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Gezer E, Özer C, Şimşek T, Yaprak Bayrak B, Turan G, Çetinarslan B, Selek A, Cantürk Z, Sözen M, Köksalan D. N-Nitrosomorpholine-induced oncocytic transformation in rat endocrine organs. Eur J Med Res 2024; 29:64. [PMID: 38245764 PMCID: PMC10799477 DOI: 10.1186/s40001-024-01654-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2023] [Accepted: 01/10/2024] [Indexed: 01/22/2024] Open
Abstract
BACKGROUND N-Nitrosomorpholine (NMO) is one of the most common N-nitroso compounds. An oncocytic transformation has been demonstrated in renal tubules of NMO-treated rats. In our study, we aimed to investigate the potential transformation of oncocytic cells in 6 endocrine organs, i.e., thyroid, adrenal and pituitary glands, pancreas, testis, and bone, of NMO-exposed rats. METHODS Thirty male rats were born and raised. Fifteen of them were given a single dose of 320 mg NMO per kg body weight, dissolved in drinking water, by a gavage tube. At the end of 52 weeks, the animals in both series were killed. Right after the killing, 6 different endocrine organs (hypophysis, thyroid, pancreas, adrenal gland, bone [femur], and testicles) of each animal were excised. RESULTS There was no evidence of oncocytic cell development in the control group. In contrast, oncocytes were observed in 8 out of 13 NMO-treated rats: 2 in the adrenal sections, 1 in the thyroid sections, 3 in the pituitary sections, and 2 in the pancreas sections. Thesticle and bone sections were completely normal. CONCLUSIONS We showed that NMO induced an oncocytic change in pancreas, thyroid, pituitary, and adrenal glands. To date, no identified specific environmental risk factors that lead to an oncocytic transformation in endocrine glands have been reported previously. Given the increasing prevalence of endocrine-disrupting chemicals in the environment, personal care products, manufactured goods, and food sources, there is a need to advance our understanding of the pathological mechanisms underlying oncocytosis in endocrine organs.
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Affiliation(s)
- Emre Gezer
- Division of Endocrinology and Metabolism, Darica Farabi Training and Research Hospital, Kocaeli, Turkey.
| | - Cüneyt Özer
- Experimental Medicine Research and Application Unit, Kocaeli University, Kocaeli, Turkey
| | - Turgay Şimşek
- Department of General Surgery, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Büşra Yaprak Bayrak
- Department of Pathology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Gupse Turan
- Department of Pathology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Berrin Çetinarslan
- Department of Endocrinology and Metabolism, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Alev Selek
- Department of Endocrinology and Metabolism, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Zeynep Cantürk
- Department of Endocrinology and Metabolism, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Mehmet Sözen
- Department of Endocrinology and Metabolism, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Damla Köksalan
- Department of Endocrinology and Metabolism, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
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2
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Lin YL, Li Y. Study on the hepatocellular carcinoma model with metastasis. Genes Dis 2020; 7:336-350. [PMID: 32884988 PMCID: PMC7452459 DOI: 10.1016/j.gendis.2019.12.008] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2019] [Revised: 12/07/2019] [Accepted: 12/31/2019] [Indexed: 02/07/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death around the world due to advanced clinical stage at diagnosis, high incidence of recurrence and metastasis after surgical treatment. It is in urgent need to create appropriate animal models to explore the mechanism, patterns, risk factors, and therapeutic strategies of HCC metastasis and recurrence. However, most of the established models lack the phenotype of invasion and metastasis in patient, or have unstable phenotype. To establish HCC models with stable metastasis phenotype requires profound understanding in cancer metastasis biology and scientific methodology. Over the past 3 decades, HCC models with stable metastasis have been extensively studied. This paper reviewed the history and development of HCC animal models and cell models, focusing on the screening and maintaining of metastatic potential and phenotype. In-depth studies using these models vastly promote the understanding of cellular and molecular mechanisms and development of therapeutic strategies on HCC metastasis.
