RESUMO
Hepatocellular carcinoma (HCC) development is associated with altered modifications in DNA methylation, changing transcriptional regulation. Emerging evidence indicates that DNA methyltransferase 1 (DNMT1) plays a key role in the carcinogenesis process. This study aimed to investigate how pirfenidone (PFD) modifies this pathway and the effect generated by the association between c-Myc expression and DNMT1 activation. Rats F344 were used for HCC development using 50 mg/kg of diethylnitrosamine (DEN) and 25 mg/kg of 2-Acetylaminofluorene (2-AAF). The HCC/PFD group received simultaneous doses of 300 mg/kg of PFD. All treatments lasted 12 weeks. On the other hand, HepG2 cells were used to evaluate the effects of PFD in restoring DNA methylation in the presence of the inhibitor 5-Aza. Histopathological, biochemical, immunohistochemical, and western blot analysis were carried out and our findings showed that PFD treatment reduced the amount and size of tumors along with decreased Glipican-3, ß-catenin, and c-Myc expression in nuclear fractions. Also, this treatment improved lipid metabolism by modulating PPARγ and SREBP1 signaling. Interestingly, PFD augmented DNMT1 and DNMT3a protein expression, which restores global methylation, both in our in vivo and in vitro models. In conclusion, our results suggest that PFD could slow down HCC development by controlling DNA methylation.
Assuntos
Carcinoma Hepatocelular , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Antígeno Nuclear de Célula em Proliferação , Piridonas , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Piridonas/farmacologia , Ratos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Humanos , Células Hep G2 , Antígeno Nuclear de Célula em Proliferação/metabolismo , Masculino , Ratos Endogâmicos F344 , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Dietilnitrosamina , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/genéticaRESUMO
Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H-NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.
Assuntos
Endopeptidases/metabolismo , Neoplasias Hepáticas Experimentais , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Tecnécio , Animais , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Compostos de Organotecnécio/farmacologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Tecnécio/química , Tecnécio/farmacocinética , Tecnécio/farmacologiaRESUMO
Hepatocellular carcinoma (HCC) arises after a long period of exposition to etiological factors that might be either independent or collectively contributing. Several rodent models resemble human HCC; however, the major limitation of these models is the lack of chronic injury that reproducibly mimics the molecular alterations as it occurs in humans. Thus, we hypothesized that chronic administration of different DEN treatments identifies the best-fit dose to induce the HCC and/or to determine whether small DEN doses act synergistically with other known hepatotoxins to induce HCC in mice. C57BL/6â¯J male mice were intraperitoneally injected twice a week for 6â¯weeks with different DEN doses ranging from 2.5 to 40â¯mg/kg body weight; then, selected doses (2.5, 5 and 20â¯mg/kg) for 6, 10, 14, and 18â¯weeks. We demonstrated that DEN at 20â¯mg/kg promoted reactive oxygen species and 4-hydroxynonenal production, cell proliferation inflammatory infiltrate, and fibrosis, which in turn induced liver cancer by week 18. These parameters were established by evaluating histopathological changes, HCC markers such as glutathione S-transferase placental-1 (Gstp1), Cytokeratin-19 (Ck19) and prostaglandin reductase-1 (Ptgr1); that of Cyp2e1, a DEN metabolizing enzyme; and the expression of the proliferation marker Ki67. While DEN at 2.5 and 5â¯mg/kg increased Gstp1 and Ck19, DEN at 20â¯mg/kg decreased them and Cyp2e1 expression and activity. In summary, our results demonstrate that DEN chronically administrated at 20â¯mg/kg induces the HCC, while DEN at 2.5 and 5â¯mg/kg could be useful in elucidating its synergistic effect with other hepatotoxic agents in mice.
