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Hernández-Galdámez HV, Fattel-Fazenda S, Flores-Téllez TNJ, Aguilar-Chaparro MA, Mendoza-García J, Díaz-Fernández LC, Romo-Medina E, Sánchez-Pérez Y, Arellanes-Robledo J, De la Garza M, Villa-Treviño S, Piña-Vázquez C. Iron-saturated bovine lactoferrin: a promising chemopreventive agent for hepatocellular carcinoma. Food Funct 2024; 15:4586-4602. [PMID: 38590223 DOI: 10.1039/d3fo05184f] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/10/2024]
Abstract
Hepatocellular carcinoma (HCC) is a tumor with minimal chance of cure due to underlying liver diseases, late diagnosis, and inefficient treatments. Thus, HCC treatment warrants the development of additional strategies. Lactoferrin (Lf) is a mammalian multifunctional iron-binding glycoprotein of the innate immune response and can be found as either a native low iron form (native-Lf) or a high iron form (holo-Lf). Bovine Lf (bLf), which shares many functions with human Lf (hLf), is safe for humans and has several anticancer activities, including chemotherapy boost in cancer. We found endogenous hLf is downregulated in HCC tumors compared with normal liver, and decreased hLf levels in HCC tumors are associated with shorter survival of HCC patients. However, the chemoprotective effect of 100% iron saturated holo-bLf on experimental hepatocarcinogenesis has not yet been determined. We aimed to evaluate the chemopreventive effects of holo-bLf in different HCC models. Remarkably, a single dose (200 mg kg-1) of holo-bLf was effective in preventing early carcinogenic events in a diethylnitrosamine induced HCC in vivo model, such as necrosis, ROS production, and the surge of facultative liver stem cells, and eventually, holo-bLf reduced the number of preneoplastic lesions. For an established HCC model, holo-bLf treatment significantly reduced HepG2 tumor burden in xenotransplanted mice. Finally, holo-bLf in combination with sorafenib, the advanced HCC first-line treatment, synergistically decreased HepG2 viability by arresting cells in the G0/G1 phase of the cell cycle. Our findings provide the first evidence suggesting that holo-bLf has the potential to prevent HCC or to be used in combination with treatments for established HCC.
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Affiliation(s)
| | - Samia Fattel-Fazenda
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), CDMX, Mexico.
| | - Teresita N J Flores-Téllez
- Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park, SK10 4TG Macclesfield, UK
| | | | - Jonathan Mendoza-García
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), CDMX, Mexico.
| | - Lidia C Díaz-Fernández
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), CDMX, Mexico.
| | - Eunice Romo-Medina
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), CDMX, Mexico.
| | - Yesennia Sánchez-Pérez
- Instituto Nacional de Cancerología (INCan), Subdirección de Investigación Básica, CDMX, Mexico
| | - Jaime Arellanes-Robledo
- Laboratorio de Enfermedades Hepáticas, Instituto Nacional de Medicina Genómica, Ciudad de México, México. Dirección de Cátedras, Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT), Ciudad de México, Mexico
| | - Mireya De la Garza
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), CDMX, Mexico.
| | - Saúl Villa-Treviño
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), CDMX, Mexico.
| | - Carolina Piña-Vázquez
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), CDMX, Mexico.
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2
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Nonomura Y, Wang X, Kikuchi T, Matsui D, Toyotake Y, Takagi K, Wakayama M. Characterization of three γ-glutamyltranspeptidases from Pseudomonas aeruginosa PAO1. J GEN APPL MICROBIOL 2023; 69:150-158. [PMID: 36653156 DOI: 10.2323/jgam.2023.01.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
The Pseudomonas aeruginosa strain, PAO1, has three putative γ-glutamyltranspeptidase (GGT) genes: ggtI, ggtII, and ggtIII. In this study, the expression of each of these genes in P. aeruginosa PAO1 was analyzed, and the properties of the corresponding GGT proteins were investigated. This is the first report on biochemical characterization of GGT paralogs from Pseudomonas species. The crude extracts prepared from P. aeruginosa PAO1 exhibited hydrolysis and transpeptidation activities of 17.3 and 65.0 mU/mg, respectively, and the transcription of each gene to mRNA was confirmed by RT-PCR. All genes were cloned, and the expression plasmids constructed were introduced into an Escherichia coli expression system. Enzyme activity of the expressed protein of ggtI (PaGGTI) was not detected in the system, while the enzyme activities of the expressed proteins derived from ggtII and ggtIII (PaGGTII and PaGGTIII, respectively) were detected. However, the enzyme activity of PaGGTII was very low and easily decreased. PaGGTII with C-terminal his-tag (PaGGTII25aa) showed increased activity and stability, and the purified enzyme consisted of a large subunit of 40 kDa and a small subunit of 28 kDa. PaGGTIII consisted of a large subunit of 37 kDa and a small subunit of 24 kDa. The maximum hydrolysis and transpeptidation activities of PaGGTII25aa were obtained at 40ºC-50ºC, and the maximum hydrolysis and transpeptidation activities of PaGGTIII were obtained at 50ºC-60ºC. These enzymes retained approximately 80% of their hydrolysis and transpeptidation activities after incubation at 50ºC for 10 min, reflecting good stability. Both PaGGTII25aa and PaGGTIII showed higher activities of hydrolysis and transpeptidation in the alkali range than in the acidic range. However, they were highly stable at a wide pH range (5-10.5).