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Affiliation(s)
- Yu-Lin Lin
- Department of Peritoneal Cancer Surgery, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, China
| | - Yan Li
- Department of Peritoneal Cancer Surgery, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, China
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3
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Thoolen B, Ten Kate FJW, Castigliego D, van Diest PJ, Malarkey DE, Elmore SA, Maronpot RR. Comparative immunohistochemical investigation of rat and human hepatocellular carcinomas. J Histotechnol 2013. [DOI: 10.1179/2046023613y.0000000026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
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4
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Expression of glutathione peroxidase 2 is associated with not only early hepatocarcinogenesis but also late stage metastasis. Toxicology 2013; 311:115-23. [PMID: 23867582 DOI: 10.1016/j.tox.2013.07.005] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2013] [Revised: 07/04/2013] [Accepted: 07/05/2013] [Indexed: 01/16/2023]
Abstract
Understanding of mechanisms of cancer progression is very important for reduction of cancer mortality. Of six rat hepatocellular carcinoma (HCC) cell lines, differing in their metastatic potential to the lung after inoculation into the tail vein of nude mice, the most metastatic featured particular overexpression of glutathione peroxidase 2 (GPX2). Therefore, we analyzed the influence of interference in highly metastatic L2 cells by siRNA transfection. Gpx2 siRNA significantly inhibited cell proliferation at 24 and 48h time points with induction of apoptosis but not cell cycle arrest. High expression of mutated p53 was detected in all HCC cell lines, with reduction in Gpx2 siRNA-transfected cells. Migration and invasion in vitro were also suppressed as compared to control siRNA-transfected cells and secretion of matrix metalloproteinase 9 was reduced. In vivo, the numbers and areas of metastatic nodules per area in the lungs were significantly reduced in the mice inoculated with Gpx2 siRNA-transfected cells as compared to control siRNA-transfected cells. In conclusion, expression of GPX2 is associated with cancer metastasis from rat HCCs both in vitro and in vivo. Together with immunohistochemical findings of elevated expression in rat and also human liver lesions, the results point to important roles in hepatocarcinogenesis.
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5
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Ogawa K, Pitchakarn P, Suzuki S, Chewonarin T, Tang M, Takahashi S, Naiki-Ito A, Sato S, Takahashi S, Asamoto M, Shirai T. Silencing of connexin 43 suppresses invasion, migration and lung metastasis of rat hepatocellular carcinoma cells. Cancer Sci 2012; 103:860-7. [PMID: 22320152 DOI: 10.1111/j.1349-7006.2012.02228.x] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2011] [Revised: 01/12/2012] [Accepted: 02/09/2012] [Indexed: 11/28/2022] Open
Abstract
To reduce cancer mortality, understanding of mechanisms of cancer metastasis is crucial. We have established six rat hepatocellular carcinoma (HCC) cell lines, which exhibit differing metastatic potential to the lung after inoculation into the tail veins of nude mice. In the present experiment, we investigated the process of cell attachment to metastatic sites and possible regulating factors. One hour after inoculation, two of two HCC cell lines with high metastatic potential and one of two HCC cell lines with low metastatic potential exhibited many attached cells in the lung. One day after inoculation, lung metastatic foci were observed only with highly-metastatic cells with elevated connexin 43 (Cx43) expression as assessed by cDNA array analysis. Furthermore, 24 or 48 h after transfection of an siRNA targeting Cx43, in vitro invasion and migration were suppressed by 68% (P < 0.001) and 36% (P < 0.05) compared with control-siRNA transfected cells, despite no differences in cellular morphology, cell proliferation or apoptotic activity. Moreover, the number of metastatic nodules per lung area in nude mice was significantly (P < 0.01) reduced. In conclusion, suppression of Cx43 expression in tumor cells reduced in vitro migration and invasion capacity and in vivo metastatic ability so that Cx43 has potential as a molecular target for prevention of cancer metastasis with Cx43 overexpressing tumors.
