Two-stage Hepatocarcinogenesis Model Research Articles

Effects of astragalus injection on different stages of early hepatocarcinogenesis in a two-stage hepatocarcinogenesis model using rats.

To clarify the suppressive effects of astragalus injection (AI) on different stages of early hepatocarcinogenesis induced by weak promotion, SD rats initiated with a single intraperitoneal (i.p.) injection of N-diethylnitrosamine (DEN) at 200 mg/kg body weight and promoted with 0.5% piperonyl butoxide (PBO) in diet were repeatedly administered AI at 5 ml/kg body weight/day in the early postinitiation (EPI) or late postinitiation (LPI) period for 2 or 8 weeks, respectively. The number and area of glutathione S-transferase placental form (GST-P)-immunoreactive (+) foci tended to increase in the DEN+PBO group compared with the DEN-alone group. Among the PBO-promoted groups, number and area of GST-P+ foci did not visibly change in the DEN+PBO+AI-EPI group compared with the DEN+PBO group. In contrast, number and area of GST-P+ foci tended to decrease in the DEN+PBO+AI-LPI group compared with the DEN+PBO group. Number of Ki67+ cells was increased in the DEN+PBO group compared with the DEN-alone group and was decreased in both AI-administered groups compared with the DEN+PBO group. Gene expression analysis revealed that the DEN+PBO+AI-LPI group showed increased transcript levels of Ccne1, Cdkn1b, Rb1, Bax, Bcl2, Casp3, and Casp9 compared with the DEN+PBO group; however, the DEN+PBO+AI-EPI group did not show changes in the transcript levels of any genes examined compared with the DEN+PBO. These results suggest that AI administration during the LPI period caused weak suppression of hepatocarcinogenesis under weak promotion with a low PBO dose by the mechanism involving facilitation of cell cycle suppression causing G1/S arrest and apoptosis via the mitochondrial pathway. In addition, the results suggest that AI administration during the EPI period has no effect on weakly promoted hepatocarcinogenesis.

Spironolactone in Combination with α-glycosyl Isoquercitrin Prevents Steatosis-related Early Hepatocarcinogenesis in Rats through the Observed NADPH Oxidase Modulation.

Administration of the diuretic, spironolactone (SR), can inhibit chronic liver diseases. We determined the effects of SR alone or in combination with the antioxidant α-glycosyl isoquercitrin (AGIQ) on hyperlipidemia- and steatosis-related precancerous lesions in high-fat diet (HFD)-fed rats subjected to a two-stage hepatocarcinogenesis model. Rats were fed with control basal diet or HFD, which was administered with SR alone or in combination with an antioxidant AGIQ in drinking water. An HFD increased body weight, intra-abdominal fat (adipose) tissue weight, and plasma lipids, which were reduced by coadministration of SR and AGIQ. SR and AGIQ coadministration also reduced hepatic steatosis and preneoplastic glutathione S-transferase placental form-positive foci, in association with decrease in NADPH oxidase (NOX) subunit p22phox-positive cells and an increase in active-caspase-3-positive cells in the foci. Hepatic gene expression analysis revealed that the coadministration of SR and AGIQ altered mRNA levels of lipogenic enzymes ( Scd1 and Fasn), antioxidant-related enzymes ( Catalase), NOX component ( P67phox), and anti-inflammatory transcriptional factor ( Pparg). Our results indicated that SR in combination with AGIQ had the potential of suppressing hyperlipidemia- and steatosis-related early hepatocarcinogenesis through the reduced expression of NOX subunits.

Differential impacts of mineralocorticoid receptor antagonist potassium canrenoate on liver and renal changes in high fat diet-mediated early hepatocarcinogenesis model rats.