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Affiliation(s)
- Yuuki Nonomura
- Department of Biotechnology, College of Life Sciences, Ritsumeikan University
| | - Xinjia Wang
- Department of Biotechnology, College of Life Sciences, Ritsumeikan University
| | - Takeshi Kikuchi
- Department of Bioinformatics, College of Life Sciences, Ritsumeikan University
| | - Daisuke Matsui
- Department of Biotechnology, College of Life Sciences, Ritsumeikan University
| | - Yosuke Toyotake
- Department of Biotechnology, College of Life Sciences, Ritsumeikan University
| | - Kazuyoshi Takagi
- Department of Applied Chemistry, College of Life Sciences, Ritsumeikan University
| | - Mamoru Wakayama
- Department of Biotechnology, College of Life Sciences, Ritsumeikan University
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Farhat F, Wasim S, Rehman L, Abidi SMA. Affinity purification, identification, and biochemical characterization of Gamma-glutamyl transpeptidase, a membrane anchored enzyme of Gigantocotyle explanatum. Parasitol Res 2023; 122:915-926. [PMID: 36719531 DOI: 10.1007/s00436-023-07786-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Accepted: 01/25/2023] [Indexed: 02/01/2023]
Abstract
Gamma-glutamyl transpeptidase is an enzyme that facilitates the transfer of glutamyl groups from glutamyl peptides to other peptides or water. Additionally, it also participates in important processes such as amino acid transport, cellular redox control, drug detoxification, apoptosis, and DNA fragmentation in a various organism. In the present study, GGT activity in Gigantocotyle explanatum was examined in order to characterize the enzyme in the helminth system. GGT is isolated using membrane solubilization and purified through affinity column chromatography (Con-A Sepharose column). Km and Vmax values, as well as the optimal pH, optimal temperature, and incubation period, are also determined using enzyme kinetics. The hetero-dimeric property of the enzyme is demonstrated by the purified GGT, which yielded two subunits of 65.5 and 55 kDa. The optimal pH and temperature are found to be 8.0 and 37 °C, respectively. While assessing the optimal incubation time of the enzyme, it was observed that the purified GGT not only retained its functional integrity up to 15 min but also reflected considerable thermostability at higher temperatures, by retaining 78% and 25% of its initial activities at 50 °C and 60 °C, respectively. One millimolar concentration of 6-Diazo-5-Oxo Nor-isoleucine (DON), a specific inhibitor of GGT, completely abolished GGT activity. These results suggest that GGT in these worms is a catalytically active enzyme with distinguishing characteristics that can be used for further study to comprehend its function in amphistome biology and in host-parasite relationships, especially since the potential therapeutic candidacy of the GGT enzyme has already been indicated in these groups of organisms.
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Affiliation(s)
- Faiza Farhat
- Section of Parasitology, Department of Zoology, Aligarh Muslim University, Uttar Pradesh, Aligarh, 202002, India.
| | - Sobia Wasim
- College of Medicine, Gachon University, Incheon, South Korea
| | - Lubna Rehman
- Department of Lymphoma/Myeloma, MD Anderson Cancer Center, The University of Texas, Houston, TX, USA
| | - S M A Abidi
- Section of Parasitology, Department of Zoology, Aligarh Muslim University, Uttar Pradesh, Aligarh, 202002, India
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Castro-Gil MP, Torres-Mena JE, Salgado RM, Muñoz-Montero SA, Martínez-Garcés JM, López-Torres CD, Mendoza-Vargas A, Gabiño-López NB, Villa-Treviño S, Del Pozo-Yauner L, Arellanes-Robledo J, Krötzsch E, Pérez-Carreón JI. The transcriptome of early GGT/KRT19-positive hepatocellular carcinoma reveals a downregulated gene expression profile associated with fatty acid metabolism. Genomics 2021; 114:72-83. [PMID: 34861383 DOI: 10.1016/j.ygeno.2021.11.035] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2021] [Revised: 11/05/2021] [Accepted: 11/26/2021] [Indexed: 01/14/2023]
Abstract
Hepatocellular carcinoma expressing hepatobiliary progenitor markers, is considered of poor prognosis. By using a hepatocarcinogenesis model, laser capture microdissection, and RNA-Sequencing analysis, we identified an expression profile in GGT/KRT19-positive experimental tumors; 438 differentially expressed genes were found in early and late nodules along with increased collagen deposition. Dysregulated genes were involved in Fatty Acid Metabolism, RXR function, and Hepatic Stellate Cells Activation. Downregulation of Slc27a5, Acsl1, and Cyp2e1, demonstrated that Retinoid X Receptor α (RXRα) function is compromised in GGT/KRT19-positive nodules. Since RXRα controls NRF2 pathway activation, we determined the expression of NRF2 targeted genes; Akr1b8, Akr7a3, Gstp1, Abcc3, Ptgr1, and Txnrd1 were upregulated, indicating NRF2 pathway activation. A comparative analysis in human HCC showed that SLC27A5, ACSL1, CYP2E1, and RXRα gene expression is mutually exclusive with KRT19 gene expression. Our results indicate that the downregulation of Slc27a5, Acsl1, Rxrα, and Cyp2e1 genes is an early event within GGT/KRT19-positive HCC.
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Affiliation(s)
| | | | - Rosa M Salgado
- Laboratory of Connective Tissue, Centro Nacional de Investigación y Atención de Quemados, Instituto Nacional de Rehabilitación "Luis Guillermo Ibarra Ibarra", CDMX, Mexico
| | - Said A Muñoz-Montero
- Department of Computational Genomics, National Institute of Genomic Medicine, CDMX, Mexico
| | | | | | | | | | - Saúl Villa-Treviño
- Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, CDMX, Mexico
| | - Luis Del Pozo-Yauner
- Department of Pathology, College of Medicine, University of South Alabama, Mobile, AL, USA
| | - Jaime Arellanes-Robledo
- Laboratory of Liver Diseases, National Institute of Genomic Medicine, CDMX, Mexico; Directorate of Cátedras, National Council of Science and Technology, CDMX, Mexico
| | - Edgar Krötzsch
- Laboratory of Connective Tissue, Centro Nacional de Investigación y Atención de Quemados, Instituto Nacional de Rehabilitación "Luis Guillermo Ibarra Ibarra", CDMX, Mexico
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García-Chávez JN, Vásquez-Garzón VR, López MG, Villa-Treviño S, Montiel R. Integration of chronological omics data reveals mitochondrial regulatory mechanisms during the development of hepatocellular carcinoma. PLoS One 2021; 16:e0256016. [PMID: 34383828 PMCID: PMC8360386 DOI: 10.1371/journal.pone.0256016] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2020] [Accepted: 07/28/2021] [Indexed: 12/13/2022] Open
Abstract
Mitochondria participate in multiple functions in eukaryotic cells. Although disruption of mitochondrial function has been associated with energetic deregulation in cancer, the chronological changes in mitochondria during cancer development remain unclear. With the aim to assess the role of mitochondria throughout cancer development, we analyzed samples chronologically obtained from induced hepatocellular carcinoma (HCC) in rats. In our analyses, we integrated mitochondrial proteomic data, mitochondrial metabolomic data and nuclear genome transcriptomic data. We used pathway over-representation and weighted gene co-expression network analysis (WGCNA) to integrate expression profiles of genes, miRNAs, proteins and metabolite levels throughout HCC development. Our results show that mitochondria are dynamic organelles presenting specific modifications in different stages of HCC development. We also found that mitochondrial proteomic profiles from tissues adjacent to nodules or tumor are determined more by the stage of HCC development than by tissue type, and we evaluated two models to predict HCC stage of the samples using proteomic profiles. Finally, we propose an omics integration pipeline to massively identify molecular features that could be further evaluated as key regulators, biomarkers or therapeutic targets. As an example, we show a group of miRNAs and transcription factors as candidates, responsible for mitochondrial metabolic modification in HCC.
