Induced Liver Tumorigenesis Research Articles

Persistent mTORC1 activation via Depdc5 deletion results in spontaneous hepatocellular carcinoma but does not exacerbate carcinogen- and high-fat diet-induced hepatic carcinogenesis in mice

The mechanistic target of rapamycin complex 1 (mTORC1) acts as a central regulator of metabolic pathways that drive cellular growth. Abnormal activation of mTORC1 occurs at high frequency in human and mouse hepatocellular carcinoma (HCC). DEP domain-containing protein 5 (DEPDC5), a component of GATOR1 complex, is a repressor of amino acid-sensing branch of the mTORC1 pathway. In the current study, we found that persistent activation of hepatic mTORC1 signaling caused by Depdc5 ablation was sufficient to induce a pathological program of liver damage, inflammation and fibrosis that triggers spontaneous HCC development. Take advantage of the combinatory treatment with a single dose of diethylnitrosamine (DEN) and chronic feeding with high-fat diet (HFD), we demonstrated that hepatic depdc5 deletion did not aggravate DEN&HFD induced liver tumorigenesis, probably due to its protective effects on diet-induced liver steatosis. In addition, we further showed that chronic rapamycin treatment did not have any apparent tumor-suppressing effects on DEN&HFD treated control mice, whereas it dramatically reduced the tumor burden in mice with hepatic Depdc5 ablation. This study provides the novel in vivo evidence for Depdc5 deletion mediated mTORC1 hyperactivation in liver tumorigenesis caused by aging or DEN&HFD treatment. Moreover, our findings also propose that pharmacological inhibition of mTORC1 signaling maybe a promising strategy to treat HCC patients with mutations in DEPDC5 gene.

P53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth.

Cholesterol is essential for membrane biogenesis, cell proliferation, and differentiation. The role of cholesterol in cancer development and the regulation of cholesterol synthesis are still under active investigation. Here we show that under normal-sterol conditions, p53 directly represses the expression of SQLE, a rate-limiting and the first oxygenation enzyme in cholesterol synthesis, in a SREBP2-independent manner. Through transcriptional downregulation of SQLE, p53 represses cholesterol production invivo and invitro, leading to tumor growth suppression. Inhibition of SQLE using small interfering RNA (siRNA) or terbinafine (a SQLE inhibitor) reverses the increased cell proliferation caused by p53 deficiency. Conversely, SQLE overexpression or cholesterol addition promotes cell proliferation, particularly in p53 wild-type cells. More importantly, pharmacological inhibition or shRNA-mediated silencing of SQLE restricts nonalcoholic fatty liver disease (NAFLD)-induced liver tumorigenesis in p53 knockout mice. Therefore, our findings reveal a role for p53 in regulating SQLE and cholesterol biosynthesis, and further demonstrate that downregulation of SQLE is critical for p53-mediated tumor suppression.

Abstract 5239: Preventive effects of pentoxifylline on hepatic tumorigenesis in a novel mouse model of NASH-related liver cancer

Abstract Overweight and obesity are becoming more prevalent worldwide. As the hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have received attention as major causes of liver disorder. In addition, hepatocellular carcinoma (HCC) associated with NASH becomes an important issue. Although no standard medicinal treatment for NASH is established, pentoxifylline (PTX), a medicine used to improve circulation, is reported to ameliorate histopathological appearance of NASH and is thought to be a candidate therapeutic agent for NASH. In the present study, we investigated effects of PTX on NAFLD/NASH and the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in monosodium glutamate (MSG)-treated mice, a novel mouse model of NASH-related hepatocarcinogenesis, and db/db obese and diabetic mice. Male MSG and db/db mice were administered DEN, and then they received drinking water containing PTX (100 mg/kg/day) throughout the experiment. At sacrifice, drinking water with PTX significantly inhibited the development of hepatic pre-neoplastic lesions compared with respective control groups. MSG mice were thought to be susceptible to liver tumorigenesis. Hepatic triglyceride contents in db/db mice were decreased by PTX administration. The serum levels of total cholesterol, triglyceride, free fatty acid, and alanine aminotransferase were all decreased by PTX treatment, as was the mRNA expression of pro-inflammatory cytokines (TNF-alpha and IL-1beta) the macrophage-inducing chemokine CCL2/MCP-1, and several lipogenic genes (FASN and SREBP1c) in the liver. In vitro studies also revealed that PTX treatment decreased the expression of several genes associated with de novo lipogenesis and CCL2/MCP-1 in hepatoma and hepatic stellate cell lines, respectively. These findings suggest that PTX prevents NAFLD/NASH-related liver tumorigenesis by attenuating chronic hepatic inflammation and decreasing lipogenic gene expression in the liver. PTX might be a promising and efficient agent for treatment of NAFLD/NASH patients, who have a higher risk for developing liver cancer. Citation Format: Yohei Shirakami, Akinori Maruta, Koki Obara, Takayasu Ideta, Tsuneyuki Miyazaki, Hiroyasu Sakai, Takahiro Kochi, Masaya Kubota, Takuji Tanaka, Masahito Shimizu, Mitsuru Seishima. Preventive effects of pentoxifylline on hepatic tumorigenesis in a novel mouse model of NASH-related liver cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5239.

