Project description:Gene-expression profiles of rat liver cirrhosis induced by diethylnitrosamine and the effect of erlotinib on liver fibrogenesis and liver cancer development Hepatocellular carcinoma (HCC) is the sixth most common solid tumor worldwide and the third leading cause of cancer-related death. Given the lack of successful treatment options, chemoprevention in high-risk patients has been proposed as an alternative strategy. Mounting evidence supports a role for epidermal growth factor (EGF) during chronic liver disease and hepatocellular transformation. We address the hypothesis that blocking the EGF-EGF receptor (EGFR) pathway may be an effective strategy for inhibiting fibrogenesis and hepatocarcinogenesis. A rat model of diethylnitrosamine (DEN)-induced cirrhosis was used to examine the effects of erlotinib on underlying chronic liver disease and HCC formation. The DEN-induced rat model closely resembles disease progression in humans both pathologically and molecularly. Erlotinib significantly prevented the development of HCC tumor nodules in a dose-dependent fashion. Further, erlotinib inhibited the activation of hepatic stellate cells and prevented fibrogenesis. Erlotinib also reduced hepatotoxicity and improved liver function. Finally, a gene expression signature predictive of poor survival in human cirrhosis patients was reversed in response to erlotinib. Our data demonstrate for the first time that EGFR inhibition prevents liver fibrogenesis. Further, our results suggest that erlotinib is a potentially effective HCC chemoprevention strategy through inhibition of cirrhosis progression which can be monitored at the molecular level. Keywords: Cirrhotic liver, Expression array, Illumina, Signatures, Outcome prediction Animals received humane care according to the criteria outlined in the M-bM-^@M-^\Guide for the Care and Use of Laboratory AnimalsM-bM-^@M-^] of the National Academy of Sciences. All animals were maintained in accordance with the institutional guidelines of the Massachusetts General Hospital Subcommittee on Research Animal Care. Male Wistar rats received weekly IP injections of diethylnitrosamine (DEN) at 50 mg/kg, 100mg/kg, or vehicle control (PBS) for 18 weeks. A subset of rats received either daily (5X a week) IP injections of 2 mg/kg erlotinib or vehicle control during weeks 13 - 18. In a separate study, the erlotinib dose was lowered to 0.5 mg/kg. The vehicle groups from the two studies were not significantly different so they were combined together for analysis. Rats were weighed at the end of each week. Animals were sacrificed at 9, 13 and 19 weeks after a one-week washout period to eliminate acute effects of DEN. At the time of sacrifice, the non-tumor liver tissues were collected in RNase-free tubes and snap-frozen in liquid nitrogen. Frozen tissues were stored at -80M-BM-0C until RNA extraction.

Project description:Gene-expression profiles of rat hepatocellular carcinoma induced by diethylnitrosamine (DEN) and the effect of erlotinib Hepatocellular carcinoma (HCC) is the sixth most common solid tumor worldwide and the third leading cause of cancer-related death. Given the lack of successful treatment options, chemoprevention in high-risk patients has been proposed as an alternative strategy. Mounting evidence supports a role for epidermal growth factor (EGF) during chronic liver disease and hepatocellular transformation. We address the hypothesis that blocking the EGF-EGF receptor (EGFR) pathway may be an effective strategy for inhibiting fibrogenesis and hepatocarcinogenesis. A rat model of diethylnitrosamine (DEN)-induced cirrhosis was used to examine the effects of erlotinib on underlying chronic liver disease and HCC formation. The DEN-induced rat model closely resembles disease progression in humans both pathologically and molecularly. Erlotinib significantly prevented the development of HCC tumor nodules in a dose-dependent fashion. Further, erlotinib inhibited the activation of hepatic stellate cells and prevented fibrogenesis. Erlotinib also reduced hepatotoxicity and improved liver function. Finally, a gene expression signature predictive of poor survival in human cirrhosis patients was reversed in response to erlotinib. Our data demonstrate for the first time that EGFR inhibition prevents liver fibrogenesis. Further, our results suggest that erlotinib is a potentially effective HCC chemoprevention strategy through inhibition of cirrhosis progression which can be monitored at the molecular level. Animals received humane care according to the criteria outlined in the M-bM-^@M-^\Guide for the Care and Use of Laboratory AnimalsM-bM-^@M-^] of the National Academy of Sciences. All animals were maintained in accordance with the institutional guidelines of the Massachusetts General Hospital Subcommittee on Research Animal Care. Male Wistar rats received weekly IP injections of diethylnitrosamine (DEN) at 50 mg/kg, 100mg/kg, or vehicle control (PBS) for 18 weeks. A subset of rats received either daily (5X a week) IP injections of 2 mg/kg erlotinib or vehicle control during weeks 13 - 18. In a separate study, the erlotinib dose was lowered to 0.5 mg/kg. The vehicle groups from the two studies were not significantly different so they were combined together for analysis. Rats were weighed at the end of each week. Animals were sacrificed at 9, 13 and 19 weeks after a one-week washout period to eliminate acute effects of DEN. At the time of sacrifice, the non-tumor liver tissues were collected in RNase-free tubes and snap-frozen in liquid nitrogen. Frozen tissues were stored at -80M-BM-0C until RNA extraction.