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Affiliation(s)
- J. Noé García-Chávez
- Langebio, Unidad de Genómica Avanzada, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Mexico
| | | | - Mercedes G. López
- Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Mexico
| | - Saúl Villa-Treviño
- Department of Cell Biology, Center for Research and Advanced Studies (CINVESTAV-IPN), Ciudad de México, Mexico
| | - Rafael Montiel
- Langebio, Unidad de Genómica Avanzada, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Mexico
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Lawson JS, Syme HM, Wheeler-Jones CPD, Elliott J. Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype. Res Vet Sci 2019; 127:99-102. [PMID: 31683198 PMCID: PMC6863388 DOI: 10.1016/j.rvsc.2019.10.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2019] [Accepted: 10/21/2019] [Indexed: 11/19/2022]
Abstract
The Crandell-Rees Feline Kidney Cell (CRFK) is an immortalised cell line derived from the feline kidney that is utilised for the growth of certain vaccinal viruses. Confusion exists as to whether CRFK are epithelial or mesenchymal in phenotype. The aim of this study was to characterise CRFK cells via immunofluorescence, enzyme cytochemistry, western blotting, RT-qPCR for S100A4 and comparison to primary feline proximal tubular epithelial cells (FPTEC) and feline cortical fibroblasts (FCF). CRFK cells were of fusiform morphology and appeared similar to FCF. CRFK expressed the mesenchymal intermediate filament (IF) protein vimentin together with two cell adhesion molecules associated with feline fibroblasts (CD29 and CD44), and lacked expression of the epithelial IF cytokeratin, myogenic IF desmin and endothelial marker von Willebrand factor (vWF). In addition, CRFK did not demonstrate brush border enzyme activity typical of FPTEC. S100A4 gene expression, implicated in both neoplastic transformation and epithelial to mesenchymal transition, was highly upregulated in CRFK in comparison to the primary feline renal cells. CRFK appear phenotypically similar to fibroblasts, rather than tubular epithelial cells, and may have undergone neoplastic transformation or epithelial-to-mesenchymal transition after extensive passaging. This finding may have potential implications for future research utilising this cell line.
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Affiliation(s)
- J S Lawson
- Comparative Biomedical Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
| | - H M Syme
- Clinical Sciences and Services, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK
| | - C P D Wheeler-Jones
- Comparative Biomedical Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK
| | - J Elliott
- Comparative Biomedical Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK
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Lawson JS, Liu HH, Syme HM, Purcell R, Wheeler-Jones CPD, Elliott J. The cat as a naturally occurring model of renal interstitial fibrosis: Characterisation of primary feline proximal tubular epithelial cells and comparative pro-fibrotic effects of TGF-β1. PLoS One 2018; 13:e0202577. [PMID: 30138414 PMCID: PMC6107233 DOI: 10.1371/journal.pone.0202577] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Accepted: 08/06/2018] [Indexed: 01/08/2023] Open
Abstract
Chronic kidney disease (CKD) is common in both geriatric cats and aging humans, and is pathologically characterised by chronic tubulointerstitial inflammation and fibrosis in both species. Cats with CKD may represent a spontaneously occurring, non-rodent animal model of human disease, however little is known of feline renal cell biology. In other species, TGF-β1 signalling in the proximal tubular epithelium is thought to play a key role in the initiation and progression of renal fibrosis. In this study, we first aimed to isolate and characterise feline proximal tubular epithelial cells (FPTEC), comparing them to human primary renal epithelial cells (HREC) and the human proximal tubular cell line HK-2. Secondly, we aimed to examine and compare the effect of human recombinant TGF-β1 on cell proliferation, pro-apoptotic signalling and genes associated with epithelial-to-mesenchymal transition (EMT) in feline and human renal epithelial cells. FPTEC were successfully isolated from cadaverous feline renal tissue, and demonstrated a marker protein expression profile identical to that of HREC and HK-2. Exposure to TGF-β1 (0-10 ng/ml) induced a concentration-dependent loss of epithelial morphology and alterations in gene expression consistent with the occurrence of partial EMT in all cell types. This was associated with transcription of downstream pro-fibrotic mediators, growth arrest in FPTEC and HREC (but not HK-2), and increased apoptotic signalling at high concentrations of TGF- β1. These effects were inhibited by the ALK5 (TGF-β1RI) antagonist SB431542 (5 μM), suggesting they are mediated via the ALK5/TGF-β1RII receptor complex. Taken together, these results suggest that TGF-β1 may be involved in epithelial cell dedifferentiation, growth arrest and apoptosis in feline CKD as in human disease, and that cats may be a useful, naturally occurring model of human CKD.
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Affiliation(s)
- Jack S. Lawson
- Comparative Biomedical Sciences, The Royal Veterinary College, London, United Kingdom
- * E-mail:
| | - Hui-Hsuan Liu
- Comparative Biomedical Sciences, The Royal Veterinary College, London, United Kingdom
| | - Harriet M. Syme
- Clinical Sciences and Services, The Royal Veterinary College, North Mymms, Hatfield, Herts, United Kingdom
| | - Robert Purcell
- Comparative Biomedical Sciences, The Royal Veterinary College, London, United Kingdom
| | | | - Jonathan Elliott
- Comparative Biomedical Sciences, The Royal Veterinary College, London, United Kingdom
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8
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Chien CS, Chen YH, Chen HL, Wang CP, Wu SH, Ho SL, Huang WC, Yu CH, Chang MH. Cells responsible for liver mass regeneration in rats with 2-acetylaminofluorene/partial hepatectomy injury. J Biomed Sci 2018; 25:39. [PMID: 29695258 PMCID: PMC5937839 DOI: 10.1186/s12929-018-0441-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2018] [Accepted: 04/20/2018] [Indexed: 12/17/2022] Open
Abstract
Background Whether hepatic progenitor cells (HPCs)/oval cells regenerate liver mass upon chronic liver injury is controversial in mice and has not been conclusively proven in humans and rats. In this study, we examined which cell type—hepatocytes or oval cells—mediates liver regeneration in the classic rat 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) injury where AAF reversibly blocks hepatocyte proliferation, thereby inducing oval cell expansion after the regenerative stimulus of PH. Methods We employed lineage tracing of dipeptidyl peptidase IV (DPPIV, a hepatocyte canalicular enzyme)-positive hepatocytes by subjecting rats with DPPIV-chimeric livers to AAF/PH, AAF/PH/AAF (continuous AAF after AAF/PH to nonselectively inhibit regenerating hepatocytes), or AAF/PH/retrorsine injury (2-dose retrorsine after AAF/PH to specifically and irreversibly block existing hepatocytes); through these methods, we determined hepatocyte contribution to liver regeneration. To determine the oval cell contribution to hepatocyte regeneration, we performed DPPIV(+) oval cell transplantation combined with AAF/PH injury or AAF/PH/retrorsine injury in DPPIV-deficient rats to track the fate of DPPIV(+) oval cells. Results DPPIV-chimeric livers demonstrated typical oval cell activation upon AAF/PH injury. After cessation of AAF, DPPIV(+) hepatocytes underwent extensive proliferation to regenerate the liver mass, whereas oval cells underwent hepatocyte differentiation. Upon AAF/PH/AAF injury where hepatocyte proliferation was inhibited by continuous AAF treatment following AAF/PH, oval cells extensively expanded in an undifferentiated state but did not produce hepatocytes. By substituting retrorsine for AAF administration following AAF/PH (AAF/PH/retrorsine), oval cells regenerated large-scale hepatocytes. Conclusions Hepatocyte self-replication provides the majority of hepatocyte regeneration, with supplementary contribution from oval cells in rats under AAF/PH injury. Oval cells expand and maintain in an undifferentiated state upon continuously nonselective liver injury, whereas they can significantly regenerate hepatocytes in a noncompetitive environment.