Metformin inhibits early stage diethylnitrosamine‑induced hepatocarcinogenesis in rats.

Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)-induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma-specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S-transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK-dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high-risk patients, including chronic hepatitis and cirrhosis.

Preventive effects of astaxanthin on diethylnitrosamine-induced liver tumorigenesis in C57/BL/KsJ-db/db obese mice.

Obesity and its related metabolic abnormalities, including oxidative stress and adipokine imbalance, are involved in liver carcinogenesis. The aim of the present study was to examine the effects of astaxanthin, a powerful biological antioxidant, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese mice. Male db/db mice were given a single i.p. injection of DEN (25 mg/kg bodyweight) at 2 weeks of age, and, subsequently, from 4 weeks of age, they were fed a diet containing 200 p.p.m. astaxanthin throughout the experiment. Twenty weeks of astaxanthin administration significantly inhibited the development of hepatocellular neoplasms (liver cell adenoma and hepatocellular carcinoma) and the hepatic expression of cyclin D1 mRNA compared with the basal diet group in DEN-treated db/db mice. Astaxanthin administration in DEN-treated experimental mice markedly reduced the derivatives of reactive oxygen metabolites/biological antioxidant potential ratio, which is a serum marker of oxidative stress, while increasing the mRNA expression of the antioxidant enzymes superoxide dismutase 2 and glutathione peroxidase 1 in the liver and white adipose tissue. The serum levels of adiponectin increased after astaxanthin administration in these mice. Dietary astaxanthin prevented the development of liver tumorigenesis in obese mice by improving oxidative stress and ameliorating serum adiponectin level. Therefore, astaxanthin may be useful in the chemoprevention of liver tumorigenesis in obese individuals.

Metformin suppresses diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-+Leprdb/+Leprdb mice.

Obesity and related metabolic disorders, such as diabetes mellitus, raise the risk of liver carcinogenesis. Metformin, which is widely used in the treatment of diabetes, ameliorates insulin sensitivity. Metformin is also thought to have antineoplastic activities and to reduce cancer risk. The present study examined the preventive effect of metformin on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-+Leprdb/+Leprdb (db/db) obese and diabetic mice. The mice were given a single injection of DEN at 2 weeks of age and subsequently received drinking water containing metformin for 20 weeks. Metformin administration significantly reduced the multiplicity of hepatic premalignant lesions and inhibited liver cell neoplasms. Metformin also markedly decreased serum levels of insulin and reduced insulin resistance, and inhibited phosphorylation of Akt, mammalian target of rapamycin (mTOR), and p70S6 in the liver. Furthermore, serum levels of leptin were decreased, while those of adiponectin were increased by metformin. These findings suggest that metformin prevents liver tumorigenesis by ameliorating insulin sensitivity, inhibiting the activation of Akt/mTOR/p70S6 signaling, and improving adipokine imbalance. Therefore, metformin may be a potent candidate for chemoprevention of liver tumorigenesis in patients with obesity or diabetes.

Krüppel-like factor 10 null mice exhibit lower tumor incidence and suppressed cellular proliferation activity following chemically induced liver tumorigenesis.

