Hepatocarcinogenesis In Rats Research Articles

Attenuation of the Wnt/β-catenin/TCF pathway by in vivo interferon-α2b (IFN-α2b) treatment in preneoplastic rat livers

Wnt/β-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, promoting cell growth and proliferation. Forkhead box containing protein class O (FOXO) transcription factors compete with TCF for β-catenin binding, particularly under cellular oxidative stress conditions. Contrary to β-catenin/TCF, β-catenin/FOXO promotes the transcription of genes involved in cell cycle arrest and apoptosis. We have previously demonstrated that in vivo interferon-α2b (IFN-α2b) administration induces apoptosis in preneoplastic livers, a mechanism mediated by reactive oxygen species (ROS) and transforming growth factor-β1 (TGF-β1). This study was aimed to assess the status of the Wnt/β-catenin/TCF pathway in a very early stage of rat hepatocarcinogenesis and to further evaluate the effects of in vivo IFN-α2b treatment on it. We demonstrated that the Wnt/β-catenin/TCF pathway is activated in preneoplastic rat livers. More important, in vivo IFN-α2b treatment inhibits Wnt/β-catenin/TCF pathway and promotes programed cell death possibly providing a link with FOXO pathway.

Application of a two-phenotype color-shift model with heterogeneous growth to a rat hepatocarcinogenesis experiment

The color-shift model (CSM) was introduced by Kopp-Schneider et al. [1] to describe formation and progression of foci of altered hepatocytes (FAH). It incorporates the field-effect hypothesis which postulates that entire colonies of altered hepatocytes simultaneously alter their phenotype. In the original CSM, FAH grow with deterministic growth rate and change their phenotype after an exponentially distributed waiting time. A modification of the original color-shift model (CSMbeta) is presented here in which the growth rate varies from focus to focus according to a beta distribution. The concept of an exponentially distributed waiting time to phenotype change is modified to the concept of a random radius at which phenotype changes and this radius is modelled as beta distributed. The original and the modified CSM are applied to data from an initiation-promotion rat hepatocarcinogenesis experiment with diethylnitrosomorpholine (DEN) and N-nitrosomorpholine (NNM), in which two phenotypes of FAH were observed in hematoxilin/eosin (H&E) stained liver sections. The Cramer-von-Mises Distance is used as a measure for the discrepancy between empirical and theoretical size distributions. Comparisons of model fit show that considerable improvement is obtained for CSMbeta compared to the original CSM.

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.

Angioprevention is Implicated in Resveratrol Chemoprevention of Experimental Hepatocarcinogenesis

Hepatocellular carcinoma (HCC), a hypervascular tumor, is one of the most common and lethal cancers worldwide. We previously showed that resveratrol, a dietary polyphenol, inhibits rat liver carcinogenesis through antioxidative and antiinfl ammatory mechanisms. As resveratrol possesses antiangiogenic properties, we hypothesize that it may exert chemoprevention of hepatocarcinogenesis by suppressing angiogenesis. The antiangiogenic effect of resveratrol was investigated by analyzing livers from our previous study in which resveratrol (50-300 mg/kg) exerted chemopreventive action against diethylnitrosamine (DENA)-induced rat liver tumorigenesis. Hepatic angiogenesis was evaluated by microvessel density (MVD) based on immunohistochemical staining of CD31-positive endothelial cells. The expression of hepatic vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by immunohistochemistry. Sixteen weeks following the administration of DENA, there was a substantial increase in hepatic MVD as compared to normal liver. A dramatic increase in hepatic VEGF and HIF-1α was observed in DENA-treated animals compared to normal counterparts. Treatment with resveratrol dose-dependently abrogated the DENA-induced increased MVD as well as the elevated expression of VEGF and HIF-1α. DENA-initiated hepatocarcinogenesis in rats exhibits substantial neovascularization possibly due to overexpression of VEGF upregulated by HIF-1α. Resveratrol exerts a remarkable angiosuppressive effect in DENA-evoked hepatocellular carcinogenesis. Resveratrol-mediated inhibition of angiogenesis could be achieved by suppressing VEGF expression through downregulation of HIF1α. These results, in conjunction with our previous fi ndings, provide evidence that angiosuppression is involved in resveratrol-mediated chemoprevention of rat liver carcinogenesis and support the potential use of this natural agent in the prevention and therapy of HCC.

Amelioration effects against N-nitrosodiethylamine and CCl(4)-induced hepatocarcinogenesis in Swiss albino rats by whole plant extract of Achyranthes aspera.