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Affiliation(s)
- Chin-Sung Chien
- Department of Pediatrics, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation. No.289, Jianguo Rd., Xindian Dist, New Taipei City, 23142, Taiwan.,Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University . No.7, Chung Shan South Rd., Zhongzheng Dist, Taipei, 10002, Taiwan
| | - Ya-Hui Chen
- Department of Pediatrics, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation. No.289, Jianguo Rd., Xindian Dist, New Taipei City, 23142, Taiwan.,Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University . No.7, Chung Shan South Rd., Zhongzheng Dist, Taipei, 10002, Taiwan.,Hepatitis Research Center, National Taiwan University Hospital. No.1, Changde St., Zhongzheng Dist, Taipei, 10048, Taiwan
| | - Hui-Ling Chen
- Hepatitis Research Center, National Taiwan University Hospital. No.1, Changde St., Zhongzheng Dist, Taipei, 10048, Taiwan
| | - Chiu-Ping Wang
- Department of Pediatrics, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation. No.289, Jianguo Rd., Xindian Dist, New Taipei City, 23142, Taiwan
| | - Shang-Hsin Wu
- Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University . No.7, Chung Shan South Rd., Zhongzheng Dist, Taipei, 10002, Taiwan
| | - Shu-Li Ho
- Hepatitis Research Center, National Taiwan University Hospital. No.1, Changde St., Zhongzheng Dist, Taipei, 10048, Taiwan
| | - Wen-Cheng Huang
- Department of Pediatrics, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation. No.289, Jianguo Rd., Xindian Dist, New Taipei City, 23142, Taiwan
| | - Chun-Hsien Yu
- Department of Pediatrics, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation. No.289, Jianguo Rd., Xindian Dist, New Taipei City, 23142, Taiwan. .,Department of Pediatrics, School of Medicine, Tzu Chi University, No.701, Sec. 3, Zhongyang Rd, Hualien, 97004, Taiwan.
| | - Mei-Hwei Chang
- Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University . No.7, Chung Shan South Rd., Zhongzheng Dist, Taipei, 10002, Taiwan.,Hepatitis Research Center, National Taiwan University Hospital. No.1, Changde St., Zhongzheng Dist, Taipei, 10048, Taiwan.,Department of Pediatrics, National Taiwan University Hospital, and College of Medicine, National Taiwan University. No.8, Chung Shan South Rd., Zhongzheng Dist, Taipei, 10041, Taiwan
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McMillin M, DeMorrow S, Glaser S, Venter J, Kyritsi K, Zhou T, Grant S, Giang T, Greene JF, Wu N, Jefferson B, Meng F, Alpini G. Melatonin inhibits hypothalamic gonadotropin-releasing hormone release and reduces biliary hyperplasia and fibrosis in cholestatic rats. Am J Physiol Gastrointest Liver Physiol 2017; 313:G410-G418. [PMID: 28751425 PMCID: PMC5792219 DOI: 10.1152/ajpgi.00421.2016] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/07/2016] [Revised: 06/23/2017] [Accepted: 07/05/2017] [Indexed: 01/31/2023]
Abstract
Melatonin is a hormone produced by the pineal gland with increased circulating levels shown to inhibit biliary hyperplasia and fibrosis during cholestatic liver injury. Melatonin also has the capability to suppress the release of hypothalamic gonadotropin-releasing hormone (GnRH), a hormone that promotes cholangiocyte proliferation when serum levels are elevated. However, the interplay and contribution of neural melatonin and GnRH to cholangiocyte proliferation and fibrosis in bile duct-ligated (BDL) rats have not been investigated. To test this, cranial levels of melatonin were increased by implanting osmotic minipumps that performed an intracerebroventricular (ICV) infusion of melatonin or saline for 7 days starting at the time of BDL. Hypothalamic GnRH mRNA and cholangiocyte secretion of GnRH and melatonin were assessed. Cholangiocyte proliferation and fibrosis were measured. Primary human hepatic stellate cells (HSCs) were treated with cholangiocyte supernatants, GnRH, or the GnRH receptor antagonist cetrorelix acetate, and cell proliferation and fibrosis gene expression were assessed. Melatonin infusion reduced hypothalamic GnRH mRNA expression and led to decreased GnRH and increased melatonin secretion from cholangiocytes. Infusion of melatonin was found to reduce hepatic injury, cholangiocyte proliferation, and fibrosis during BDL-induced liver injury. HSCs supplemented with BDL cholangiocyte supernatant had increased proliferation, and this increase was reversed when HSCs were supplemented with supernatants from melatonin-infused rats. GnRH stimulated fibrosis gene expression in HSCs, and this was reversed by cetrorelix acetate cotreatment. Increasing bioavailability of melatonin in the brain may improve outcomes during cholestatic liver disease.NEW & NOTEWORTHY We have previously demonstrated that GnRH is expressed in cholangiocytes and promotes their proliferation during cholestasis. In addition, dark therapy, which increases melatonin, reduced cholangiocyte proliferation and fibrosis during cholestasis. This study expands these findings by investigating neural GnRH regulation by melatonin during BDL-induced cholestasis by infusing melatonin into the brain. Melatonin infusion reduced cholangiocyte proliferation and fibrosis, and these effects are due to GNRH receptor 1-dependent paracrine signaling between cholangiocytes and hepatic stellate cells.