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Affiliation(s)
- Kumiko Ogawa
- Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
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6
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High mobility group box associated with cell proliferation appears to play an important role in hepatocellular carcinogenesis in rats and humans. Toxicology 2008; 255:160-70. [PMID: 19041683 DOI: 10.1016/j.tox.2008.10.023] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2008] [Revised: 10/20/2008] [Accepted: 10/21/2008] [Indexed: 11/22/2022]
Abstract
To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.
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7
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Güller M, Toualbi-Abed K, Legrand A, Michel L, Mauviel A, Bernuau D, Daniel F. c-Fos overexpression increases the proliferation of human hepatocytes by stabilizing nuclear Cyclin D1. World J Gastroenterol 2008; 14:6339-46. [PMID: 19009649 PMCID: PMC2766115 DOI: 10.3748/wjg.14.6339] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of stable c-Fos overexpression on immortalized human hepatocyte (IHH) proliferation.
METHODS: IHHs stably transfected with c-Fos (IHH-Fos) or an empty vector (IHH-C) were grown in medium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins (Cyclin D1, E, A) cyclin dependent kinases (cdk) cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, total and phosphorylated GSK-3β and epidermal growth factor receptor (EGF-R) were assayed by Western blotting. Analysis of Cyclin D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction (PCR) analysis. Stability of Cyclin D1 was studied by cycloheximide blockade experiments.
RESULTS: Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorporation following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpressing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK-3β, a kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein compared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH-Fos by treatment with AG1478, a specific EGF-R tyrosine kinase inhibitor, prevented the phosphorylation of GSK-3β induced by serum stimulation and decreased Cyclin D1 stability in the nucleus.
CONCLUSION: Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular carcinoma.
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8
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Abstract
Recent studies have drawn attention to cytokines as important modulators of hepatocyte cell death during acute and chronic liver disease. Through interaction with cell surface receptors, they activate specific intracellular pathways that influence cell fate in different manners. For example, tumor necrosis factor not only induces proapoptotic signals via the caspase cascade but also activates intracellular survival pathways, namely the nuclear factor (NF)-kappaB pathway. In this article, we will focus on the function of the NF-kappaB pathway in liver physiology and pathology. Especially, recent data based on experiments with genetically modified mice will be discussed, which demonstrated important and controversial functions of this pathway e.g. in cytokine-mediated hepatocyte apoptosis, ischemia-reperfusion injury, liver regeneration and the development of hepatocellular carcinoma. Moreover, the role of the interleukin-6 pathway and its possible protective function in the context of liver failure will be summarized.
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Affiliation(s)
- Tom Luedde
- EMBL Mouse Biology Program, Monterotondo, Italy
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9
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Yang YA, Zhang GM, Feigenbaum L, Zhang YE. Smad3 reduces susceptibility to hepatocarcinoma by sensitizing hepatocytes to apoptosis through downregulation of Bcl-2. Cancer Cell 2006; 9:445-57. [PMID: 16766264 PMCID: PMC2708973 DOI: 10.1016/j.ccr.2006.04.025] [Citation(s) in RCA: 119] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/11/2005] [Revised: 02/23/2006] [Accepted: 04/06/2006] [Indexed: 12/17/2022]
Abstract
In the liver, derangement of TGF-beta signaling is associated with an increased incidence of hepatocellular carcinoma (HCC), but the mechanism is not clear. We report here that forced expression of a major TGF-beta signaling transducer, Smad3, reduces susceptibility to HCC in a chemically induced murine model. This protection is conferred by Smad3's ability to promote apoptosis by repressing Bcl-2 transcription in vivo through a GC-rich element in the Bcl-2 promoter. We also show that the proapoptotic activity of Smad3 requires both input from TGF-beta signaling and activation of p38 MAPK, which occurs selectively in the liver tumor cells. Thus, Smad3 enables the tumor suppression function of TGF-beta by serving as a physiological mediator of TGF-beta-induced apoptosis.