Objective:The prevalence of oxidative stress may be implicated in the etiology of many pathological conditions. Protective antioxidant action imparted by many plant extracts and plant products make them a promising therapeutic drug for free-radical-induced pathologies. In this study, we assessed the antioxidant potential and suppressive effects of Achyranthes aspera by evaluating the hepatic diagnostic markers on chemical-induced hepatocarcinogenesis.Materials and Methods:The in vivo model of hepatocarcinogenesis was studied in Swiss albino rats. Experimental rats were divided into five groups: control, positive control (NDEA and CCl4), A. aspera treated (100, 200, and 400 mg/kg b.w.). At 20 weeks after the administration of NDEA and CCl4, treated rats received A. aspera extract (AAE) at a dose of 100, 200, and 400 mg/kg once daily route. At the end of 24 weeks, the liver and relative liver weight and body weight were estimated. Lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and reduced glutathione (GSH) were assayed. The hepatic diagnostic markers namely serum glutamic oxaloacetic transminase (AST), serum glutamic pyruvate transminase (ALT), serum alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), and bilirubin (BL) were also assayed, and the histopathological studies were investigated in control, positive control, and experimental groups.Results:The extract did not show acute toxicity and the per se effect of the extract showed decrease in LPO, demonstrating antioxidant potential and furthermore no change in the hepatic diagnosis markers was observed. Administration of AAE suppressed hepatic diagnostic and oxidative stress markers as revealed by decrease in NDEA and CCl4 -induced elevated levels of SGPT, SGOT, SALP, GGT, bilirubin, and LPO. There was also a significant elevation in the levels of SOD, CAT, GPx, GST, and GSH as observed after AAE treatment. The liver and relative liver weight were decreased after treatment with AAE in comparison to positive control group. The architecture of hepatic tissue was normalized upon treatment with extract at different dose graded at 100, 200, and 400 mg/kg. b.w. in comparison to positive control group.Conclusion:These results suggest that A. aspera significantly alleviate hepatic diagnostic and oxidative stress markers which signify its protective effect against NDEA and CCl4-induced two-stage hepatocarcinogenesis.

Inhibition by licorice flavonoid oil of glutathione S-transferase–positive foci in the medium-term rat hepatocarcinogenesis bioassay

Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of hydrophobic licorice polyphenols in medium-chain triglycerides. Recently, it was reported that licorice and its derivatives have anticarcinogenic activity in some types of tumors. However, the anticarcinogenic activity has not been identified in the liver, which is a major target organ for carcinogenesis in human. Therefore, we hypothesized that LFO has antihepatocarcinogenic activity, and we tested this hypothesis using the rat medium-term liver bioassay for carcinogens. Six-week-old male F344 rats (15 animals/group) received N-diethylnitrosamine (200 mg/kg by intraperitoneal injection) to initiate carcinogenesis. From the second week after initiation, animals received a 6-week regimen of either LFO concentrate solution (0, 150, 300, or 600 mg/kg) intragastrically or phenobarbital sodium salt in the diet (500 ppm) as a positive control. During the third week after initiation, animals were subjected to a two-thirds partial hepatectomy. During the eighth week of the treatment period, liver samples were taken from animals and examined immunohistochemically for expression of glutathione S-transferase placental form. No increase in the number of glutathione S-transferase placental form-positive liver foci was observed in all LFO groups compared with the negative control (solvent) group, and the number of foci in the 600 mg/kg LFO group was significantly lower than that in the negative control group. These results indicate that LFO concentrate solution has a significant inhibitory effect on liver carcinogenesis at 600 mg/kg.

Fucosylated Glycans Associated With Development and Metastasis of Hepatocellular Carcinoma Cells*

Dynamic fucosylation of glycoprotein especially 80 ku which bound to UEA and LCA during the course of rat hepatocarcinogenesis was investigated. In patient hepatocellular carcinoma, more UEA-and LCA-bound proteins were also observed in patients with high metastatic potential than those with low metastatic potential. Fucosylated glycans constitute important adhesion molecules such as Lewis antigens. A differential expression pattern of Lewis antigens was further confirmed on various metastasis potential hepatocellular carcinoma cells (HCC). high metastatic hepatocellular carcinoma cell line (HMCC97H) expressed much more Lewis x and b than low metastasis HMCC97L cells. Moreover, surface Lewis x, or b expression level declined significantly after the cells were treated by retinoic acid. Not only in experimental metastasis foci, but in HCC as well, both α1,3/1,2 and α1,6 fucosyltransferase activities were quite high. After retinoic acid treatment, α1,3/1,2 fucosyltransferase activities were significantly inhibited, Lewis x on epidermal growth factor receptor reduced, and the EGFR was less phosphorylated. These results suggested that fucosylated glycans such as Lewis x played an important role in HCC development and metastasis.