Assuntos
Carcinogênese/efeitos dos fármacos , Dietilnitrosamina/administração & dosagem , Dietilnitrosamina/efeitos adversos , Neoplasias Hepáticas/induzido quimicamente , Fígado/efeitos dos fármacos , Animais , Carcinogênese/metabolismo , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Fibrose/induzido quimicamente , Fibrose/metabolismo , Inflamação/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hepatocellular carcinoma causes ~10% of all cancer-related deaths worldwide, usually emerging in a background of liver fibrosis/cirrhosis (70%-90% of cases). Chemically-induced mouse models for fibrosis-associated hepatocarcinogenesis are widely-applied, resembling the corresponding human disease. Nonetheless, a long time is necessary for the development of preneoplastic/neoplastic lesions. Thus, we proposed an early fibrosis-associated hepatocarcinogenesis model for male and female mice separately, focusing on reducing the experimental time for preneoplastic/neoplastic lesions development and establishing standard models for both sexes. Then, two-week old susceptible C3H/HeJ male and female mice (n = 8 animals/sex/group) received a single dose of diethylnitrosamine (DEN, 10 or 50 mg/Kg). During 2 months, mice received 3 weekly doses of carbon tetrachloride (CCl4, 10% corn oil solution, 0.25 to 1.50 µL/g b.wt.) and they were euthanized at week 17. DEN/CCl4 protocols for males and females displayed clear liver fibrosis, featuring collagen accumulation and hepatic stellate cell activation (α-SMA). In addition, liver from males displayed increased CD68+ macrophage number, COX-2 protein expression and IL-6 levels. The DEN/CCl4 models in both sexes impaired antioxidant defense as well as enhanced hepatocyte proliferation and apoptosis. Moreover, DEN/CCl4-treated male and female developed multiple preneoplastic altered hepatocyte foci and hepatocellular adenomas. As expected, the models showed clear male bias. Therefore, we established standard and suitable fibrosis-associated hepatocarcinogenesis models for male and female mice, shortening the experimental time for the development of hepatocellular preneoplastic/neoplastic lesions in comparison to other classical models.
Assuntos
Carcinoma Hepatocelular , Cirrose Hepática , Neoplasias Hepáticas Experimentais , Animais , Tetracloreto de Carbono , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Dietilnitrosamina , Suscetibilidade a Doenças , Feminino , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos Endogâmicos C3H , Caracteres SexuaisRESUMO
INTRODUCTION AND AIM: To investigate the effect of mTOR inhibitor Rapamycin combined with transcatheter arterial embolization (TAE) on the growth, metastasis, and prognosis of hepatocellular carcinoma (HCC) in rat model. MATERIAL AND METHOD: McARH7777 cells were used to construct rat models of HCC, which were randomly divided into Model, Rapamycin, TAE, and Rapamycin + TAE groups. Quantitative reverse transcription-PCR (qRT-PCR) and Western Blot were used to detect the expression of Epithelial-Mesenchymal Transition (EMT)-related molecules, and immunohistochemical staining to determine the expression of EMTrelated proteins, angiogenic factors as well as microvessel density (MVD)-CD34. RESULTS: The hepatic tumor volume of rats in the other three groups were all significantly smaller than the Model group on the 7th, 14th, and 21st day after treatment and the combination treatment was apparently more effective than either treatment alone. Besides, both the number and the size of metastatic nodules of HCC rats after combination treatment were remarkably reduced. In addition, compared with rats in the Rapamycin + TAE group, N-cadherin, Vimentin, HIF-1α, VEGF, and MVD-CD34 were obviously enhanced, while E-cadherin was lowered in those TAE group, which were the complete opposite to the Rapamycin group. Besides, the median survival time of rats in the Rapamycin + TAE group was evidently longer than the resting groups. CONCLUSION: Rapamycin combined with TAE may effectively suppress the EMT formation and angiogenesis, thereby inhibiting the growth and lung metastasis of HCC rats, which provides a new idea for countering the recurrence and metastasis of HCC.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Carcinoma Hepatocelular/terapia , Proliferação de Células/efeitos dos fármacos , Embolização Terapêutica , Neoplasias Hepáticas Experimentais/terapia , Neoplasias Pulmonares/terapia , Sirolimo/farmacologia , Animais , Antígenos CD34/metabolismo , Caderinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Neovascularização Patológica , Proteínas do Tecido Nervoso/metabolismo , Ratos Endogâmicos BUF , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vimentina/metabolismoRESUMO
Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-ß1) and thyroid hormones (TH) regulation. INTRODUCTION AND AIM: Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-ß1) and thyroid hormones (TH) regulation. Aim. The aim of our study is to establish the statins mechanism of action and the potential key molecules involved in an in vivo and in vitro HCC model. MATERIALS AND METHODS: We used two models: in vivo (in rats) using diethylnitrosamine (DEN) and hexachlorobenzene (HCB) to develop HCC. We analyzed cell proliferation parameters (proliferating cel nuclear antigen, PCNA) and cholesterol metabolism (hydroxy-methylglutaryl-CoA reductase, HMGCoAR). In vitro (Hep-G2 cells) we evaluated the effects of different doses of Atorvastatin (AT) and Simvastatin (SM) on HCB induced proliferation and analyzed proliferative parameters, cholesterol metabolism, TGF-ß1 mRNA, c-Src and TH levels. RESULTS: In vivo, we observed that cell proliferation significantly increased as well as cholesterol serum levels in rats treated with HCB. In vitro, we observed the same results on PCNA as in vivo. The statins prevented the increase in HMG-CoAR mRNA levels induced by HCB, reaching levels similar to controls at maximum doses: AT (30 µM), and SM (20 µM). Increases in PCNA, TGF-ß1, and pc-Src, and decreases in deiodinase I mRNA levels induced by HCB were not observed when cells were pre-treated with AT and SM at maximum doses. CONCLUSION: Statins can prevent the proliferative HCB effects on Hep-G2 cells. TGF-ß1, c-Src and TH may be the statins molecular targets in hepatocarcinogenesis.