Liver cancer is the third most common cancer, and the incidence as well as the mortality rate of liver cancer are on the increase. There are many signaling pathways that are involved in hepatic tumorigenesis. One of these pathways, the transforming growth factor-β (TGF-β)/Smad pathway with KLF10, has been reported to suppress cellular proliferation in most cases. However, the actual functions of KLF10 in various pathophysiological conditions are still fragmentary and unclear. In the present study, the practical role of KLF10 in DEN-induced hepatic carcinogenesis, was elucidated using KLF10 null mice. In the necropsy and histopathological analysis, KLF10 KO mice exhibited lower tumor incidence and PCNA labeling indices than these values in the wild-type mice. Additional analyses revealed that the mRNA and protein levels of Smad3, TGF-β1, TGF-β RI and p15 were increased in the tumor tissues of the KLF10 KO mice, while those of cMyc and cyclin D1 were downregulated. The level of phospho-Smad3 was also significantly higher in the tumor tissues of the KLF10 KO mice. All together, the KLF10 KO condition may reinforce the TGF-β‑Smad signaling pathway and confer tumor-suppressor effects against chemically induced liver tumorigenesis.

Metformin prevents liver tumorigenesis induced by high-fat diet in C57Bl/6 mice

The prevalence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is increasing with the growing epidemics of obesity and diabetes. NAFLD encompasses a clinicopathologic spectrum of disease ranging from isolated hepatic steatosis to NASH, which is a more aggressive form of fatty liver disease, to cirrhosis and, finally, hepatocellular carcinoma (HCC). The exact mechanism behind the development of HCC in NASH remains unclear; however, it has been established that hepatic steatosis is the important risk factor in the development of HCC. Metformin has recently drawn attention because of its potential antitumor effect. Here, we investigated the effects of metformin on high-fat diet (HFD)-induced liver tumorigenesis, using a mouse model of NASH and liver tumor. Metformin prevented long-term HFD-induced liver tumorigenesis in C57Bl/6 mice. Of note, metformin failed to protect against liver tumorigenesis in mice that had already begun to develop NAFLD. Metformin improved short-term HFD-induced fat accumulation in the liver, associated with the suppression of adipose tissue inflammation. Collectively, these results suggest that metformin may prevent liver tumorigenesis via suppression of liver fat accumulation in the early stage, before the onset of NAFLD, which seems to be associated with a delay in the development of inflammation of the adipose tissue.

Abstract 1616: Preventive effects of EGCG on diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice

Abstract Background and aims: Obesity and related metabolic abnormalities, including insulin resistance and a state of chronic inflammation, increase the risk of hepatocellular carcinoma (HCC). Abnormal activation of the IGF/IGF-1R axis is also involved in obesity-related liver tumorigenesis. In the present study, we examined the effects of (-)-epigallocatechin gallate (EGCG), a major biologically active component of green tea, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese mice. Methods: Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks and then they received drinking water containing 0.1% EGCG for 34 weeks. Results: At sacrifice, drinking water with EGCG significantly inhibited the development of liver cell adenomas in comparison to the control EGCG-untreated group. EGCG inhibited the phosphorylation of the IGF-1R, ERK, Akt, GSK-3β, Stat3, and JNK proteins in the livers of experimental mice. The serum levels of insulin, IGF-1, IGF-2, free fatty acid, and TNF-α were all decreased by drinking EGCG. EGCG also decreased the expression of TNF-α, IL-6, IL-1α, and IL-18 mRNAs in the livers. In addition, EGCG improved liver steatosis and activated the AMPK protein in the liver. Conclusion: EGCG prevents obesity-related liver tumorigenesis by inhibiting the IGF/IGF-1R axis, improving hyperinsulinemia, and attenuating chronic inflammation. EGCG, therefore, may be useful in the chemoprevention of liver tumorigenesis in obese individuals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1616. doi:1538-7445.AM2012-1616

Abstract 844: Acyclic retinoid inhibits diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice

Abstract Background and aims: Obesity and the related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Malfunctioning of retinoid X receptor (RXR)-α due to phosphorylation by Ras/MAPK also plays a critical role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), which targets RXRα, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese and diabetic mice. Methods: Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks, after which they were fed a diet containing 0.03% or 0.06% of ACR throughout the experiment. Results: In mice treated with either dose of ACR for 34 weeks, the development of liver cell adenomas was significantly inhibited as compared to basal diet-fed mice. ACR markedly inhibited the activation of Ras and phosphorylation of the ERK, RXRα, and Stat3 proteins in the livers of experimental mice. It also increased the expression of RARβ and p21CIP1 mRNA, while decreasing the expression of cyclin D1, c-Fos, and c-Jun mRNA in the liver, thereby restoring RXRα function. Administration of ACR improved liver steatosis and activated the AMPK protein. The serum levels of insulin were decreased by ACR treatment, whereas the QUICKI values were increased, indicating improved insulin sensitivity. The serum levels of TNF-α and the expression levels of TNF-α, IL-6, and IL-1β mRNA in the livers of DEN-treated db/db mice were decreased by ACR treatment, suggesting attenuation of the chronic inflammation induced by excessive fatty deposits. Conclusion: ACR may be, therefore, useful in the chemoprevention of obesity-related HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 844. doi:10.1158/1538-7445.AM2011-844

Preventive Effects of (−)-Epigallocatechin Gallate on Diethylnitrosamine-Induced Liver Tumorigenesis in Obese and Diabetic C57BL/KsJ-db/db Mice

Obesity and related metabolic abnormalities, including insulin resistance and a state of chronic inflammation, increase the risk of hepatocellular carcinoma. Abnormal activation of the insulin-like growth factor (IGF)/ IGF-1 receptor (IGF-1R) axis is also involved in obesity-related liver tumorigenesis. In the present study, we examined the effects of (-)-epigallocatechin gallate (EGCG), a major biologically active component of green tea, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese mice. Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks and then they received drinking water containing 0.1% EGCG for 34 weeks. At sacrifice, drinking water with EGCG significantly inhibited the development of liver cell adenomas in comparison with the control EGCG-untreated group. EGCG inhibited the phosphorylation of the IGF-1R, ERK (extracellular signal-regulated kinase), Akt, GSK-3β (glycogen synthase kinase-3β), Stat3, and JNK (c-Jun NH(2)-terminal kinase) proteins in the livers of experimental mice. The serum levels of insulin, IGF-1, IGF-2, free fatty acid, and TNF-α were all decreased by drinking EGCG, which also decreased the expression of TNF-α, interleukin (IL)-6, IL-1β, and IL-18 mRNAs in the livers. In addition, EGCG improved liver steatosis and activated the AMP-activated kinase protein in the liver. These findings suggest that EGCG prevents obesity-related liver tumorigenesis by inhibiting the IGF/IGF-1R axis, improving hyperinsulinemia, and attenuating chronic inflammation. EGCG, therefore, may be useful in the chemoprevention of liver tumorigenesis in obese individuals. Cancer Prev Res; 4(3); 396-403. ©2011 AACR.

Acyclic Retinoid Inhibits Diethylnitrosamine-Induced Liver Tumorigenesis in Obese and Diabetic C57BLKS/J- +Leprdb/+Leprdb Mice

Obesity and the related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Malfunctioning of retinoid X receptor (RXR) α due to phosphorylation by Ras/MAPK also plays a critical role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), which targets RXRα, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BLKS/J- +Lepr(db)/+Lepr(db) (db/db) obese mice. Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks, after which they were fed a diet containing 0.03% or 0.06% of ACR throughout the experiment. In mice treated with either dose of ACR for 34 weeks, the development of liver cell adenomas was significantly inhibited as compared with basal diet-fed mice. ACR markedly inhibited the activation of Ras and phosphorylation of the ERK (extracellular signal-regulated kinase) and RXRα proteins in the livers of experimental mice. It also increased the expression of RAR β and p21(CIP1) mRNA while decreasing the expression of cyclin D1, c-Fos, and c-Jun mRNA in the liver, thereby restoring RXRα function. Administration of ACR improved liver steatosis and activated the AMPK protein. The serum levels of insulin decreased by ACR treatment, whereas the quantitative insulin sensitivity check index (QUICKI) values increased, indicating improved insulin sensitivity. The serum levels of TNF-α and the expression levels of TNF- α, IL-6, and IL-1 β mRNA in the livers of DEN-treated db/db mice were decreased by ACR treatment, suggesting attenuation of the chronic inflammation induced by excessive fatty deposits. ACR may be, therefore, useful in the chemoprevention of obesity-related HCC.