Astaxanthin intervention ameliorates cyclophosphamide-induced oxidative stress, DNA damage and early hepatocarcinogenesis in rat: Role of Nrf2, p53, p38 and phase-II enzymes

Cyclophosphamide, an alkylating agent, disturbs the oxidant and antioxidant balance that is associated with several unwanted toxic effects and induction of secondary cancers. Astaxanthin is a powerful antioxidant and possess several beneficial effects against various human diseases and physiological disorders. The present study was aimed to investigate the effects of astaxanthin against cyclophosphamide-induced oxidative stress, DNA damage, cell death and induction of GST-P foci in rat liver. Further attempt has been made to study the influence of astaxanthin on antioxidant response element (ARE) and the transcription factor Nrf2 (nuclear factor E 2-related factor 2) in the induction of phase-II enzymes NAD(P)H: quinine oxidoreductase-1(NQO-1) and Hemoxygenase-1 (HO-1). Both pre- and post-treatment with astaxanthin (25 mg/kg) decreased cyclophosphamide-induced oxidative stress and DNA damage in the liver as evident from the restoration in malondialdehyde and glutathione level as well as modified comet assay parameters. Significant decrease in the number as well as area of GST-P foci in rat hepatocytes was observed with astaxanthin post-treatment. Treatment with astaxanthin significantly decreased the expression of p53 and p38 as compared to cyclophosphamide treated group. It was further observed that the level of Nrf2 and phase-II enzymes, i.e. NQO-1 and HO-1 were increased with astaxanthin treatment. The present study confirms that astaxanthin is a potent antioxidant and attenuates oxidative stress, DNA damage, cell death as well as induction of early hepatocarcinogenesis in rat induced by cyclophosphamide. Our results provide the evidence that one of the mechanism of chemoprotection offered by astaxanthin is mediated through Nrf2–ARE pathway.

Histone acetylation and steroid receptor coactivator expression during clofibrate‐induced rat hepatocarcinogenesis

Peroxisome proliferators (PPs), non-genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid beta-oxidation system, including bifunctional enzyme (BE) and 3-ketoacyl-CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague-Dawley rats, NC- and KS-type. The KS-type rats showed enhanced susceptibility to ethyl-alpha-chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS-type and preneoplastic foci developed after 6-8 weeks of treatment, whereas no foci developed in the NC-type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator-activated receptor (PPAR) alpha levels between them. Among various coactivators for PPARalpha, only steroid receptor coactivator (SRC)-3 level was higher in the KS-type. To investigate the association between PPARalpha and SRC-3 or other proteins, nuclear extracts from CF-treated livers were applied to a PPARalpha column. In the KS-type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC-3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARalpha associated to the BE enhancer in CF-treated KS-type, but not in the NC-type. Histone H3 acetylation was increased 11-fold in the KS-type by CF treatment but not in the NC-type. As BE and TH are responsible for acetyl-CoA production and SRC-3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS-type livers is due to acetylation-mediated transcriptional activation and epigenetic mechanisms might be involved in CF-induced rat hepatocarcinogenesis.

Chemopreventive effect of bacoside A on N-nitrosodiethylamine-induced hepatocarcinogenesis in rats

Chemoprevention is an effective approach to control hepatocarcinogenesis. Bacoside A, the active constituent of Bacopa monniera Linn., is anticipated to play a role in chemoprevention of liver cancer. In the present study, we investigated the chemopreventive effect of bacoside A against N-nitrosodiethylamine-induced hepatocarcinogenesis in an animal model. Administration of carcinogen showed a significant elevation in the levels of lipid peroxidation, serum tumor marker enzymes and liver injury marker enzymes with subsequent decrease in the levels of both hemolysate and liver antioxidant status. Bacoside A co-treatment maintained the N-nitrosodiethylamine-induced alterations at near normal level. Histopathological and electron microscopic study of the liver tissue also supports the above biochemical observations. From our findings we conclude that bacoside A is effective to prevent DEN-induced hepatocellular carcinoma by quenching lipid peroxidation and enhancing antioxidant status through free radical scavenging mechanism and having potential of protecting endogenous enzymatic and non-enzymatic antioxidant activity.