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MESH Headings
- Animals
- Apoptosis
- Carcinoma, Hepatocellular/chemically induced
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Cells, Cultured
- Disease Susceptibility
- Down-Regulation
- Hepatocytes/metabolism
- Hepatocytes/pathology
- Liver Neoplasms, Experimental/chemically induced
- Liver Neoplasms, Experimental/metabolism
- Liver Neoplasms, Experimental/pathology
- Mice
- Mice, Transgenic
- Promoter Regions, Genetic
- Protein Transport
- Proto-Oncogene Proteins c-bcl-2/genetics
- Proto-Oncogene Proteins c-bcl-2/physiology
- Signal Transduction
- Smad3 Protein/biosynthesis
- Smad3 Protein/genetics
- Smad3 Protein/physiology
- Transcription, Genetic
- Transforming Growth Factor beta/physiology
- p38 Mitogen-Activated Protein Kinases/metabolism
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Affiliation(s)
- Yu-An Yang
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892
| | - Gen-Mu Zhang
- Laboratory Animal Science Program, National Cancer Institute at Frederick, Frederick, Maryland 21702
| | - Lionel Feigenbaum
- Laboratory Animal Science Program, National Cancer Institute at Frederick, Frederick, Maryland 21702
| | - Ying E. Zhang
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892
- Correspondence:
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Malarkey DE, Parker JS, Turman CA, Scott AM, Paules RS, Collins J, Maronpot RR. Microarray data analysis of mouse neoplasia. Toxicol Pathol 2005; 33:127-35. [PMID: 15805064 DOI: 10.1080/01926230590888315] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Microarray gene expression analysis offers great promise to help us understand the molecular events of experimental carcinogenesis, but have such promises been fulfilled? Studies of gene expression profiles of rodent are being published and demonstrate that yes, indeed, gene array data is furthering our understanding of tumor biology. Recent studies have identified differentially expressed genes in rodent mammary, colon, lung, and liver tumors. Although relatively few genes on the rodent arrays have been fully characterized, information has been generated to better identify signatures of histologic type and grade, understand invasion and metastasis, identify candidate biomarkers of early development, identify gene networks in carcinogenesis, understand responses to therapy, and decifer overlap with molecular events in human cancers. Data from mouse lung, mammary gland, and liver tumor studies are reviewed as examples of how to approach and interpret gene array data. Methods of gene array data analysis were also applied for discovery of genes involved in the regression of mouse liver tumors induced by chlordane, a nongenotoxic murine hepatocarcinogen. Promises are beginning to be fulfilled and it is clear that pathologists and toxicologists, in collaboration with molecular biologists, bioinformatists,and other scientists are making great strides in the design, analysis, and interpretation of microarray data for cancer studies.
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Affiliation(s)
- David E Malarkey
- Laboratory of Experimental Pathology, Research Triangle Park, North Carolina 27709, USA.
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11
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Ferlin MG, Chiarelotto G, Gasparotto V, Dalla Via L, Pezzi V, Barzon L, Palù G, Castagliuolo I. Synthesis and in vitro and in vivo antitumor activity of 2-phenylpyrroloquinolin-4-ones. J Med Chem 2005; 48:3417-27. [PMID: 15857148 DOI: 10.1021/jm049387x] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
In our search for potential new anticancer drugs, we designed and synthesized a series of tricyclic compounds containing the antimitotic 2-phenylazaflavone chromophore fused to a pyrrole ring in a pyrroloquinoline structure. Compounds 8, 18, 19, 22, 23, 25 and 26, when tested against a panel of fourteen human tumor cell lines, showed poor in vitro cytotoxic activity, whereas 20, 21 and 24 showed significant activity (IC(50) 0.7 to 50 microM). Steroid hormone-sensitive ovary, liver, breast and adrenal gland adenocarcinoma cell lines displayed the highest sensitivity (IC(50) 0.7 to 8 microM). Compound 24 blocked cells in the G(2)/M phase of the cell cycle and induced a significant increase in apoptotis. Compounds 20, 21 and 24 proved to alter microtubule assembly and stability, displaying a cytoplasmic microtubule network similar to that caused by Vincristine. In vivo, administration of compound 24 to Balb/c mice inhibited the growth of a syngenic hepatocellular carcinoma.