Project description:The present study aimed at proposing a novel chemically-induced cirrhosis-associated rat hepatocarcinogenesis model, involving the characterization of histological, biochemical and molecular features. Male Wistar rats received a single dose of diethylnitrosamine (DEN, 200 mg/Kg body weight [b.wt.]), and were submitted to several cycles of thioacetamide (TAA, 200 mg/Kg b.wt.), during 23 weeks. Blood and liver were collected from untreated and DEN/TAA-treated groups. Liver samples were processed for global gene expression (cDNA microarray), histopathological (HE) and collagen content (picrosirius red) evaluations, immunohistochemical (Ki-67, GST-P and α-SMA), biochemical (catalase, glutathione peroxidase and glutathione-S-transferase) and gelatin zymography (MMP-2 and 9) analysis. Using a very stringent analysis (FDR<0.01 and fold change>3), gene expression array evidenced 359 differentially expressed genes upon DEN/TAA regimen. Gene Ontology and functional analyses showed several upregulated genes involved in extracellular matrix organization, mainly collagen type I α1 and 2 (Col1α1, Col1α2) and tissue inhibitor of metalloproteinase 1 and 2 (Timp1 and Timp2) genes. In addition, glutathione S-transferase, pi 1 and 2 (Gstp1 and Gstp2) genes were markedly upregulated. In contrast, functional analyses also revealed the downregulation of antioxidant response genes, as catalase, glutathione peroxidase 1 and glutathione S-transferase mu type 3 (Cat, Gpx1 and Gstm3). In agreement with gene expression data, our model presented extensive liver cirrhosis with increased α-SMA expression and collagen deposition, as well as marked development of preneoplastic GST-P positive hyperplastic lesions and some neoplasms. Besides, we observed a decrease in total glutathione peroxidase, total glutatione-S-tranferase and catalase activities. The characterization of a suitable cirrhosis-associated hepatocarcinogenesis model could provide insights into molecular characteristics of the human disease and be applied to evaluate potential preventive and therapeutic approaches.

Project description:Background: Hepatocellular carcinoma (HCC) is increasing in incidence and treatment is often unsuccessful. Thus, prevention in high-risk patients with established cirrhosis has been proposed as an alternative strategy. It has been suggested that the increased inflammation and fibrogenesis observed during cirrhosis predisposes the liver to future malignant transformation to HCC through a “field effect”. A growing body of evidence indicates that the green tea polyphenol, (-)-epigallocatechin gallate (EGCG), can reduce inflammation and fibrogenesis during chronic liver injury. Here, we test the hypothesis that EGCG administered in the setting of cirrhosis can prevent future HCC development. Methods: A rat model of diethylnitrosamine (DEN)-induced cirrhosis was used to examine the efficacy of EGCG for inhibition of HCC formation in these cirrhotic livers. DEN (50 mg/kg) was administered weekly throughout the study while 0.02% EGCG was given in drinking water beginning at the onset of cirrhosis. At the end of the study, rats were sacrificed, livers were sectioned and stained to analyze disease progression and tumor nodules were counted. Liver function tests were performed to determine liver injury and overall liver function. Finally, genome-wide gene expression profiling on the surrounding, non-tumoral liver tissue was used to monitor the “field effect” in response to EGCG. Results: EGCG significantly (p < 0.01) prevented the development of HCC tumor nodules from on average 18.8 in vehicle controls to on average 9.1 in EGCG-treated animals. EGCG also reduced liver injury and improved liver function as assessed by serum chemistry tests. Finally, a gene expression signature predictive of poor survival and HCC development in human cirrhosis patients was reversed in response to EGCG. Conclusions: Our data are consistent with the growing body of evidence suggesting that EGCG has protective effects in liver disease. Further, our results suggest that EGCG is a potentially effective HCC prevention strategy that can be monitored using gene expression signatures.

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Transcriptome profiles of liver from cirrhotic rat treated with nizatidine

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