Higher susceptibility to aflatoxin B1‐related hepatocellular carcinoma in glycine N‐methyltransferase knockout mice

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N-methyltransferase (GNMT) interacts with aflatoxin B(1) (AFB(1)) and reduces both AFB(1)-DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt-/-) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt-/- mice are susceptible to AFB(1) carcinogenesis, and that the absence of Gnmt expression may accelerate AFB(1)-induced liver tumorigenesis. We inoculated Gnmt-/- and wild-type mice intraperitoneally with AFB(1) at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt-/- mice were diagnosed as having HCC, ∼6 months earlier than AFB(1)-treated wild-type mice. Results from microarray and real-time PCR analyses indicate that five detoxification pathway-related genes were downregulated in AFB(1)-treated Gnmt-/- mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB(1)-related HCC in Gnmt-/- mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.

Resveratrol Suppresses Oxidative Stress and Inflammatory Response in Diethylnitrosamine-Initiated Rat Hepatocarcinogenesis

Hepatocellular carcinoma (HCC), one of the most frequent and deadliest cancers, has been increasing considerably in the United States. In the absence of a proven effective therapy for HCC, novel chemopreventive strategies are urgently needed to lower the current morbidity and mortality of HCC. Recently, we have reported that resveratrol, a compound present in grapes and red wine, significantly prevents diethylnitrosamine (DENA)-induced liver tumorigenesis in rats, although the mechanism of action is not completely understood. In the present study, we have examined the underlying mechanisms of resveratrol chemoprevention of hepatocarcinogenesis by investigating the effects of resveratrol on oxidative damage and inflammatory markers during DENA-initiated rat liver carcinogenesis. There was a significant increase in hepatic lipid peroxidation and protein oxidation in carcinogen control animals compared with their normal counterparts at the end of the study (20 weeks). Elevated expressions of inducible nitric oxide synthase and 3-nitrotyrosine were noticed in the livers of the same animals. Dietary resveratrol (50-300 mg/kg) administered throughout the study reversed all the aforementioned markers in a dose-responsive fashion in rats challenged with DENA. Resveratrol also elevated the protein and mRNA expression of hepatic nuclear factor E2-related factor 2 (Nrf2). Results of the present investigation provide evidence that attenuation of oxidative stress and suppression of inflammatory response mediated by Nrf2 could be implicated, at least in part, in the chemopreventive effects of this dietary agent against chemically induced hepatic tumorigenesis in rats. The outcome of this study may benefit the development of resveratrol in the prevention and intervention of human HCC.

Hepatocyte IKKβ/NF-κB Inhibits Tumor Promotion and Progression by Preventing Oxidative Stress-Driven STAT3 Activation

The NF-kappaB activating kinase IKKbeta suppresses early chemically induced liver tumorigenesis by inhibiting hepatocyte death and compensatory proliferation. To study IKKbeta's role in late tumor promotion and progression, we developed a transplant system that allows initiated mouse hepatocytes to form hepatocellular carcinomas (HCC) in host liver after a long latency. Deletion of IKKbeta long after initiation accelerated HCC development and enhanced proliferation of tumor initiating cells. These effects of IKKbeta/NF-kappaB were cell autonomous and correlated with increased accumulation of reactive oxygen species that led to JNK and STAT3 activation. Hepatocyte-specific STAT3 ablation prevented HCC development. The negative crosstalk between NF-kappaB and STAT3, which is also evident in human HCC, is a critical regulator of liver cancer development and progression.

Dietary supplementation with branched‐chain amino acids suppresses diethylnitrosamine‐induced liver tumorigenesis in obese and diabetic C57BL/KsJ‐db/db mice