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Affiliation(s)
- Maria Grazia Ferlin
- Department of Pharmaceutical Sciences, and Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy. mariagrazia.ferlin@ unipd.it
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12
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Song B, Tang JW, Wang B, Cui XN, Hou L, Sun L, Mao LM, Zhou CH, Du Y, Wang LH, Wang HX, Zheng RS, Sun L. Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip. World J Gastroenterol 2005; 11:1463-72. [PMID: 15770722 PMCID: PMC4305688 DOI: 10.3748/wjg.v11.i10.1463] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: In order to obtain lymphogenous metastasis-associated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential.
METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e., biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip® MOE430A (containing 22690 transcripts, including 14500 known mouse genes and 4371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics.
RESULTS: Out of the 14500 known genes investigated, 110 (0.8%) were up regulated at least 23 fold. Among the total 4371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis, the 110 genes were further classified into two groups: differential biological process profile and molecular function profile.
CONCLUSION: Using high-throughput gene chip method, a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.
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Affiliation(s)
- Bo Song
- Department of Pathology, Dalian Medical University, Dalian 116027, Liaoning Province, China
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14
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Abstract
AIM: Inactivation of p53 gene is one of the most frequent genetic alterations in carcinogenesis. The mutation status of p53 gene was analyzed, in order to understand the effect of p53 mutation on chemical hepatocarcinogenesis of rats.
METHODS: During hepatocarcinogenesis of rats induced by 3’-methyl-4- dimethylaminoazobenzene (3’-Me-DAB), prehepatocarcinoma and hepatocarcinoma foci were collected by laser capture microdissection (LCM), and quantitatively analyzed for levels of p53 mRNA by LightCyclerTM real-time RT-PCR and for mutations in p53 gene exons 5-8 by direct sequencing.
RESULTS: Samples consisting of 44 precancerous foci and 24 cancerous foci were collected by LCM. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the prehepatocarcinoma lesion, more than ten times that of adjacent normal tissue, and gradually decreased from week 6 to week 24. The expression of p53 mRNA in adjacent normal tissue was significantly lower than that in prehepatocarcinoma. Similar to prehepatocarcinoma, p53 mRNA in cancer was markedly higher than that in adjacent normal tissue at week 12, and was closer to normal at week 24. Direct p53 gene sequencing showed that 35.3% (24/68) (9 precancer, 15 cancer) LCM samples exhibited point mutations, 20.5% of prehepatocarcinoma LCM samples presented missense mutations at exon 6/7 or/and 8, and was markedly lower than 62.5% of hepatocarcinoma ones (P < 0.01). Mutation of p53 gene formed the mutant hot spots at 5 codons. Positive immunostaining for p53 protein could be seen in prehepatocarcinoma and hepatocarcinoma foci at 24 weeks.
CONCLUSION: p53 gene mutation is present in initial chemical hepatocarcinogenesis, and the mutation of p53 gene induced by 3’-Me-DAB is an important factor of hepatocarcinogenesis.
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Affiliation(s)
- Wei-Guo Deng
- Department of Obstetrics and Gynecology, First Hospital, Jilin University, 1 Xinmin Street, Changchun 130021, Jilin Province, China
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15
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Fu Y, Deng W, Kawarada Y, Kawagoe M, Ma YZ, Li X, Guo N, Kameda T, Terada K, Sugiyama T. Mutation and expression of the p53 gene during chemical hepatocarcinogenesis in F344 rats. BIOCHIMICA ET BIOPHYSICA ACTA 2003; 1628:40-9. [PMID: 12850271 DOI: 10.1016/s0167-4781(03)00113-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Inactivation of the p53 gene is one of the most frequent genetic alterations in carcinogenesis. We studied gene mutations, the mRNA expression of p53, and the accumulation of p53 protein in chemical hepatocarcinogenesis in rats. Samples consisting of 44 precancerous foci and 18 cancerous foci were collected by laser capture microdissection (LCM), and analyzed for mutations in rat p53 gene exons 5-8 by PCR-single-strand conformational polymorphism (PCR-SSCP). We found that 25 PCR-SSCP bands of exons 6/7 and 8 were altered in 22/62 (35.4%) LCM samples. Direct p53 gene sequencing showed that 20/62 (9 precancer, 11 cancer) (32.3%) LCM samples exhibited 34 point mutations. Ten LCM samples exhibited double or triple mutations in exons 6/7 and 8 simultaneously. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the precancerous lesion, 20 times that of adjacent normal tissue, and returned to normal by week 23. Similar to precancer, p53 mRNA in cancer was five times as high as that of adjacent normal tissue at week 12, and was closer to normal at week 23. When p53 mRNA declined from a high to low, positive immunostaining for the p53 protein began to be seen in precancerous and cancerous foci, suggesting that the p53 protein had accumulated in these foci. Results show that p53 gene mutation is present in initial chemical hepatocarcinogenesis and p53 mRNA concentration is clearly elevated before gene mutation. Once the p53 gene has mutated, mRNA concentration progressively declines, suggesting that mutation leads to inactivation of the p53 gene.