Mineralocorticoid receptor (MR)/NADPH oxidase (NOX) signaling is involved in the development of obesity, insulin resistance, and renal diseases; however, the role of this signaling on steatotic preneoplastic liver lesions is not fully elucidated. We determined the effects of the MR antagonist potassium canrenoate (PC) on MR/NOX signaling in hepatic steatosis and preneoplastic glutathione S-transferase placental form (GST-P)-positive liver foci. Rats were subjected to a two-stage hepatocarcinogenesis model and fed with basal diet or high fat diet (HFD) that was co-administered with PC alone or in combination with the antioxidant alpha-glycosyl isoquercitrin (AGIQ). PC reduced obesity and renal changes (basophilic tubules that expressed MR and p22phox) but did not affect blood glucose tolerance and non-alcoholic fatty liver disease activity score (NAS) in HFD-fed rats. However, the drug increased the area of GST-P-positive liver foci that expressed MR and p22phox as well as increased expression of NOX genes (p22phox, Poldip2, and NOX4). PC in combination with AGIQ had the potential of inhibiting the effects of PC on the area of GST-P-positive liver foci and the effects were associated with increasing expression of an anti-oxidative enzyme (Catalase). The results suggested that MR/NOX signaling might be involved in development of preneoplastic liver foci and renal basophilic changes in HFD-fed rats; however, the impacts of PC were different in each organ.

Identification of epigenetically downregulated Tmem70 and Ube2e2 in rat liver after 28-day treatment with hepatocarcinogenic thioacetamide showing gene product downregulation in hepatocellular preneoplastic and neoplastic lesions produced by tumor promotion

The present study identified genes showing promoter region hypermethylation by CpG island microarrays in the liver of rats treated with hepatocarcinogen thioacetamide (TAA) for 28days. Among 47 hypermethylated genes, Hist1h2aa, Tmem70, Ube2e2, and Slk were confirmed to show hypermethylation by methylation-specific quantitative polymerase chain reaction (PCR) and pyrosequencing analyses as well as downregulation of transcript levels by real-time reverse transcription-PCR analysis in the livers of rats treated with TAA. All gene products of the 4 selected genes showed decreased immunoreactivity forming negative liver cell foci in a subpopulation of glutathione S-transferase placental form (GST-P)+ foci in TAA-promoted rat livers in a two-stage hepatocarcinogenesis model. Among them, TMEM70 and UBE2E2 showed increased incidences of negative foci in GST-P+ foci by promotion of all examined TAA, β-naphthoflavone, piperonyl butoxide, fenbendazole and phenobarbital, while HIST1H2AA and SLK did not respond to all promotive treatments. In the late stage of tumor promotion by TAA, the incidence of GST-P+ proliferative lesions with downregulation of TMEM70 or UBE2E2 was higher in adenomas and carcinomas than liver cell foci. TMEM70 plays a role in mitochondrial oxidative phosphorylation, and UBE2E2 participates in the stabilization of cell cycle regulatory proteins. Therefore, our results indicate that aberrant epigenetic gene downregulation suggestive of a metabolic shift of cellular respiration from oxidative phosphorylation to glycolysis and aberrant cell cycle regulation facilitating cell proliferation from as early as 28days after hepatocarcinogen treatment contribute to tumor development.

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.

Effect of resveratrol analogue, DMU-212, on antioxidant status and apoptosis-related genes in rat model of hepatocarcinogenesis.

The aim of the study was to examine whether antioxidant properties of 3,4,4',5-tetramethoxystilbene (DMU-212) contribute to its anticarcinogenic activity and whether DMU-212 affects the expression of apoptosis-related genes. Two-stage model of hepatocarcinogenesis was used; male Wistar rats were challenged with N-nitrosodiethylamine (NDEA), 200 mg/kg body weight (b.w.), intraperitoneal, then phenobarbital (PB) in drinking water (0.05%) was administered. Simultaneously, DMU-212 was given per os at a dose 20 or 50 mg/kg b.w. two times a week for 16 weeks. DMU-212 caused a moderate decrease in hepatic thiobarbituric acid reactive substances and protein carbonyls concentration elevated in rats treated with NDEA/PB. The activity of antioxidant enzymes examined reduced by NDEA/PB treatment was not restored in rats coadministered with DMU-212. Effects of DMU-212 on messenger RNA (mRNA) expression of antioxidant enzymes in rats challenged with NDEA/PB were diversified; no changes in their protein expression were noted in any of the groups. The expression of 17,000 genes was analyzed by Affymetrix® Rat Gene 1.1 ST Array; 15 apoptosis-related genes were selected and validated by RT-q PCR. The combined treatment with NDEA/PB and DMU-212 increased the mRNA level of some genes driving mitochondria-mediated apoptosis, whereas the mRNA expression of some anti-apoptotic genes triggering receptor-mediated apoptosis was reduced. The expression of genes encoding caspases-4, -8, -9, and -12 was also increased in rats treated with DMU-212. Although antioxidant effect of DMU-212 in rats challenged with NDEA/PB was moderate, its potential anticarcinogenic properties were demonstrated as evidenced by modulation of apoptosis-related genes.