Obesity and related metabolic abnormalities, including insulin resistance, are risk factors for hepatocellular carcinoma in non-alcoholic steatohepatitis as well as in chronic viral hepatitis. Branched-chain amino acids (BCAA), which improve insulin resistance, inhibited obesity-related colon carcinogenesis in a rodent model, and also reduced the incidence of hepatocellular carcinoma in obese patients with liver cirrhosis. In the present study, we determined the effects of BCAA on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in obese C57BL/KsJ-db/db (db/db) mice with diabetes mellitus. Male db/db mice were given tap water containing 40 ppm DEN for an initial 2 weeks and thereafter they received a basal diet containing 3.0% of BCAA or casein, which served as a nitrogen content-matched control of BCAA, throughout the experiment. Supplementation with BCAA significantly reduced the total number of foci of cellular alteration, a premalignant lesion of the liver, and the expression of insulin-like growth factor (IGF)-1, IGF-2, and IGF-1 receptor in the liver when compared to the casein supplementation. BCAA supplementation for 34 weeks also significantly inhibited both the development of hepatocellular neoplasms and the proliferation of hepatocytes in comparison to the basal diet or casein-fed groups. Supplementation with BCAA improved liver steatosis and fibrosis and inhibited the expression of alpha-smooth muscle actin in the DEN-treated db/db mice. The serum levels of glucose and leptin decreased by dietary BCAA, whereas the value of the quantitative insulin sensitivity check index increased by this agent, indicating the improvement of insulin resistance and hyperleptinemia. In conclusion, oral BCAA supplementation improves insulin resistance and prevents the development of liver tumorigenesis in obese and diabetic mice.

The constitutive active/androstane receptor facilitates unique phenobarbital-induced expression changes of genes involved in key pathways in precancerous liver and liver tumors.

Our overall goal is to elucidate progressive changes, in expression and methylation status, of genes which play key roles in phenobarbital (PB)-induced liver tumorigenesis, with an emphasis on their potential to affect signaling through critical pathways involved in the regulation of cell growth and differentiation. PB-elicited unique expression changes of genes, including some of those identified previously as exhibiting regions of altered DNA methylation, were discerned in precancerous liver tissue and/or individual liver tumors from susceptible constitutive active/androstane receptor (CAR) wild-type (WT) compared with resistant CAR knockout (KO) mice. Many of these function in crucial cancer-related processes, for example, angiogenesis, apoptosis, cell cycle, DNA methylation, Hedgehog signaling, invasion/metastasis, Notch signaling, and Wnt signaling. Furthermore, a subset of the uniquely altered genes contained CAR response elements (CAREs). This included Gadd45b, a coactivator of CAR and inhibitor of apoptosis, and two DNA methyltransferases (Dnmt1, Dnmt3a). The presence of CAREs in Dnmts suggests a potential direct link between PB and altered DNA methylation. The current data are juxtaposed with the effects of PB on DNA methylation and gene expression which occurred uniquely in liver tumor-prone B6C3F1 mice, as compared with the resistant C57BL/6, following 2 or 4 weeks of treatment. Collectively, these data reveal a comprehensive view of PB-elicited molecular alterations (i.e., changes in gene expression and DNA methylation) that can facilitate hepatocarcinogenesis. Notably, candidate genes for initial "fingerprints" of early and late stages of PB-induced tumorigenesis are proposed.

Identification of Genes that May Play Critical Roles in Phenobarbital (PB)-Induced Liver Tumorigenesis due to Altered DNA Methylation

Aberrant DNA methylation plays important roles in tumorigenesis, and the nongenotoxic rodent tumor promoter phenobarbital (PB) alters methylation patterns to a greater extent in liver tumor susceptible as compared to resistant mice (Watson and Goodman, 2002). Unique hepatic regions of altered DNA methylation (RAMs) were identified in sensitive B6C3F1, as compared to resistant C57BL/6, mice at 2 or 4 weeks of PB treatment using a novel approach involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis (Bachman et al., 2006b). PCR products representing 90 of 170 (53%) total unique B6C3F1 RAMs at 2 or 4 weeks were cloned and subjected to BLAST-like alignment tool searches that resulted in 51 gene matches; some of these have documented oncogenic or tumor suppressor roles. Importantly, uniquely hypomethylated genes play roles in angiogenesis (e.g., chymase 1, tyrosine kinase nonreceptor 2, and possibly ephrin B2 and triple functional domain, PTPRF interacting) and invasion and metastasis, including those involved in the epithelial-mesenchymal transition (transcription factor 4, transforming growth factor beta receptor II, and ral guanine nucleotide dissociation stimulator). Common cellular targets and regulators of the genes representing unique B6C3F1 RAMs were uncovered, indicating that they might act in concert to more efficiently promote tumorigenesis. Genes not previously associated with mouse liver tumorigenesis exhibited altered methylation at these very early times following PB treatment. We hypothesize that at least some of the unique PB-induced B6C3F1 RAMs represent key events facilitating transformation, which is consistent with a causative role of altered DNA methylation during early stages of tumorigenesis.