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Affiliation(s)
- Yan Fu
- Department of Biochemistry, Akita University School of Medicine, 1-1-1 Hondo, 010-8543, Akita, Japan
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16
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Eferl R, Ricci R, Kenner L, Zenz R, David JP, Rath M, Wagner EF. Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell 2003; 112:181-92. [PMID: 12553907 DOI: 10.1016/s0092-8674(03)00042-4] [Citation(s) in RCA: 378] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The transcription factor c-Jun mediates several cellular processes, including proliferation and survival, and is upregulated in many carcinomas. Liver-specific inactivation of c-Jun at different stages of tumor development was used to study its role in chemically induced hepatocellular carcinomas (HCCs) in mice. The requirement for c-jun was restricted to early stages of tumor development, and the number and size of hepatic tumors was dramatically reduced when c-jun was inactivated after the tumor had initiated. The impaired tumor development correlated with increased levels of p53 and its target gene noxa, resulting in the induction of apoptosis without affecting cell proliferation. Primary hepatocytes lacking c-Jun showed increased sensitivity to TNF-alpha-induced apoptosis, which was abrogated in the absence of p53. These data indicate that c-Jun prevents apoptosis by antagonizing p53 activity, illustrating a mechanism that might contribute to the early stages of human HCC development.
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Affiliation(s)
- Robert Eferl
- Research Institute of Molecular Pathology (IMP), Dr. Bohrgasse 7, A-1030, Vienna, Austria
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17
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Oh JY, Jeong JS, Kim YJ, Nam KJ, Park BH, Kwon EY, Kim YH, Hwang TH. Ultrasonographic evidence of phenotypic instability during hepatocarcinogenesis in N-nitrosomorpholine-treated rats. Exp Mol Pathol 2002; 73:67-73. [PMID: 12127056 DOI: 10.1006/exmp.2002.2434] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Carcinogen-induced hepatoma in immunocompetent animal models has shown a progress similar to the clinical course of human hepatoma. Ultrasonography (US) was used for consecutive evaluation of the phenotypic changes in Sprague-Dawley rats exposed for 8 weeks to N-nitrosomorpholine (NNM, 200 mg/L). Three distinctive US findings were ascites, coarseness (defined as small and heterogeneously widespread increased echogenecity), and nodularity (defined as a >0.6-cm-sized echogenic region and clearly showing a tumor-like mass). Abdominal ascites was observed in 5 of 26 rats at week 8 NNM posttreatment and the number of rats showing ascites gradually increased. Coarseness (22 of 26 rats) and nodularity (1 of 18) appeared at weeks 8 and 17 NNM posttreatment, respectively. The gross and histological findings indicated that coarseness and nodularity shown in US reflected fibrosis and hepatocellular carcinoma or cholangiofibroma, respectively. The computer-aided quantification of coarseness and nodularity showed that the regression-linked phenotypic instability was present in coarseness but not in nodularity. We conclude that the heterogeneity of preneoplasia in NNM-treated rats might be induced by phenotypic instability rather than random initiating events of preneoplastic lesion.