Assuntos
Antineoplásicos/farmacologia , Atorvastatina/farmacologia , Carcinoma Hepatocelular/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Sinvastatina/farmacologia , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Dietilnitrosamina , Feminino , Células Hep G2 , Hexaclorobenzeno , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: NF-kB (nuclear factor kappa B) is a transcription factor composed of two subunits, p50 and p65, which plays a key role in the inflammatory process. Melatonin has oncostatic, antiangiogenic and antimetastatic properties, and some recent studies have indicated an inhibitory effect of melatonin on NF-kB in some types of cancer. This work aims to investigate the effects of melatonin treatment on the expression of NFkB in breast and liver cancer models. METHOD: The breast cancer xenographic model was performed using female Balb/c nude athymic mice injected with MDA-MB-231 cells. The animals were treated with 40 mg/Kg of melatonin for 21 days. Volume of the tumors was measured with a digital caliper. Hepatocarcinoma model was developed by using the HepG2 cells in vitro, treated with 1 mM melatonin for 24 h. The expression of NF-kB protein was verified by immunohistochemistry and immunocytochemistry and quantified by optical densitometry, in vivo study and in vitro study, respectively. NF-kB gene expression was performed by quantitative RT-PCR. RESULTS: The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size (P=0.0022). There was a decrease in NF-kB protein staining (P=0.0027) and gene expression (P=0.0185) in mice treated with melatonin. The opposite results were observed for the hepatocarcinoma model. HepG2 cells treated with melatonin showed an increase in the NF-kB immunostaining when compared to control cells (P=0.0042). CONCLUSION: Our results indicated that the treatment with melatonin was able to decrease both gene and protein expressions of NF-kB in breast cancer cells and, conversely, increase the transcription factor protein expression in hepatocarcinoma cells. These data highlighted a double role in the expression of NF-kB, depending on the cell type. Further studies are needed to better elucidate the action of melatonin in NF-kB, since this transcription factor acts on different signaling pathways that are fundamental for carcinogenesis.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Melatonina/farmacologia , NF-kappa B/biossíntese , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Melatonina/administração & dosagem , Melatonina/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , NF-kappa B/genética , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
PURPOSE: Glycerol usage is increasing in food industry for human and animal nutrition. This study analyzed the impact of glycerol metabolism when orally supplemented during the early stage of rat liver carcinogenesis. METHODS: Wistar rats were subjected to a 2-phase model of hepatocarcinogenesis (initiated-promoted, IP group). IP animals also received glycerol by gavage (200 mg/kg body weight, IPGly group). RESULTS: Glycerol treatment reduced the volume of preneoplastic lesions by decreasing the proliferative status of liver foci, increasing the expression of p53 and p21 proteins and reducing the expression of cyclin D1 and cyclin-dependent kinase 1. Besides, apoptosis was enhanced in IPGly animals, given by an increment of Bax/Bcl-2 ratio, Bad and PUMA mitochondrial expression, a concomitant increase in cytochrome c release and caspase-3 activation. Furthermore, hepatic levels of glycerol phosphate and markers of oxidative stress were increased in IPGly rats. Oxidative stress intermediates act as intracellular messengers, inducing p53 activation and changes in JNK and Erk signaling pathways, with JNK activation and Erk inhibition. CONCLUSION: The present work provides novel data concerning the preventive actions of glycerol during the development of liver cancer and represents an economically feasible intervention to treat high-risk individuals.