Proanthocyanidin exposure to B6C3F1 mice significantly attenuates dimethylnitrosamine-induced liver tumor induction and mortality by differentially modulating programmed and unprogrammed cell deaths

Proanthocyanidins are of current interest as chemopreventive agents. The potential of the pre-, post- and co-exposure of proanthocyanidin-rich grape seed extract (GSPE) in preventing, reducing and/or delaying dimethylnitrosamine ( N-nitrosodimethylamine, DMN)-induced liver tumorigenesis, carcinogenesis and mortality in male B6C3F1 mice was determined. Animals were divided into six groups: I—control, II—GSPE alone, III—DMN alone, IV—GSPE + DMN, V—DMN exposure (3 months) followed by GSPE diet (9 months) and VI—GSPE diet (3 months) + DMN (3 months) + control diet (6 months). DMN exposure (0–8 weeks: 5 mg/kg; 8–12 weeks: 10 mg/kg, i.p.) was limited to a total period of 3 months. GSPE was incorporated in laboratory chow (ADI: 100 mg/kg b.w.). Animals were sacrificed at 3 month intervals, and serum chemistry, liver histopathology, integrity of hepatic genomic DNA, antioxidant status, and rates of apoptotic and necrotic cell deaths were determined. DMN-induced liver tumor formation (85%) and animal lethality (38%) were powerfully antagonized by co-administration of GSPE + DMN (tumor positive: 45%; death: 11%). More than 75% of the DMN-treated animals had numerous tumors (five or more), which were significantly reduced in the GSPE + DMN group (35%). GSPE also negatively influenced other protocols specifically designed to test initiation and progression phases. Thus, GSPE was instrumental in modulating metabolic cascades and regulated orchestration of cell death processes involved during the multistage tumorigenic process. These results unraveled that long-term exposure to proanthocyanidin-rich grape seed extract may serve as a potent barrier to all three stages of DMN-induced liver carcinogenesis and tumorigenesis by selectively altering oxidative stress, genomic integrity and cell death patterns in vivo.

INHIBITORY EFFECT OF TRANSIENT ADMINISTRATION OF FLUTAMIDE (SCH-13521, 4'-NITRO-3'-TRIFLUOROMETHYLISOBUTYRANILIDE) ON DIETHYL-NITROSAMINE (DEN) INDUCED LIVER-TUMORS IN MALE B6C3F1 MICE

In order to understand the role of androgen in DEN induced liver tumorigenesis in mice, we studied the influence of anti-androgenic compound, flutamide (SCH 13521, 4'-Nitro-3'-trifluoromethylisobutyranilide) on liver tumors, in B6C3F1 male mice. At 6 weeks of age mice were treated with DEN, (0.2 muM/g body weight). Transient administration of flutamide (0.5 mg/g food) in diet to male mice for (i) three weeks immediately after diethylnitrosamine (DEN) treatment, for (ii) three weeks starting at the age of 25 weeks and (iii) starting at 25 weeks of age until end of experiment inhibited liver tumors incidence to 63%, 50% and 53%, respectively, compared to that of DEN alone, 81.3% (significantly different). However, continuous administration of this drug throughout the experimental period (58 weeks) produced 96.9% liver tumor incidence. The time-dependent sacrifice of DEN alone treated mice at 25, 30, 38, 44 and 58 weeks of age produced liver tumor incidence of 0, 12.5, 31.5, 43.8 and 81.3%, respectively (r = 0.99). Mean tumor number and size were also correlatively increased with the prolongation of latency and highest value was noted at 58 weeks of age. Implantation of testosterone pellet after castration significantly increased cytosolic androgen receptor levels but decreased in mice treated with flutamide. Three weeks administration of flutamide to male B6C3F1 mice enhanced nuclear BrdU incorporated cells in the liver. Our results suggested that flutamide reduces cytosolic androgen receptor levels and increases DNA synthesis. These factors may in part be responsible for the liver tumor inhibitory effect of flutamide in male mice.