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Affiliation(s)
- Jong-Young Oh
- Institute of Medical Science, Department of Radiology, Medical College of Dong-A University, Busan 602-714, South Korea
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Tang ZY, Sun FX, Tian J, Ye SL, Liu YK, Liu KD, Xue Q, Chen J, Xia JL, Qin LX, Sun SL, Wang L, Zhou J, Li Y, Ma ZC, Zhou XD, Wu ZQ, Lin ZY, Yang BH. Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential. World J Gastroenterol 2001; 7:597-601. [PMID: 11819839 PMCID: PMC4695559 DOI: 10.3748/wjg.v7.i5.597] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Metastatic human HCC model is needed for the studies on mechanism and interven tion of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of hu man HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metasta sis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesio nmolecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis, antisense approach, metallopro teinase inhibitor, differentiation inducer, etc. It is concluded that the establ ishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.
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Affiliation(s)
- Z Y Tang
- Liver Cancer Institute of Fudan University (previous Liver Cancer Institute of Shanghai Medical University)136 Yixueyuan Road, Zhongshan Hospital, Shanghai 200032,China.
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Tang ZY. Hepatocellular carcinoma--cause, treatment and metastasis. World J Gastroenterol 2001; 7:445-54. [PMID: 11819809 PMCID: PMC4688653 DOI: 10.3748/wjg.v7.i4.445] [Citation(s) in RCA: 302] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/13/2001] [Revised: 07/20/2001] [Accepted: 07/27/2001] [Indexed: 02/06/2023] Open
Abstract
In the recent decades, the incidence of hepatocellular carcinoma (HCC) has been found to be increasing in males in some countries. In China, HCC ranked second of cancer mortality since 1990s. Hepatitis B and C viruses (HBV and HCV) and dietary aflatoxin intake remain the major causative factors of HCC. Surgery plays a major role in the treatment of HCC, particularly for small HCC. Down-staging unresectable huge HCC to smaller HCC and followed by resection will probably be a new approach for further study. Liver transplantation is indicated for small HCC, however, some issues remain to be solved. Different modes of regional cancer therapy for HCC have been tried. Systemic chemotherapy has been disappointing in the past but the future can be promising. Biotherapy, such as cytokines, differentiation inducers, anti-angiogenic agents, gene therapy and tumor vaccine will probably play a role, particularly in the prevention of tumor recurrence. HCC invasiveness is currently the major target of study. Tremendous works have been done at the molecular level, which will provide clues for biomarker of HCC progression as well as targets for intervention.
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Affiliation(s)
- Z Y Tang
- Liver Cancer Institute of Fudan University, 136 Yixueyuan Road, Zhongshan Hospital, Shanghai 200032, China.
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Ogawa K, Nakanishi H, Takeshita F, Futakuchi M, Asamoto M, Imaida K, Tatematsu M, Shirai T. Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice. Int J Cancer 2001; 91:797-802. [PMID: 11275982 DOI: 10.1002/1097-0215(200002)9999:9999<::aid-ijc1140>3.0.co;2-#] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
For better understanding of cancer metastasis, we have established an in vivo model for induction of highly metastatic hepatocellular carcinomas (HCC) in male F344 rats. From 1 tumor, 4 cell lines with differing metastatic potential (C1, C2, C6, C5F) were established by subcloning using the limited-dilution cloning technique. Two other lines, N1 and L2, arose from another primary HCC and a lung metastatic lesion, respectively. Although cell adhesion of each cell line in culture medium was different, tumors developing in the subcutis of nude mice after transplantation were all moderately differentiated HCC with a trabecular pattern. On subcutaneous injection into nude mice, all 6 cell lines proved to be tumorigenic in the injection site and C5F was highly metastatic to the lung. With injection into the tail vein, N1 and L2 formed frequent metastases in the lung as well as in lymph nodes. Using intraperitoneal injection, C1, C6, N1 and L2 showed marked disseminated growth in the abdominal cavity with bloody ascitis. Northern blot analysis revealed expression of known metastasis-related genes, KAI1 and heparanase, to be decreased in C5F, but no differences in expression of nm23-H1 were evident. A point mutation in the GSK-3beta phosphorylation site of the beta-catenin gene was found in L2. These transplantable HCC cell lines that have different metastatic ability should be useful for elucidation of mechanisms of metastasis.