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Affiliation(s)
- Matthew McMillin
- Research, Central Texas Veterans Health Care System, Temple, Texas
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Sharon DeMorrow
- Research, Central Texas Veterans Health Care System, Temple, Texas
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Shannon Glaser
- Research, Central Texas Veterans Health Care System, Temple, Texas
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Julie Venter
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Konstantina Kyritsi
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Tianhao Zhou
- Research, Central Texas Veterans Health Care System, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Stephanie Grant
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Thao Giang
- Research, Central Texas Veterans Health Care System, Temple, Texas
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - John F Greene
- Department of Pathology, Baylor Scott & White Health, Temple, Texas; and
| | - Nan Wu
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Brandi Jefferson
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
| | - Fanyin Meng
- Research, Central Texas Veterans Health Care System, Temple, Texas
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
- Research Foundation, Baylor Scott & White Health, Temple, Texas
| | - Gianfranco Alpini
- Research, Central Texas Veterans Health Care System, Temple, Texas;
- Digestive Disease Research Center, Baylor Scott & White Health, Temple, Texas
- Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center and Baylor Scott & White Health, Temple, Texas
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10
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Crawford DR, Ilic Z, Guest I, Milne GL, Hayes JD, Sell S. Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice. Carcinogenesis 2017; 38:717-727. [PMID: 28535182 DOI: 10.1093/carcin/bgx048] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.
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Affiliation(s)
- Dana R Crawford
- Albany Medical Center, Department of Immunology and Microbial Disease, 43 New Scotland Avenue, Albany, NY 12208, USA
| | - Zoran Ilic
- Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA
| | - Ian Guest
- Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA
| | - Ginger L Milne
- Vanderbilt University School of Medicine, Department of Medicine and Pharmacology, Nashville, TN 37323, USA
| | - John D Hayes
- Division of Cancer Research, Medical Research Institute, University of Dundee, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK
| | - Stewart Sell
- Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA
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11
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The Hippo signaling functions through the Notch signaling to regulate intrahepatic bile duct development in mammals. J Transl Med 2017; 97:843-853. [PMID: 28581486 PMCID: PMC5901959 DOI: 10.1038/labinvest.2017.29] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2016] [Revised: 12/22/2016] [Accepted: 02/06/2017] [Indexed: 12/24/2022] Open
Abstract
The Hippo signaling pathway and the Notch signaling pathway are evolutionary conserved signaling cascades that have important roles in embryonic development of many organs. In murine liver, disruption of either pathway impairs intrahepatic bile duct development. Recent studies suggested that the Notch signaling receptor Notch2 is a direct transcriptional target of the Hippo signaling pathway effector YAP, and the Notch signaling is a major mediator of the Hippo signaling in maintaining biliary cell characteristics in adult mice. However, it remains to be determined whether the Hippo signaling pathway functions through the Notch signaling in intrahepatic bile duct development. We found that loss of the Hippo signaling pathway tumor suppressor Nf2 resulted in increased expression levels of the Notch signaling pathway receptor Notch2 in cholangiocytes but not in hepatocytes. When knocking down Notch2 on the background of Nf2 deficiency in mouse livers, the excessive bile duct development induced by Nf2 deficiency was suppressed by heterozygous and homozygous deletion of Notch2 in a dose-dependent manner. These results implicated that Notch signaling is one of the downstream effectors of the Hippo signaling pathway in regulating intrahepatic bile duct development.
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12
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Velasco-Loyden G, Pérez-Martínez L, Vidrio-Gómez S, Pérez-Carreón JI, Chagoya de Sánchez V. Cancer chemoprevention by an adenosine derivative in a model of cirrhosis-hepatocellular carcinoma induced by diethylnitrosamine in rats. Tumour Biol 2017; 39:1010428317691190. [DOI: 10.1177/1010428317691190] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Hepatocellular carcinoma is one of the most common cancers, and approximately 80% develop from cirrhotic livers. We have previously shown that the aspartate salt of adenosine prevents and reverses carbon tetrachloride–induced liver fibrosis in rats. Considering the hepatoprotective role of this adenosine derivative in fibrogenesis, we were interested in evaluating its effect in a hepatocarcinogenesis model induced by diethylnitrosamine in rats, where multinodular cancer is preceded by cirrhosis. Rats were injected with diethylnitrosamine for 12 weeks to induce cirrhosis and for 16 weeks to induce hepatocarcinogenesis. Groups of rats were treated with aspartate salt of adenosine from the beginning of carcinogen administration for 12 or 18 weeks total, and another group received the compound from weeks 12 to 18. Fibrogenesis was estimated and the proportion of preneoplastic nodules and tumors was measured. The apoptotic and proliferation rates in liver tissues were evaluated, as well as the expression of cell signaling and cell cycle proteins participating in hepatocarcinogenesis. The adenosine derivative treatment reduced diethylnitrosamine-induced collagen expression and decreased the proportion of nodules positive for the tumor marker γ-glutamyl transferase. This compound down-regulated the expression of thymidylate synthase and hepatocyte growth factor, and augmented the protein level of the cell cycle inhibitor p27; these effects could be part of its chemopreventive mechanism. These findings suggest a hepatoprotective role of aspartate salt of adenosine that could be used as a therapeutic compound in the prevention of liver tumorigenesis as described earlier for hepatic fibrosis.
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Affiliation(s)
- Gabriela Velasco-Loyden
- Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular (IFC), Universidad Nacional Autónoma de México (UNAM), México City, México
| | - Lidia Pérez-Martínez
- Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular (IFC), Universidad Nacional Autónoma de México (UNAM), México City, México
| | - Susana Vidrio-Gómez
- Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular (IFC), Universidad Nacional Autónoma de México (UNAM), México City, México
| | - Julio Isael Pérez-Carreón
- Laboratorio de Bioquímica y Estructura de Proteínas, Instituto Nacional de Medicina Genómica (INMEGEN), México City, México
| | - Victoria Chagoya de Sánchez
- Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular (IFC), Universidad Nacional Autónoma de México (UNAM), México City, México
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13
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Sakolish C, Mahler GJ. A novel microfluidic device to model the human proximal tubule and glomerulus. RSC Adv 2017. [DOI: 10.1039/c6ra25641d] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
A novel multi-channel microfluidic device to model human glomerular filtration and proximal tubular re-uptake.