Assuntos
Anticarcinógenos/uso terapêutico , Apoptose , Suplementos Nutricionais , Glicerol/uso terapêutico , Neoplasias Hepáticas Experimentais/prevenção & controle , Estresse Oxidativo , Lesões Pré-Cancerosas/prevenção & controle , Animais , Anticarcinógenos/sangue , Anticarcinógenos/metabolismo , Biomarcadores/sangue , Carcinogênese , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glicerol/sangue , Glicerol/metabolismo , Peroxidação de Lipídeos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/sangue , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação , Lesões Pré-Cancerosas/sangue , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Ratos Wistar , Carga TumoralRESUMO
Hexachlorobezene (HCB), a fungicide widely distributed in the environment, promotes the development of hepatocellular preneoplastic lesions (PNL) and tumors in rodents. In contrast, vitamin D3 (VD3) supplementation presents a potential role for the prevention/treatment of chronic liver diseases. Thus, we investigated whether VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis. Female Balb/C mice were injected a single dose of diethylnitrosamine (DEN, 50 mg/kg) at postnatal day 15. From day 40 onwards, mice were fed with a standard diet containing 0.02% HCB alone or supplemented with VD3 (10,000 or 20,000 IU/Kg diet) for 20 weeks. Untreated mice were fed just standard diet. After this period, mice were euthanized and liver and serum samples were collected. Compared to the untreated group, DEN/HCB treatment decreased total hepatic glutathione levels and glutathione peroxidase (GSH-Px) activity while increased lipid peroxidation, p65 protein expression, cell proliferation/apoptosis and the PNL development. In contrast, dietary VD3 supplementation enhanced vitamin D receptor (VDR) protein expression, total glutathione levels and GSH-Px activity while diminished lipid hydroperoxide levels. Also, VD3 supplementation decreased p65 protein expression, hepatocyte proliferation, the size and the liver area occupied by PNL. Therefore, our findings indicate that VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis.
Assuntos
Colecalciferol/administração & dosagem , Suplementos Nutricionais/análise , Fungicidas Industriais/toxicidade , Hexaclorobenzeno/toxicidade , Neoplasias Hepáticas Experimentais/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colecalciferol/análise , Feminino , Fungicidas Industriais/metabolismo , Glutationa/metabolismo , Hexaclorobenzeno/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/etiologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos dos fármacos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is characterized by multistage formation. The presence of ductular reaction, cytokeratin 7 positivity (PCK7), and increased levels of gamma glutamyltransferase (γGT) has been observed during liver carcinogenesis and contribute to tumor progression. Our goal was to evaluate the ductular reaction in multistage carcinogenesis and to correlate PCK7 and γGT levels with tumor incidence, histological characteristics, liver DNA damage index, and the expression of oxidative stress proteins. HCC was induced in 24 male Wistar rats weighing 145-150 g by chronic and intermittent exposure to 50 or 100 mg/kg diethylnitrosamine (DEN). Six control animals received only vehicle. Blood was collected to determine hepatic enzyme levels. Animals were divided into three groups: control (CO), precancerous lesions (PL), and advanced HCC. Liver samples were obtained for immunohistochemical analyses and the measurement of protein expression. Statistical analyses included Tukey's test and Pearson's correlation analyses. We observed an extensive ductular reaction in advanced HCC and a strong correlation between PCK7 and levels of γGT and the poor prognosis and aggressiveness of HCC. The extent of PCK7 and high γGT levels were associated with overexpression of inducible nitric oxide synthase (iNOS) and heat shock factor protein 1 (HSF-1). However, PCK7 and γGT levels were negatively correlated with protein expression of nuclear factor erythroid 2-related factor 2 (NRF2) and inducible heat shock protein 70 (iHSP70). These findings suggest that ductular reaction is involved in the progression of multistage hepatocarcinogenesis.