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Affiliation(s)
- K Ogawa
- First Department of Pathology, Nagoya City University, Medical School, 1 Kawasumi, Mizuho-cho, Nagoya 467-8601, Japan.
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Futakuchi M, Hirose M, Ogiso T, Kato K, Sano M, Ogawa K, Shirai T. Establishment of an in vivo highly metastatic rat hepatocellular carcinoma model. Jpn J Cancer Res 1999; 90:1196-202. [PMID: 10622528 PMCID: PMC5926015 DOI: 10.1111/j.1349-7006.1999.tb00695.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
We previously found by chance that N-nitrosomorpholine (NMOR) given after a multi-carcinogenic treatment induces liver carcinomas with 56% lung metastasis, and it was confirmed that hepatocellular carcinoma (HCC) with 100% lung metastasis was produced by 24-week treatment with NMOR and additional treatment with diethylnitrosamine (DEN). In the present study, we modified the duration of NMOR to establish an animal model with a simple experimental protocol and an appropriate experimental duration which would facilitate further study of the mechanisms of metastasis and antimetastatic agents. The results revealed DEN exposure followed by a 16-week treatment with NMOR to be a most efficient method for the induction of HCC metastasizing to the lung. Loss of cadherin, demonstrated immunohistochemically, occurred in an early stage of carcinogenesis, and this was reflected in malignant conversion of primary lesions. This model, with its essential similarities to malignant tumor behavior in man, should find application not only for elucidation of the mechanisms underlying metastasis, but also in the development of anti-metastatic agents.
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Affiliation(s)
- M Futakuchi
- First Department of Pathology, Nagoya City University Medical School, Nagoya.
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Wirnitzer U, Töpfer R, Rosenbruch M. Altered p53 expression in early stages of chemically induced rodent hepatocarcinogenesis. Toxicol Pathol 1998; 26:636-45. [PMID: 9789950 DOI: 10.1177/019262339802600507] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Most available data on the involvement of p53 in rodent carcinogenesis are based on results of the end point of chemically or virally induced carcinogenesis, i.e., tumors. To investigate the role of altered p53 expression in early stages of rodent hepatocarcinogenesis in a systematic way, we treated male Wistar rats for 6 wk, for 13 wk, and for 6 wk followed by a 7-wk recovery period with chemicals classified as genotoxic (200 ppm acetylaminofluorene [AAF], 100 ppm N-nitrosomorpholine [MMN], 200 ppm benzo(a)pyrene), as tumor promoters and carcinogenic in experimental animals (5 ppm ethinylestradiol, 500 ppm phenobarbitone, 3,000 ppm clofibric acid), as carcinogenic in animal experiments (600 ppm thioacetamide), as noncarcinogenic (200 ppm thyroxine), and as tumor promoters in experimental animals (20,000 ppm tryptophan, 120,000 ppm fructose). Immunohistochemical assessment of altered p53 expression on liver sections with polyclonal serum (CM5) resulted in positive staining in 17/21 benzo(a)pyrene-, 1/18 thioacetamide-, 2/21 clofibric acid-, 2/21 phenobarbitone-, 7/19 ethinylestradiol-, 1/21 tryptophan-, 3/19 thyroxine-, and 1/21 fructose-treated rats and in 2/19 controls. These data support earlier results obtained from analogous investigations with a high incidence of altered p53 expression after NNM and AAF treatment. Thus, altered p53 expression appears to be an early and frequent event in rodent carcinogenesis induced by genotoxic chemicals in contrast to most epigenetically acting chemicals.
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Affiliation(s)
- U Wirnitzer
- Institute of Toxicology, Pharma Research Center, Wuppertal, Germany
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