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14
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Otieno MA, Bhaskaran V, Janovitz E, Callejas Y, Foster WB, Washburn W, Megill JR, Lehman-McKeeman L, Gemzik B. Mechanisms for Hepatobiliary Toxicity in Rats Treated with an Antagonist of Melanin Concentrating Hormone Receptor 1 (MCHR1). Toxicol Sci 2016; 155:379-388. [DOI: 10.1093/toxsci/kfw216] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
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15
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Dixon D, Sleight S, Aust S, Rezabek M. Tumor-Promoting, Initiating, and Hepatotoxic Effects of 3,4,3',4'-Tetrabromobiphenyl (34-TBB) in Rats. ACTA ACUST UNITED AC 2016. [DOI: 10.3109/10915818809019543] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Female, 180–200 g Sprague-Dawley rats were used to determine if 3,4,3',4'-tetrabromobi-phenyl (34-TBB) is a promoter or initiator in a two-stage hepatocarcinogenesis assay. To test for promotion, rats were partially hepatectomized (PH) 24 hr before initiation (day 1) with 10 mg of diethylnitrosamine (DEN)/kg body weight given intraperitoneally (IP). Thirty days later, promotion was with 34-TBB (0.1,1 or 5 mg/kg) or phenobarbital (PB) (500 mg/kg) in diets for 180 days. To test for initiation, rats were PH and were initiated on day 1 with 34-TBB (1, 5, or 10 mg/kg) orally or DEN (10 mg/kg) IP. On day 31, promotion was with 500 mg of PB/kg of diet for 180 days. Noninitiated and non-PH rats were used to assess the histological and ultrastructural tissue changes associated with administration of 34-TBB in the diet for 180 days. Tumor promotion-initiation were assessed by counting and measuring hepatic enzyme-altered foci (EAF) with gamma-glutamyl transpeptidase (GGT) activity. Congener 34-TBB acts as a promoter in experimental hepatocarcinogenesis in rats, as evidenced by increased numbers of GGT-positive EAF. Also, 34-TBB may have initiation potential, as suggested by increased numbers of EAF in rats initiated with 34-TBB and promoted by PB. Dietary administration of 34-TBB for 180 days is not severely toxic in rats, as evidenced by mild histological and ultrastructural changes and minimal alterations in organ and body weights. Congener 34-TBB does not accumulate in liver and adipose tissue of rats.
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Affiliation(s)
- D. Dixon
- Department of Pathology Michigan State University East Lansing, MI 48824
| | - S.D. Sleight
- The Rockefeller University, Laboratory Animal Research Center, New York, New York
| | - S.D. Aust
- Department of Biochemistry, Michigan State University, East Lansing, Michigan
| | - M.S. Rezabek
- The Rockefeller University, Laboratory Animal Research Center, New York, New York
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16
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Evans MG, Sleight SD. Effects of Simultaneous Dietary Exposure to 2,2′,4,4′,5,5′-Hexabromobiphenyl and 3,3′,4,4′,5,5′-Hexachlorobiphenyl on Hepatic Tumor Promotion in Rats. ACTA ACUST UNITED AC 2016. [DOI: 10.3109/10915818909018078] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Female Sprague-Dawley rats were partially hepatectomized, initiated with diethylnitrosamine (DEN), and fed diets containing 2,2′,4,4′,5,5′-hexabromobiphenyl (245-HBB), 3,3′,4,4′,5,5′-hexachlorobiphenyl (345-HCB), or combinations of 245-HBB and 345-HCB to determine the tumor-promoting ability of these compounds in a two-stage (initiation/promotion) hepatocar-cinogenesis system. Tumor-promoting ability was assessed by measuring hepatic foci positive for gamma glutamyl transpeptidase (GGT) activity. Concentrations of 10 or 100 mg 245-HBB/kg of diet caused significantly increased numbers of GGT-positive hepatic foci. When 245-HBB and 345-HCB were fed simultaneously, an additive effect on tumor-promoting ability was observed at dietary concentrations of 10 mg/kg 245-HBB and 0.1 mg/kg 345-HCB. However, an inhibitory effect on tumor promotion occurred when dietary concentrations of 100 mg/kg 245-HBB and 1.0 mg/kg 345-HCB were fed simultaneously. These results suggest that the tumor-promoting ability of simultaneous exposure to 245-HBB and 345-HCB can be additive or inhibitory depending upon the concentration of each congener in the diet.
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Affiliation(s)
- M. G. Evans
- Harvard Medical School New England Regional Primate Research Center One Pine Hill Drive Southborough, Massachusetts 01772
| | - S. D. Sleight
- Harvard Medical School New England Regional Primate Research Center One Pine Hill Drive Southborough, Massachusetts 01772
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17
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Pereira MA, Herren SL, Britt AL, Khoury MM. Initiation/promotion bioassay in rat liver: use of gamma glutamyltranspeptidase-positive foci to indicate carcinogenic activity. Toxicol Pathol 2016; 10:11-18. [DOI: 10.1177/019262338201000205] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Gamma Glutamyltranspeptidase (GGTase)-positive foci have been used to indicate activity in an initiation/promotion bioassay in rat liver. This rat liver foci bioassay has been proposed for inclusion in tier 2 of a three tier decision tree approach to carcinogenesis testing where it would function to confirm carcinogenic activity. The assay was sensitive to hepatocarcinogens and some non-hepatocarcinogens and was able to distinguish between tumor initiators and tumor promoters. The induction of GGTase-positive foci by methylating agents was associated with the formation of O6-methylguanine and not N-7 methylguanine, which would indicate a mutagenic origin for the foci. The foci once induced did not regress over the life time of the animal. Zonal induction of GGTase activity was induced by some promoters which confounded the scoring of foci incidence. The results to date indicate that the rat liver foci bioassay warrants further validation for inclusion in tier 2 and emphasizes the need to demonstrate the predictive and precursor relationship of GGTase-positive foci to cancer.
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Affiliation(s)
- Michael A. Pereira
- Toxicology and Microbiology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, 26 W. St. Clair Street, Cincinnati, Ohio 45268
| | - Sydna L. Herren
- Toxicology and Microbiology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, 26 W. St. Clair Street, Cincinnati, Ohio 45268
| | - Alfred L. Britt
- Department of Laboratory Animal Medicine, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
| | - Marlene M. Khoury
- Department of Laboratory Animal Medicine, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267
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18
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Schulte-Hermann R, Timmermann-Trosiener I, Schuppler J. Response of liver foci in rats to hepatic tumor promoters. Toxicol Pathol 2016; 10:63-68. [PMID: 28094714 DOI: 10.1177/019262338201000209] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Putative preneoplastic foci (islands) of altered hepatocytes were induced by single doses of initiating hepatocarcinogens and identified by various histochemical and morphological markers. DNA synthesis, measured by 3H-thymidine and autoradiography, as well as mitotic activity were found to be higher in the foci than in normal hepatocytes. Without promotion this proliferative advantage is partially counterbalanced by reversion of altered cells to the normal phenotype and/or increased incidence of cell death by apoptosis, so that the islands show little growth. Prolonged administration of promoters such as phenobarbital, hexachlorocyclohexane etc. resulted in the appearance of more and larger foci, which exhibited steady growth. Island enlargement was due to an upsurge of cell proliferation (in the initial stage only) and to interference of the promoter with phenotypic reversion and/or apoptosis of island cells. Island multiplication during promotion was essentially due to induction of the histochemical marker (γ-GT) in “latent” islands. Promoter withdrawal led to rapid reductions in size and number of foci suggesting that the effect of promoters on the phenotype of island cells is reversible under the conditions employed. Finally we offer the hypothesis that non-mutagenic compounds may produce liver tumors in experimental animals by promotion of “spontaneous” preneoplastic lesions.