Essential role of constitutive androstane receptor in Ginkgo biloba extract induced liver hypertrophy and hepatocarcinogenesis

Ginkgo biloba extract (GBE) is commonly used as a herbal supplement. The National Toxicology Program (NTP) study of GBE reported clear evidence of hepatocarcinogenicity in mice. To clarify the mode of action (MOA) for hepatocarcinogenesis by GBE, we investigated the involvement of the constitutive androstane receptor (CAR) in hepatocarcinogenesis induced by GBE using CAR-knockout (CARKO) and wild type (WT) mice. We used the same lot of GBE that was used for the NTP study. In 1-week GBE dietary treatment, hepatocellular DNA replication was increased in WT mice but not in CARKO mice. In 4- or 13-week treatment, greater hepatic Cyp2b10 induction and hepatocellular hypertrophy were observed in WT mice, whereas these effects of GBE were much smaller in CARKO mice. In a two-stage hepatocarcinogenesis model initiated by diethylnitrosamine, 27-week treatment with GBE resulted in an increase of eosinophilic altered foci and adenomas in WT mice. By contrast, foci and adenomas were clearly less evident in CARKO mice. These results indicate that GBE-induced hepatocarcinogenesis is mainly CAR-mediated. Since CAR-mediated MOA for hepatocarcinogenesis in rodents is considered to be qualitatively implausible for humans, our findings would be helpful to evaluate the carcinogenic characterization of GBE to humans.

マウス肝二段階発がんモデルを用いたpiperonyl butoxide (PBO) による発がん促進時の肝臓における網羅的DNAメチル化変動解析

マウス肝二段階発がんモデルを用いたpiperonyl butoxide (PBO) による発がん促進時の肝臓における網羅的DNAメチル化変動解析

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-β/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)+ liver cell foci and the numbers of Ki-67+ proliferating cells within GST-P+ foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P+ foci (area) to 48.9–49.4% and Ki-67+ cells to 55.5–61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN– foci, phospho-Akt substrate+ foci, phospho-Smad3– foci and Smad4– foci in GST-P+ foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN– foci in GST-P+ foci to 59.8–72.2% and Smad4– foci to 62.4–71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate+ foci in GST-P+ foci to 75.2–75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-β/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.

3,4,5,4′-trans-tetramethoxystilbene (DMU-212) modulates the activation of NF-κB, AP-1, and STAT3 transcription factors in rat liver carcinogenesis induced by initiation-promotion regimen

It has been reported that methylated analog of resveratrol, 3,4,5,4′-trans-tetramethoxystilbene (DMU-212), demonstrates strong antiproliferative, and proapoptotic activity. The aim of this study was to evaluate the effect of DMU-212 on the activation of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and signal transducer and activator of transcription 3 (STAT3) transcription factors, using a two-stage model of rat hepatocarcinogenesis (HCC) in Wistar rats. Initiation was performed by a single intraperitoneal injection of N-nitrosodiethylamine (NDEA) (200 mg/kg) followed by promotion with phenobarbital (PB) (0.05 %) in drinking water. DMU-212 was administered by gavage in a dose of 20 or 50 mg/kg b.w. two times a week for 16 weeks. There was a significant increase in the activation of all investigated hepatic transcription factors in the NDEA/PB-induced rats. The activation of NF-κB induced by NDEA/PB treatment was suppressed by DMU-212 as evidenced by a reduction of p65 and p50 subunits translocation, DNA binding capacity, increased retention of IκB, and the reduced IKK activity. Moreover, DMU-212 reduced the level of iNOS protein induced by NDEA/PB. Treatment with DMU-212 alone increased the constitutive AP-1 subunits c-Jun and c-Fos levels and c-Jun binding to TRE consensus site. The combined treatment diminished c-Fos level and DNA binding. At a dose of 50 mg/kg, DMU-212 decreased also the STAT3 activation induced by NDEA/PB. These data indicate that DMU-212 may suppress pro-inflammatory transcription factors, particularly NF-κB, and in consequence iNOS expression in rat model of HCC which makes DMU-212 a good candidate for the development of HCC chemopreventive agent.Electronic supplementary materialThe online version of this article (doi:10.1007/s11010-014-1983-9) contains supplementary material, which is available to authorized users.