Supplementation of selenium reduces chemical hepatocarcinogenesis in male Sprague-Dawley rats

Selenium is an essential micronutrient mineral found mainly in soils and has been shown to prevent certain cancers in humans and animals. However, the dose and effects of selenium on liver cancer are controversial. The aim of this study was to investigate the effects of sodium selenite (4 mg/kg in drinking water) on chemically induced hepatocarcinogenesis in rats. Hepatocarcinogenesis was induced by a single intraperitoneal injection of diethyl nitrosamine (DEN) (200 mg/kg body weight) and 2 weeks later, the carcinogenic effect was promoted by 2-acetylaminofluorene (2-AAF) (0.02%). 44 Sprague-Dawley rats were divided into 6 groups: negative control, positive control (DEN+2-AAF), pre-selenium group (sodium selenite for 4 weeks, then DEN+2-AAF), pre-selenium control group (sodium selenite for 4 weeks, no DEN or 2-AAF), post-selenium group (sodium selenite for 8 weeks after 4 weeks of DEN injection) and post-selenium control group (sodium selenite for 8 weeks, no DEN or 2-AAF). Hematoxylin and eosin plus Gordon and Sweet’s methods were used to stain liver tissues. The results showed that the number and sizes of hepatic nodules in pre- and post-selenium treatment groups significantly decreased ( P<0.05) compared with the positive control. Microscopic analysis of pre- and post-selenium groups showed that the majority of nodules were hyperplastic with preserved liver architecture, whereas the positive control was full of neoplastic nodules with a completely disrupted liver architecture. Hence, pre- and post-selenium treatments can reduce the extent of liver cancer on chemically induced hepatocarcinogenesis in rats.

Biochemical studies on the effect of curcumin and embelin during N-nitrosodiethylamine/phenobarbital induced-hepatocarcinogenesis in wistar rats

The biochemical effects of administration of embelin a benzoquinone derivative from Embelia ribes and curcumin (diferuloyl methane) isolated from Curcuma longa against a 2 step hepatocarcinogenic regimen comprising of N-nitrosodiethylamine (DENA)/phenobarbital (PB) was studied in wistar strain male albino rats with respect to lipid profile, renal function tests and levels of blood glucose. Rats administered DENA/PB showed hyper cholesterolaemia, hyper triglyceridaemia, elevated low-density lipoproteins (LDL), free fatty acids (FFA), very-low-density lipoproteins (VLDL) levels and decreased urea levels. Pre- and co-treatment with embelin and curcumin for 14 weeks significantly prevented the biochemical alterations induced by DENA/PB. Results of our study suggest the protective and hypolipidemic effects of embelin and curcumin during chemically- induced hepatocarcinogenesis in wistar rats.

Effect of antisense oligomer in controlling c-raf.1 overexpression during diethylnitrosamine-induced hepatocarcinogenesis in rat

In ras-mediated signal transduction pathway, c-raf.1 is believed to have predominant oncogenic potential and has been found to be highly expressed in certain human and animal malignancies including hepatocellular carcinoma. In the present study, anticancer efficacy of antisense c-raf.1 oligomer on the inhibition of c-raf.1 mRNA overexpression during hepatocarcinogenesis was determined. Initially antiproliferating effect of the antisense oligomers was studied in vitro by measuring the rate of tritiated thymidine incorporation into DNA in rat hepatocellular carcinoma cells in culture medium. Based on the findings, the antisense treatment was carried out in rat hepatocarcinogenesis model-initiated with diethylnitrosamine and promoted using 2-acetylaminoflourene. Different drug-metabolizing enzymes, lipid peroxidation, liver morphology and histopathological studies along with c-raf.1 gene expression by in situ hybridization were performed. c-raf.1 antisense oligomers exhibited an inhibitory effect (approximately 68%) on cancer cell proliferation in vitro. Gross and microscopic examination of liver showed fewer (29%) and smaller hyperplastic nodules and preneoplastic lesions (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control group. Treatment of antisense c-raf.1 oligomers enhanced cytochrome P-450 content (81%) and reduced glutathione S-transferase activity (33%), UDP glucuronosyltransferase activity (74%) and MDA concentration (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control animals. The oligomer treatment also resulted in less expression in terms of c-raf.1 expressed lesion count as compared to carcinogen control group. The study demonstrates that the antisense oligomer targeted against c-raf.1 mRNA inhibits the overexpression of c-raf.1 gene during hepatocellular carcinoma in rats.