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Affiliation(s)
- R. Schulte-Hermann
- Institut für Toxikologie und Pharmakologie Universität Marburg, Pilgrimstein 2 355 Marburg a.d.Lahn, West-Germany
| | - I. Timmermann-Trosiener
- Institut für Toxikologie und Pharmakologie Universität Marburg, Pilgrimstein 2 355 Marburg a.d.Lahn, West-Germany
| | - J. Schuppler
- Research Laboratories, Schering, Berlin-Bergkamen
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19
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Popp JA, Leonard TB. The Use of In vivo Hepatic Initiation-Promotion Systems in Understanding the Hepatocarcinogenesis of Technical Grade Dinitrotoluene. Toxicol Pathol 2016. [DOI: 10.1177/019262338201000232] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
A 2 year bioassay sponsored by the Chemical Industry Institute of Toxicology demonstrated the potent hepatocarcinogenicity of technical grade dinitrotoluene (DNT) which contains 76% 2,4-DNT, 18% 2,6-DNT and less than 3% of each 2,3; 2,5; 3,4 and 3,5-DNT isomers. In contrast, a 2 year bioassay of 2,4-DNT sponsored by the National Cancer Institute did not result in the appearance of hepatic neoplasms above the spontaneous incidence. Using previously described in vivo hepatic initiation-promotion systems, an evaluation of the initiating and promoting activity of individual DNT isomers was undertaken to provide an understanding for the differences between the two bioassays. Weak hepatocyte initiating activity was identified in technical grade DNT and purified 2,6-DNT. In contrast, 2,3; 2,4; 2,5; 3,4 and 3,5 isomers had no detectable initiating activity. When fed following a diethylnitrosamine initiating regimen, technical grade DNT, purified 2,4 and 2,6-DNT isomers had demonstrable promoting activity. Therefore, the hepatic neoplasms resulting from technical grade DNT feeding apparently resulted from the initiating activity of 2,6-DNT followed by the promoting effect of both the 2,4 and 2,6-DNT isomers. The lack of hepatic neoplasms following chronic feeding of 2,4-DNT was apparently due to the lack of hepatic initiating activity.
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Affiliation(s)
- James A. Popp
- Department of Pathology Chemical Industry Institute of Toxicology P. O. Box 12137 Research Triangle Park, NC 27709
| | - Thomas B. Leonard
- Department of Pathology Chemical Industry Institute of Toxicology P. O. Box 12137 Research Triangle Park, NC 27709
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20
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de la Iglesia FA, Farber E. Hypolipidemics Carcinogenicity and Extrapolation of Experimental Results for Human Safety Assessments. Toxicol Pathol 2016. [DOI: 10.1177/019262338201000218] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Dyslipoproteinemias represent a group of disorders closely related to alterations of cholesterol and triglycerides. The alterations of these lipids are considered important risk factors in coronary heart disease and indicate the need for clinically effective and safe drugs. Hypolipidemic agent therapy, however, does not appear without risk since the administration of these agents is by necessity, on a long-term basis. In the conduct of animal safety studies with some hypolipidemics, hyperplastic nodules or tumors developed in the liver of rodents. Data from the literature seem to indicate that the tumor response in rodents varies with the type of hypolipidemic drug administered. This paper summarizes the studies with the new lipid-regulating agent gemfibrozil. Aside from conventional long-term studies in rodents, the ultrastructural aspects of the liver were analyzed in several species and genotoxicity assays and short-term tests for hepatocarcinogenicity were conducted. Thus, it was possible to obtain an overview of these biological phenomena in order to allow for safety extrapolations. The biological behavior of these liver nodules showed that gemfibrozil and clofibrate-induced hepatocytes had not undergone malignant transformation. Further, the phenomenon of peroxisome proliferation, a characteristic event that follows hypolipidemic administration in rodents, was not confirmed in primate or human liver. Peroxisome proliferation has been linked to the process of hepatocarcinogenesis in rodents, although genotoxicity assays were negative and initiation/promotion tests failed to elicit tumors or nodules in a system where hepatocarcinogens manifest their activity. Thus, hypolipidemics such as gemfibrozil or Clofibrate may possess low tumorigenic potential with low risk due to the lack of correlation between these tests. Nevertheless, these agents are indicated for specific lipoprotein phenotype alteration with the resulting clinical benefits.
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Affiliation(s)
- Felix A. de la Iglesia
- Department of Pathology and Experimental Toxicology, Warner-Lambert/Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan
| | - Emmanuel Farber
- Department of Pathology, Banting Institute, University of Toronto, Toronto, Ontario, Canada
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21
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Popp JA, Leonard TB. The Use of In vivo Hepatic Initiation-Promotion Systems in Understanding the Hepatocarcinogenesis of Technical Grade Dinitrotoluene. Toxicol Pathol 2016; 10:190-194. [DOI: 10.1177/019262338201000220] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
A 2 year bioassay sponsored by the Chemical Industry Institute of Toxicology demonstrated the potent hepatocarcinogenicity of technical grade dinitrotoluene (DNT) which contains 76% 2,4-DNT, 18% 2,6-DNT and less than 3% of each 2,3; 2,5; 3,4 and 3,5-DNT isomers. In contrast, a 2 year bioassay of 2,4-DNT sponsored by the National Cancer Institute did not result in the appearance of hepatic neoplasms above the spontaneous incidence. Using previously described in vivo hepatic initiation-promotion systems, an evaluation of the initiating and promoting activity of individual DNT isomers was undertaken to provide an understanding for the differences between the two bioassays. Weak hepatocyte initiating activity was identified in technical grade DNT and purified 2,6-DNT. In contrast, 2,3; 2,4; 2,5; 3,4 and 3,5 isomers had no detectable initiating activity. When fed following a diethylnitrosamine initiating regimen, technical grade DNT, purified 2,4 and 2,6-DNT isomers had demonstrable promoting activity. Therefore, the hepatic neoplasms resulting from technical grade DNT feeding apparently resulted from the initiating activity of 2,6-DNT followed by the promoting effect of both the 2,4 and 2,6-DNT isomers. The lack of hepatic neoplasms following chronic feeding of 2,4-DNT was apparently due to the lack of hepatic initiating activity.