Global DNA methylation screening of liver in piperonyl butoxide-treated mice in a two-stage hepatocarcinogenesis model

Disruptive epigenetic gene control has been shown to be involved in carcinogenesis. To identify key molecules in piperonyl butoxide (PBO)-induced hepatocarcinogenesis, we searched hypermethylated genes using CpG island (CGI) microarrays in non-neoplastic liver cells as a source of proliferative lesions at 25 weeks after tumor promotion with PBO using mice. We further performed methylation-specific polymerase chain reaction (PCR), real-time reverse transcription PCR, and immunohistochemical analysis in PBO-promoted liver tissues. Ebp4.1, Wdr6 and Cmtm6 increased methylation levels in the promoter region by PBO promotion, although Cmtm6 levels were statistically non-significant. These results suggest that PBO promotion may cause altered epigenetic gene regulation in non-neoplastic liver cells surrounding proliferative lesions to allow the facilitation of hepatocarcinogenesis. Both Wdr6 and Cmtm6 showed decreased expression in non-neoplastic liver cells in contrast to positive immunoreactivity in the majority of proliferative lesions produced by PBO promotion. These results suggest that both Wdr6 and Cmtm6 were spared from epigenetic gene modification in proliferative lesions by PBO promotion in contrast to the hypermethylation-mediated downregulation in surrounding liver cells. Considering the effective detection of proliferative lesions, these molecules could be used as detection markers of hepatocellular proliferative lesions and played an important role in hepatocarcinogenesis.

Inhibitory effect of α-lipoic acid on thioacetamide-induced tumor promotion through suppression of inflammatory cell responses in a two-stage hepatocarcinogenesis model in rats

To investigate the protective effect of α-lipoic acid (a-LA) on the hepatocarcinogenic process promoted by thioacetamide (TAA), we used a two-stage liver carcinogenesis model in N-diethylnitrosamine (DEN)-initiated and TAA-promoted rats. We examined the modifying effect of co-administered a-LA on the liver tissue environment surrounding preneoplastic hepatocellular lesions, with particular focus on hepatic macrophages and the mechanism behind the decrease in apoptosis of cells surrounding preneoplastic hepatocellular lesions during the early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)+ liver cell foci and the numbers of proliferating and apoptotic cells in the liver. Co-administration with a-LA suppressed these effects. TAA also increased the numbers of ED2+, cyclooxygenase-2+, and heme oxygenase-1+ hepatic macrophages as well as the number of CD3+ lymphocytes. These effects were also suppressed by a-LA. Transcript levels of some inflammation-related genes were upregulated by TAA and downregulated by a-LA in real-time RT-PCR analysis. Outside the GST-P+ foci, a-LA reduced the numbers of apoptotic cells, active caspase-8+ cells and death receptor (DR)-5+ cells. These results suggest that hepatic macrophages producing proinflammatory factors may be activated in TAA-induced tumor promotion. a-LA may suppress tumor-promoting activity by suppressing the activation of these macrophages and the subsequent inflammatory responses. Furthermore, a-LA may suppress tumor-promoting activity by suppressing the DR5-mediated extrinsic pathway of apoptosis and the subsequent regeneration of liver cells outside GST-P+ foci.