Cytokeratin 8/18 overexpression and complex formation as an indicator of GST-P positive foci transformation into hepatocellular carcinomas

Screening of the proteome of microdissected glutathione S-transferase placental form (GST-P) positive foci and normal-appearing liver on anionic (Q10), and cationic (CM10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays demonstrated significant overexpression of cytokeratin 8 (CK8; m/z 54,020), cytokeratin 18 (CK18; m/z 47,760), microsomal cytochrome 5A (m/z 15,224) and histone type 2 H2aa3 (m/z 15,964) in the livers of rats initiated with diethylnitrosamine (DEN) followed by 10 weeks on phenobarbital (PB) at a dose of 500 ppm. Furthermore, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with promotion of hepatocarcinogenesis by PB and the development of hepatocellular carcinomas. The data were confirmed by immunohistochemistry and real-time Q-PCR and profound overexpression of CK8 and CK18 (CK8/18) proteins and mRNAs were detected in several large size GST-P positive foci and liver tumors. A strong correlation between CK8/18 positive foci development and multiplicity of hepatocellular carcinomas was further observed. Moreover, elevation of CK8/18 was strongly associated with induction of cell proliferation in GST-P positive foci and tumors. In conclusion, our data imply that CK8/18 overexpression, those two cytokeratins complex formation associated with histone type 2 H2aa3 up-regulation and intermediate filament reorganization may drive neoplastic transformation of GST-P positive foci during rat hepatocarcinogenesis leading to the formation of hepatocellular carcinomas.

N-Nitrosodiethylamine과 사염화탄소로 유발된 흰쥐의 간암발생에 대한 인진호탕 추출액의 효과

본 실험은 N-nitrosodiethylamine(NDEA)와 사염화탄소(CCl₄)로 유발된 흰쥐의 간암 생성에 대한 인진호탕의 효과를 조사하였다. 실험은 8주 동안 실시하였고, 세 군으로 분류하였다. 정상군(Nor), 간암을 유발한 대조군(Con), 간암 유발 후 인진호탕 추출액(260 ㎎/㎏/day)을 투여한 실험군(IJH)으로 구분하였다. 체중은 정상군에 비하여 대조군에서 유의적으로 감소하였지만 실험군에서는 증가하였다. NDEA로 유발된 흰쥐의 혈청 AST, ALT, LDH, ALP와 AFP의 수준은 증가하였다. 반대로, 인진호탕 추출액을 처리했을 때, 혈청 AST, ALT, LDH, ALP와 AFP의 수준이 감소하였다. 흰쥐의 간에서 bcl-2 mRNA의 수준은 대조군에 비해 실험군에서 증가되었다. 그러나 c-myc mRNA의 수준은 대조군에 비해 감소하였다. 또한 NDEA로 손상된 간세포 조직의 공포화가 인진호탕 추출액의 투여로 감소하는 것을 관찰할 수 있었다. 이상의 결과로 보아 인진호탕 추출액 투여가 NDEA와 사염화탄소로 유도된 흰쥐의 간암 생성을 억제 또는 지연하는 것으로 사료된다.

Molecular Expression Analysis of β-Naphthoflavone-induced Hepatocellular Tumors in Rats

The present study was performed to characterize molecular expression levels of preneoplastic and neoplastic lesions induced by beta-naphthoflavone (BNF), an aryl hydrocarbon receptor (AhR) agonist in rat hepatocarcinogenesis. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and two weeks later, they were fed a diet containing 0% or 1% BNF for twenty-eight weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and sacrificed at week 30. Histopathologically, BNF increased the incidence and multiplicity of altered foci (1.7-fold and 3.3-fold) and hepatocellular adenomas (HCAs) (4.0-fold and 4.7-fold). Immunohistochemically, BNF increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in altered foci (2.3-fold) and HCAs (6.7-fold) compared with the surrounding tissue and decreased the staining of cell cycle regulators (P21, C/EBPalpha). In addition, loss of reactivity for AhR-regulated (CYP1A1, CYP1B1) molecules and increased reactivity of Nrf-2-regulated (AKR7, GPX2) molecules were also observed in proliferative lesions. Furthermore, increased staining of histone deacetylase (HDAC1) in the nucleus was prominent in HCAs. The differential expression patterns were confirmed at mRNA levels by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results suggest that enhanced cell proliferation and protection against oxidative stress play an important role in BNF-induced hepatocarcinogenesis in rats.

