD (2 mg/kg body weight) were intraperitoneally injected weekly and daily, respectively. Administration of SSD alone had no side effects. The liver nodule formation, tumorous invasion to surrounding organs and increased cellular atypia induced by DEN were markedly reduced by SSD in the SSD + DEN group compared with the DEN group. On the other hand, immunohistochemical staining demonstrated that the expression of COX-2 and C/EBPβ proteins was significantly increased in tumor cells and macrophages of liver tissue from DEN-treated rats, whereas the expression of the two proteins was markedly lowered in the SSD + DEN group. Overall, our results suggest that SSD prevents DEN-induced hepatocarcinogenesis in rats through inhibition of C/EBPβ and COX-2, providing indispensable experimental evidence for the clinical application of SSD as a novel chemopreventive agent against HCC in the future.

Preventive effect of JTE-522, a selective cyclooxygenase-2 inhibitor, on DEN-induced hepatocarcinogenesis in rats

Background Chemopreventive effect of a selective cyclooxygenase-2 (COX-2) inhibitor JTE-522 on diethylnitrosamine (DEN)-induced hepatocarcinogenesis was evaluated in Wistar rats. Methods Animals in the control group (G1) were injected with phosphate buffered saline (PBS), those in hepatocellular carcinoma (HCC) group (G2) were injected with DEN with regular foods for 14 weeks, and those in the treatment groups were injected with DEN for 14 weeks fed with JTE-522 for 7 (G3) and 14 weeks (G4), respectively. Proliferation and precancerous lesions were evaluated by expression levels of proliferating cell nuclear antigen (PCNA) and glutathione S-transferase-P (GST-P), respectively by immunohistochemistry and Western blot analysis. Apoptosis and oxidative stress were evaluated by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) and 8-hydroxy-2’-deoxyguanosine (8-OHdG) staining, respectively. Results After 14 weeks of the treatment, HCC was developed in G2, G3, and G4 showing no significant differences in gross appearance and histology of the liver among the three groups. There were no significant differences in the expression levels of PCNA and numbers of TUNEL and 8-OHdG positive cells in the liver among the three groups. However, GST-P positive area was significantly suppressed in G3 and G4 compared to G2. Conclusion Our data revealed that JTE-522 had a modest inhibitory effect on hepatocarcinogenesis in rats in a manner independent of induction of apoptosis and inhibition of oxidative stress.

Protective Role of Lycopene Against Diethylnitrosamine Induced Experimental Hepatocarcinogenesis

Lycopene was considered as a major carotenoid in the human diet for only the last few centuries. Recently lycopene has been found to possess chemoprotective effect against gastrointestinal tract, urinary bladder, prostate and breast cancers. In the present study, the protective effect of lycopene, the natural extract from tomato pomace against diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) formation in rats was investigated. Four groups of male albino rats at the age of 6 weeks were studied: 1) control (C), 2) DEN, i.p. injected 5 times with doses of 200 mg/kg b.w., one dose every two days. 3) Lycopene, orally given 0.12 mg/rat/day. 4) Lycopene + DEN, the treatment with lycopene started 7 days before DEN injection and continued till the end of the experimental period. Rats in group 2 and 4 were treated with Phenobarbital (PB) at a dose of 500 ppm in the drinking water as a tumor promoter. PB treatment started with the first injection of DEN and continued till the end of the experimental period. The results indicated that DEN caused HCC nodules as evidenced by a remarkable significant increase in α-L-fucosidase and metalloproteinases (MMPs) enzyme activity in both serum and cytosol. Increased activity of these enzymes is a marker for both preneoplastic and carcinoma lesions. Administration of lycopene prior to DEN injection protected rats from DEN-induced HCC as evidenced by a significant descent in α-L-fucosidase and MMPs enzymes activity in both serum and cytosol. The histological investigation of liver tissue confirmed these results. DEN group showed proliferated hyperplastic and anaplastic hepatocellular nodules surrounded with thin layer of fibroblastic cells, desmoplasia and inflammatory cells infiltrations, focal hepatic haemorrhages and necrosis. Meanwhile, lycopene + DEN group showed no histopathological changes. These results indicate that lycopene effectively inhibits DEN-induced hepatocarcinogenesis in rats.

Protective Role of Lycopene Against Diethylnitrosamine Induced Experimental Hepatocarcinogenesis

Lycopene was considered as a major carotenoid in the human diet for only the last few centuries. Recently lycopene has been found to possess chemoprotective effect against gastrointestinal tract, urinary bladder, prostate and breast cancers. In the present study, the protective effect of lycopene, the natural extract from tomato pomace against diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) formation in rats was investigated. Four groups of male albino rats at the age of 6 weeks were studied: 1) control (C), 2) DEN, i.p. injected 5 times with doses of 200 mg/kg b.w., one dose every two days. 3) Lycopene, orally given 0.12 mg/rat/day. 4) Lycopene + DEN, the treatment with lycopene started 7 days before DEN injection and continued till the end of the experimental period. Rats in group 2 and 4 were treated with Phenobarbital (PB) at a dose of 500 ppm in the drinking water as a tumor promoter. PB treatment started with the first injection of DEN and continued till the end of the experimental period. The results indicated that DEN caused HCC nodules as evidenced by a remarkable significant increase in α-L-fucosidase and metalloproteinases (MMPs) enzyme activity in both serum and cytosol. Increased activity of these enzymes is a marker for both preneoplastic and carcinoma lesions. Administration of lycopene prior to DEN injection protected rats from DEN-induced HCC as evidenced by a significant descent in α-L-fucosidase and MMPs enzymes activity in both serum and cytosol. The histological investigation of liver tissue confirmed these results. DEN group showed proliferated hyperplastic and anaplastic hepatocellular nodules surrounded with thin layer of fibroblastic cells, desmoplasia and inflammatory cells infiltrations, focal hepatic haemorrhages and necrosis. Meanwhile, lycopene + DEN group showed no histopathological changes. These results indicate that lycopene effectively inhibits DEN-induced hepatocarcinogenesis in rats.