Effect of enzymatically modified isoquercitrin on preneoplastic liver cell lesions induced by thioacetamide promotion in a two-stage hepatocarcinogenesis model using rats

To investigate the protective effect of enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and thioacetamide (TAA)-promoted rats. We examined the modifying effect of co-administration with EMIQ on the liver tissue environment including hepatic macrophages and lymphocytes and on the induction mechanism of preneoplastic cell apoptosis during early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)+ liver cell foci and the numbers of proliferating and apoptotic cells in randomly selected areas in liver sections. Co-administration with EMIQ suppressed these effects. TAA also increased the numbers of ED2+, cyclooxygenase-2+, and heme oxygenase-1+ liver cells, as well as the number of CD3+ lymphocytes. These effects were also suppressed by EMIQ. EMIQ increased liver levels of thiobarbituric acid-reactive substance and 8-hydroxydeoxyguanosine, and TUNEL+ apoptotic cells, death receptor 5 (DR5)+ cells and 4-hydroxy-2-nonenal+ cells within GST-P+ foci. Outside the GST-P+ foci, EMIQ decreased the numbers of apoptotic cells and DR5+ cells. These results suggest that TAA-induced tumor promotion involves activation of hepatic macrophages producing proinflammatory factors. EMIQ may suppress the TAA-induced tumor-promoting activity by an anti-inflammatory mechanism mediated by suppressing the activation of these macrophages. Furthermore, EMIQ may suppress tumor-promoting activity differentially between the inside and outside of GST-P+ foci. Within GST-P+ foci, EMIQ facilitates the apoptosis of preneoplastic cells through the upregulation of DR5. Outside the GST-P+ foci, EMIQ suppresses apoptosis and the subsequent regeneration of non-transformed liver cells.

A novel antihepatitis drug, bicyclol, prevents liver carcinogenesis in diethylnitrosamine-initiated and phenobarbital-promoted mice tumor model.

Bicyclol, an antihepatitis drug developed by Chinese scientists, has been shown to prevent the malignant transformation induced by 3-methylcholanthrene and 12-O-tetradecanoylphorbol-13-acetate in WB-F344 rat liver epithelial cells. This study provides further evidence on its role as a chemopreventive agent in experimental mice with diethylnitrosamine- (DEN-) initiated and phenobarbital- (PB-) promoted liver carcinoma. Liver tissue and serum were collected. In the two-stage model of hepatocarcinogenesis in mice, oral administration of bicyclol (100, 200 mg/kg) before DEN injection showed significant reduction in the incidence of hepatocellular foci, nodules, or carcinoma. Histopathological examination revealed that there was no hepatocellular carcinoma (HCC) and hepatoma formation in the mice pretreated with bicyclol (200 mg/kg) at week 20, while the mice treated with DEN/PB developed 33.3% HCC and 55.6% hepatoma. Furthermore, the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and α-fetal protein (AFP) in serum significantly increased in the DEN/PB model group in comparison with the control group. Pretreatment with bicyclol showed a marked reduction in the above condition. Bicyclol also decreased the expression of AFP and proliferating cell nuclear antigen level in the liver tissue and attenuated the decrease in body weight. In this study, we also found that 10 weeks after stopping the administration of PB and drugs, the control and bicyclol-treated (200 mg/kg) animals showed no HCC and hepatoma formation at the time of termination whereas DEN/PB-induced mice developed 100% hepatoma and 50% HCC. These results further indicate that bicyclol has the chemopreventive potential for liver carcinogenesis induced by carcinogens.

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model

To clarify the involvement of signaling of transforming growth factor (TGF)-β during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6weeks after starting promotion) and late-stage promotion (57weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 — identified as Smad3 by later immunoblot analysis — increased in the subpopulation of GST-P+ foci, while Smad4, a nuclear transporter of Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P+ lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P+ foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P− foci induced by promotion with agonists of peroxisome proliferator-activated receptor-α did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction of GST-P+ proliferative lesions.

Tumor promotion by copper-overloading and its enhancement by excess iron accumulation involving oxidative stress responses in the early stage of a rat two-stage hepatocarcinogenesis model