Effects of dietary supplementations of tomato extract vs. lycopene on nonalcoholic steatohepatitis related hepatocarcinogenesis in rats

Higher intake of tomatoes enriched in lycopene (LY) has been associated with lower risk for a variety of cancers. We recently showed that high‐fat diet (HFD) induced nonalcoholic steatohepatitis (NASH) and promoted diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis in a rat model (Wang Y et al., Int. J. Cancer 2009). Using the same model, we investigated the efficacy of equivalent dosage of dietary LY from either tomato extract (TE) or purified LY against NASH related early hepatocarcinogenesis. Six groups of rats (n=8/group) were injected with DEN and then fed either Lieber‐DeCarli control diet or HFD with or without TE or LY for 6 weeks. Results showed similar plasma and hepatic LY concentrations between TE and LY groups, moreover, both TE and LY supplementations in the HFD groups significantly reduced altered hepatic foci multiplicity, lipid peroxidation, expressions of PCNA and cyclinD1, phosphorylated‐ERK, and nuclear NF‐kB p65 subunit in the liver, as compared with the HFD alone. However, TE, but not LY, inhibited HFD‐upregulated inflammatory foci and mRNA levels of proinflammatory cytokines (TNFalpha, IL‐1beta, IL‐12, CD14) in the liver. These data indicate that both TE and LY inhibit NASH related early hepatocarcinogenesis whereas TE provides an additional anti‐inflammatory potency due to a possible synergistic effect of multiple constituents in tomatoes (Supported by NIH grant R01CA104932).Grant Funding SourceNIH grant R01CA104932

Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticarcinogenic agent. However, its short half-life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of "resistant hepatocyte" model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (p < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB-induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways.

Expression and Correlation of Hypoxia-inducible Factor-1α, Vascular Endothelial Growth Factor and Microvessel Density in Experimental Rat Hepatocarcinogenesis

An experimental rat hepatocellular carcinoma (HCC) model was established using diethylnitrosamine and N-nitrosomorpholine to induce carcinogenesis in Sprague-Dawley rats. During hepatocarcinogenesis, seven rats were sacrificed at 0, 4, 8, 12 and 16 weeks and 10 rats were sacrificed at 20 weeks. The levels of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) protein and mRNA were examined by immunohistochemistry, Western blot and semi-quantitative reverse transcriptase-polymerase chain reaction at different stages in the rat HCC model. Twenty weeks after induction of hepatocarcinogenesis, the expression of HIF-1alpha and VEGF protein and mRNA significantly increased compared with week 0. Microvessel density (MVD) increased considerably once liver cancer developed. There was a significant positive correlation between MVD and both HIF-1alpha and VEGF, and between HIF-1alpha and VEGF levels. These results suggest that HIF-1alpha and VEGF play important roles in tumour occurrence and development during rat hepatocarcinogenesis, possibly through promoting tumour angiogenesis.

Morin regulates the expression of NF-κB-p65, COX-2 and matrix metalloproteinases in diethylnitrosamine induced rat hepatocellular carcinoma

Morin—a bioflavonoid is a naturally available dietary agent believed to impede cancer promotion and progression. The present study was conducted to decipher, in vivo, the role of morin on the expression of matrix metalloproteinases, cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB)-p65 during diethylnitrosamine (DEN)-induced hepatocarcinogenesis in Wistar albino rats. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that administration of DEN (200 mg/kg bodyweight in drinking water) to experimental animals caused inflammation of the liver due to up-regulation of NF-κB-p65 and COX-2. RT-PCR and immunoblot analysis also revealed that the oral supplementation of morin (500 ppm in diet) to DEN-induced hepatocellular carcinoma rats down-regulated the expression of COX-2 and NF-κB-p65, thereby preventing inflammation and angiogenesis mediated hepatocellular carcinogenesis. Further, immunohistological analysis for NF-κB-p65 nuclear localization confirms the above observations. Gelatin zymography was performed for matrix metalloproteinase MMP-2 and MMP-9 expression to confirm their role in angiogenesis in DEN induced hepatocellular carcinoma and its modulation by morin. Both MMP-2 and MMP-9 levels were found to be increased in DEN-induced animals when compared to control. MMP-2 and MMP-9 levels were down-regulated in morin post-treated animals when compared to DEN-induced animals favouring prevention of angiogenesis. In conclusion, our findings indicate that morin possessed anti-inflammatory and anti-cancer properties favouring suppression of DEN-induced hepatocellular carcinoma.