Dietary Supplementation of the Citrus Antioxidant Auraptene Inhibits N,N-Diethylnitrosamine-Induced Rat Hepatocarcinogenesis

Objectives: We have previously reported that an antioxidant, auraptene (AUR), isolated from citrus fruit effectively inhibits chemically induced carcinogenesis in digestive tracts, such as the oral cavity, esophagus and large bowel. In this study, we investigated the modifying effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in male F344 rats in two different experiments to determine whether the compound exerts a cancer-chemopreventive action in other organs. Methods: In the first experiment, animals were fed diets containing AUR at dose levels of 100 and 500 ppm for 7 weeks 1 week before, during, and 1 week after the start of liver carcinogenesis induced by DEN (40 ppm in drinking water for 5 weeks) to predict the modulatory effect on hepatocarcinogenesis. After 7 weeks, the numbers of hepatocellular enzyme-altered foci (EAF; cm²) which stained positive for the placental form of glutathione S-transferase (GST-P) and transforming growth factor (TGF)-α were determined on immunohistochemically stained sections. In the second experiment conducted to confirm the findings, animals subjected to DEN treatment were fed AUR-containing diets (100 and 500 ppm) during either the initiation stage (‘initiation’ feeding for 7 weeks) or post-initiation phase (‘post-initiation’ feeding for 25 weeks) of DEN-induced hepatocarcinogenesis. Results: In the first experiment, feeding with AUR at both doses during DEN exposure decreased the mean numbers of GST-P-positive and TGF-α-positive EAF/cm², and the reduction in the number of TGF-α-positive EAF by feeding 500 ppm AUR was statistically significant (p < 0.005). In the second experiment, the ‘initiation’ feeding with 500 ppm AUR significantly inhibited the incidence (33 vs. 83%, p = 0.000511) and multiplicity (0.67 ± 1.09 vs. 1.96 ± 1.85, p < 0.005) of liver cell carcinoma. Also, the ‘post-initiation’ feeding with AUR at both doses significantly reduced the development of hepatocellular carcinoma (100 ppm: incidence, 15%, p = 0.000006; multiplicity: 0.25 ± 0.64, p < 0.001; 500 ppm: incidence, 11%, p = 0.000002; multiplicity, 0.26 ± 0.81, p < 0.001). In addition, AUR feeding reduced cell proliferation and the apoptotic index in liver cell neoplasms. Conclusions: The results suggest that the citrus antioxidant AUR is a potential chemopreventive agent against DEN-induced hepatocarcinogenesis in rats.

Membrane and cytosolic protein phosphorylation patterns in the early stages of DEN-induced hepatocarcinogenesis in rats fed a high or low protein diet.

The membrane and cytosolic protein phosphorylation patterns in the early stages of diethylnitrosamine-induced rat liver carcinogenesis, promoted by 2-acetylaminofluorene in the diet plus partial hepatectomy (DEN-AAF-PH), were analyzed by two-dimensional gel electrophoresis in animals fed a low protein (5% casein) diet, or the original high protein (24% casein) diet, in order to modulate the development of GST-P-positive preneoplastic lesions. Compared with untreated controls, membrane and cytosolic protein phosphorylation patterns changed only slightly in low protein-fed rats 7 days post-hepatectomy, with no appearance of enzyme-altered hyperplastic foci in the liver sections. By contrast, high protein-fed animals demonstrated GST-P-positive preneoplastic lesions 7 days post-hepatectomy and several acidic and more basic high M(r) phosphorylated membrane (between 97 and 116 kDa) as well as cytosolic (between 97 and 200 kDa) proteins could be detected. In the presence of enzyme-altered hepatocytes in the liver sections, low protein-fed rats demonstrated at 60 days post-hepatectomy cytosolic protein phosphorylation patterns remarkably similar to those shown by 24% casein-fed animals at 7 days post-hepatectomy, suggesting close correlation between protein phosphorylation patterns and development of preneoplastic lesions during the early stages of DEN-AAF-PH liver carcinogenesis. This may arise by a constitutive activation of one or more signal transduction pathways, possibly involving protein kinase C, during liver tumour promotion.

Effect of dietary fat on diethylnitrosamine induced hepatocarcinogenesis in Wistar rats.

In a diethylnitrosamine (DEN) induced hepatoma model, the effect of fish oil, a rich source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and ground nut oil, a rich source of oleic acid (OA) and linoleic acid (LA), was studied. Both 20% fish oil and ground nut oil inhibited DEN-induced hepatocarcinogenesis in rats. Contrary to general belief, an inverse correlation was observed between the development of neoplastic nodules and the amount of lipid peroxides formed in the high fat (20% fish oil and ground nut oil) diet groups, indicating that the higher the lipid peroxides formed, the lower the incidence of liver tumours in DEN-treated animals. Results of the analysis of liver fatty acid composition of the phospholipid fraction suggested that DEN and fish oil treatment can inhibit the activity of the delta-6 and 5-desaturases. It is concluded from the results of the present study that a 'high fat' diet and lipid peroxidation can modulate DEN-induced hepatocarcinogenesis in rats.