To investigate liver tumor promotion mechanisms of copper (Cu)- and iron (Fe)-overloading, immunolocalization of metal-related biomolecules and lipid peroxidation end products was examined in preneoplastic liver cell foci that expressed glutathione S-transferase placental form (GST-P) in early-stage tumor promotion over 6 weeks in a rat two-stage hepatocarcinogenesis model. Gene expression and concentrations of thiobarbituric acid-reactive substance (TBARS) in the liver were also analyzed. Cu-overloading alone exerted a weak promoting activity, which was enhanced by Fe-overloading. By Cu-overloading, GST-P(+) foci that co-expressed transferrin receptors or downregulated ceruloplasmin increased, suggesting preneoplastic lesion-specific enhancement of oxidative cellular stress. Cu-overloading also increased transcripts of antioxidant enzymes (Gstm3 and Gst Yc2 subunit), cell proliferation, and numbers of single liver cells expressing GST-P or heme oxygenase-1 (HO-1) in the liver, suggesting that oxidative stress induces single-cell toxicity, with the ensuing regeneration contributing to tumor promotion. Fe-overloading increased liver TBARS and HO-1-expressing Kupffer cells, the latter suggesting protection against inflammatory stimuli causing fluctuating proinflammatory cytokine mRNA levels. By co-overloading of Cu and Fe, Cu-overload-related single liver cell toxicity and regeneration increased, as did cytokine imbalances involving increased cyclooxygenase-2-producing Kupffer cells and accumulation of malondialdehyde within GST-P(+) foci. These results suggest an involvement of oxidative stress responses in Cu-induced tumor promotion and Fe-induced enhancement by increasing cytokine imbalances and GST-P(+) foci-specific lipid peroxidation.

Induction of GST-P-positive proliferative lesions facilitating lipid peroxidation with possible involvement of transferrin receptor up-regulation and ceruloplasmin down-regulation from the early stage of liver tumor promotion in rats

To elucidate the role of metal-related molecules in hepatocarcinogenesis, we examined immunolocalization of transferrin receptor (Tfrc), ceruloplasmin (Cp) and metallothionein (MT)-1/2 in relation to liver cell foci positive for glutathione-S-transferase placental form (GST-P) in the early stage of tumor promotion by fenbendazole (FB), phenobarbital, piperonyl butoxide or thioacetamide in a rat two-stage hepatocarcinogenesis model. To estimate the involvement of oxidative stress responses to the promotion, immunolocalization of 4-hydroxy-2-nonenal, malondialdehyde and acrolein was similarly examined. Our findings showed that MT-1/2 immunoreactivity was not associated with the cellular distribution of GST-P and proliferating cell nuclear antigen, suggesting no role of MT-1/2 in hepatocarcinogenesis. We also found enhanced expression of Tfrc after treatment with strong tumor-promoting chemicals. With regard to Cp, the population showing down-regulation was increased in the GST-P-positive foci in relation to tumor promotion. Up-regulation of Tfrc and down-regulation of Cp was maintained in GST-P-positive neoplastic lesions induced after long-term promotion with FB, suggesting the expression changes occurring downstream of the signaling pathway involved in the formation of GST-P-positive lesions. Furthermore, enhanced accumulation of lipid peroxidation end products was observed in the GST-P-positive foci by promotion. Post-initiation treatment with peroxisome proliferator-activated receptor alpha agonists did not enhance any such distribution changes in GST-P-negative foci. The results thus suggest that facilitation of lipid peroxidation is involved in the induction of GST-P-positive lesions by tumor promotion from an early stage, and up-regulation of Tfrc and down-regulation of Cp may be a signature of enhanced oxidative cellular stress in these lesions.

Crosstalk between PTEN/Akt2 and TGFβ signaling involving EGF receptor down‐regulation during the tumor promotion process from the early stage in a rat two‐stage hepatocarcinogenesis model

The present study investigated the involvement of signaling of phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/protein kinase B (Akt) and transforming growth factor-beta (TGFbeta) as well as receptor tyrosine kinases in the tumor promotion processes in a two-stage hepatocarcinogenesis model using male F344 rats. The cellular localization of related molecules was examined in liver cell foci expressing glutathione S-transferase placental form (GST-P) at the early stage of tumor promotion by fenbendazole (FB), piperonyl butoxide, or thioacetamide. Distribution in the liver cell foci and neoplastic lesions positive for GST-P was also examined at the later stage of FB promotion. In contrast to the initiation-alone cases, subpopulations of GST-P-positive foci induced by promotion for 6 weeks, regardless of the promoting chemicals used, enhanced down-regulation of PTEN and up-regulation of phosphorylated (active) Akt2 and phosphorylated substrate(s) of Akt-kinase activity. Also, up-regulation of TGFbeta receptor I and down-regulation of epidermal growth factor receptor (EGFR) were enhanced in the subpopulation of GST-P-positive foci in all promoted cases. A similar pattern of cellular distribution of these molecules was also observed in the neoplastic lesions at the late stage. These results suggest a crosstalk between Akt2 and TGFbeta signaling that involves a mechanism requiring EGFR down-regulation during the entire tumor promotion process starting from the early stage. In particular, a shift in subcellular localization of phosphorylated substrate(s) of Akt from the cell membrane in liver cell foci to the cytoplasm in carcinomas was observed, suggesting an alteration of the function or activity of the corresponding molecule(s).

Resveratrol-mediated chemoprevention of diethylnitrosamine-initiated hepatocarcinogenesis: Inhibition of cell proliferation and induction of apoptosis

Hepatocellular carcinoma (HCC) is one of the most common cancers and lethal diseases. In view of the limited treatment and a grave prognosis of liver cancer, preventive control has been emphasized. Resveratrol, a polyphenol found in grape skins, peanuts, berries and red wine, has been shown to possess potent growth inhibitory effects against various human cancer cells. Although resveratrol has been found to exhibit chemopreventive actions in experimentally induced skin, breast, colon and esophagus rodent tumors, chemopreventive potential of this dietary constituent has not been explored well against experimental liver cancer. We evaluated the inhibitory effect of resveratrol using a two-stage model of rat hepatocarcinogenesis in Sprague–Dawley rats. Initiation was performed by a single intraperitoneal injection of diethylnitrosamine (DENA, 200 mg/kg), followed by promotion with phenobarbital (0.05%) in drinking water. The rats had free access to food supplemented with resveratrol equivalent to 50, 100 or 300 mg/kg body weight/day. Resveratrol treatment was started 4 weeks prior to the initiation and continued for 20 weeks. Resveratrol dose-dependently reduced the incidence, total number and multiplicity of visible hepatocyte nodules. Mean nodular volume and nodular volume as percentage of liver volume were also inhibited upon resveratrol treatment. Histopathological examination of liver tissue confirmed the protective effect of resveratrol. Immunohistochemical detection of cell proliferation and assay of apoptosis indicated a decrease in cell proliferation and increase of apoptotic cells in the livers of resveratrol-supplemented rats. Resveratrol also induced the expression of pro-apoptotic protein Bax, reduced anti-apoptotic Bcl-2 expression, with a concurrent increase in Bax/Bcl-2 ratio with respect to DENA control. The present study provides evidence, for the first time, that resveratrol exerts a significant chemopreventive effect on DENA-initiated hepatocarcinogenesis through inhibition of cell proliferation and induction of apoptosis. Resveratrol-induced apoptogenic signal during rat liver carcinogenesis may be mediated through the downregulation of Bcl-2 and upregulation of Bax expression. Due to a favorable toxicity profile, resveratrol can potentially be developed as a chemopreventive drug against human HCC.

Hepatocarcinogenic Susceptibility of Fenofibrate and Its Possible Mechanism of Carcinogenicity in a Two-Stage Hepatocarcinogenesis Model of rasH2 Mice

Fenofibrate (FF) has previously been shown to induce hepatocellular neoplasia in a conventional mouse bioassay (NDA 1993), but there has been no report to examine the carcinogenic susceptibility of rasH2 mice to this chemical. In the present study, male rasH2 mice were subjected to a two-thirds partial hepatectomy (PH), followed by an N-diethylnitrosamine (DEN) initiation twenty-four hours after PH, and given a diet containing 0, 1200, or 2400 ppm FF for seven weeks. The incidences of preneoplastic foci were significantly increased in mice from the FF-treated groups. Immunohistochemistry revealed that significant increases in proliferating cell nuclear antigen (PCNA)-positive cells and cytokeratin 8/18 positive foci were observed in FF-treated groups. In addition, the transgene and several downstream molecules such as c-myc, c-jun, activating transcription factor 3 (ATF3), and cyclin D1 were overexpressed in these groups. These results suggest that the hepatocarcinogenic activity of rasH2 mice to FF can be detected in this hepatocarcinogenesis model and that up-regulation of genes for the ras/MAPK pathway and cell cycle was probably involved in the hepatocarcinogenic mechanism of rasH2 mice.