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Affiliation(s)
- James A. Popp
- Department of Pathology Chemical Industry Institute of Toxicology P. O. Box 12137 Research Triangle Park, NC 27709
| | - Thomas B. Leonard
- Department of Pathology Chemical Industry Institute of Toxicology P. O. Box 12137 Research Triangle Park, NC 27709
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22
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de la Iglesia FA, Farber E. Hypolipidemics Carcinogenicity and Extrapolation of Experimental Results for Human Safety Assessments. Toxicol Pathol 2016; 10:152-170. [DOI: 10.1177/019262338201000228] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Dyslipoproteinemias represent a group of disorders closely related to alterations of cholesterol and triglycerides. The alterations of these lipids are considered important risk factors in coronary heart disease and indicate the need for clinically effective and safe drugs. Hypolipidemic agent therapy, however, does not appear without risk since the administration of these agents is by necessity, on a long-term basis. In the conduct of animal safety studies with some hypolipidemics, hyperplastic nodules or tumors developed in the liver of rodents. Data from the literature seem to indicate that the tumor response in rodents varies with the type of hypolipidemic drug administered. This paper summarizes the studies with the new lipid-regulating agent gemfibrozil. Aside from conventional long-term studies in rodents, the ultrastructural aspects of the liver were analyzed in several species and genotoxicity assays and short-term tests for hepatocarcinogenicity were conducted. Thus, it was possible to obtain an overview of these biological phenomena in order to allow for safety extrapolations. The biological behavior of these liver nodules showed that gemfibrozil and clofibrate-induced hepatocytes had not undergone malignant transformation. Further, the phenomenon of peroxisome proliferation, a characteristic event that follows hypolipidemic administration in rodents, was not confirmed in primate or human liver. Peroxisome proliferation has been linked to the process of hepatocarcinogenesis in rodents, although genotoxicity assays were negative and initiation/promotion tests failed to elicit tumors or nodules in a system where hepatocarcinogens manifest their activity. Thus, hypolipidemics such as gemfibrozil or clofibrate may possess low tumorigenic potential with low risk due to the lack of correlation between these tests. Nevertheless, these agents are indicated for specific lipoprotein phenotype alteration with the resulting clinical benefits.
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Affiliation(s)
- Felix A. de la Iglesia
- Department of Pathology and Experimental Toxicology, Warner-Lambert/Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan and Department of Pathology, Banting Institute, University of Toronto, Toronto, Ontario, Canada
| | - Emmanuel Farber
- Department of Pathology and Experimental Toxicology, Warner-Lambert/Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan and Department of Pathology, Banting Institute, University of Toronto, Toronto, Ontario, Canada
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23
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Furtado KS, de Oliveira Andrade F, Campos A, Rosim MP, Vargas-Mendez E, Henriques A, De Conti A, Scolastici C, Barbisan LF, Carvalho RF, Moreno FS. β-ionone modulates the expression of miRNAs and genes involved in the metastatic phenotype of microdissected persistent preneoplastic lesions in rats submitted to hepatocarcinogenesis. Mol Carcinog 2016; 56:184-196. [PMID: 27061051 DOI: 10.1002/mc.22483] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2015] [Revised: 02/25/2016] [Accepted: 03/18/2016] [Indexed: 12/19/2022]
Abstract
MicroRNAs (miRNAs) are post-transcriptional gene expression regulators which expression is frequently altered in hepatocellular carcinoma (HCC). β-ionone (βI) is noted for its ability to inhibit persistent preneoplastic lesions (pPNLs) in liver rats. We evaluated the expression of miRNAs involved in carcinogenesis and possible targets modulated by βI, in pPNLs and surrounding of microdissected tissues. Rats subjected to resistant hepatocyte model were treated during promotion stage with βI (16 mg/100 g body weight) or corn oil (CO; 0.25 mL/100 g body weight; controls). Five animals receive no treatment (NT). In CO group, 38 and 29 miRNAs showed reduced expression relative to NT (P < 0.05) in pPNLs and surrounding, respectively. No miRNAs showed increased expression in surrounding of the CO compared to NT group; however, 30 miRNAs showed increased expression (P ≤ 0.05) in pPNLs of the CO group. There was no difference between βI and CO groups (P > 0.05) in the expression of miRNAs in surrounding. In pPNLs βI increased expression of miR-122 and miR-34a (P ≤ 0.05) and reduced of Igf2 (P ≤ 0.05), target of the latter, compared to CO. Additionally, βI decreased the expression of miR-181c and its target Gdf2 (P ≤ 0.05). βI reduced the expression of miR-181b and miR-708 (P ≤ 0.05) and increased the expression of their respective target mRNAs Timp3 and Mtss1 (P ≤ 0.05), relative to CO group. Modulation of miRNAs target genes by βI was confirmed in vitro. βI is a promising chemopreventive agent in the initial stages of hepatocarcinogenesis, as it modulates the expression of the miRNAs and target genes that can alter the metastatic phenotype of HCC. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Kelly Silva Furtado
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Fábia de Oliveira Andrade
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Adriana Campos
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Mariana Papaléo Rosim
- Laboratory of Nutrigenomics and Programming, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Ernesto Vargas-Mendez
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Aline Henriques
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Aline De Conti
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Clarissa Scolastici
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
| | - Luis Fernando Barbisan
- Laboratory of Experimental Chemical Carcinogenesis, Department of Morphology, Botucatu, Institute of Biosciences, São Paulo State University, São Paulo, Brazil
| | - Robson Francisco Carvalho
- Laboratory of Striated Muscle Biology, Department of Morphology, Botucatu, Institute of Biosciences, São Paulo State University, São Paulo, Brazil
| | - Fernando Salvador Moreno
- Laboratory of Diet, Nutrition, and Cancer, Faculty of Pharmaceutical Sciences, Department of Food and Experimental Nutrition, University of São Paulo, São Paulo, SP, Brazil
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Pacheco-Rivera R, Fattel-Fazenda S, Arellanes-Robledo J, Silva-Olivares A, Alemán-Lazarini L, Rodríguez-Segura M, Pérez-Carreón J, Villa-Treviño S, Shibayama M, Serrano-Luna J. Double staining of β-galactosidase with fibrosis and cancer markers reveals the chronological appearance of senescence in liver carcinogenesis induced by diethylnitrosamine. Toxicol Lett 2016; 241:19-31. [DOI: 10.1016/j.toxlet.2015.11.011] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2015] [Revised: 11/06/2015] [Accepted: 11/07/2015] [Indexed: 01/04/2023]
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25
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Talbot NC, Wang L, Garrett WM, Caperna TJ, Tang Y. Establishment and characterization of feeder cell-dependent bovine fetal liver cell lines. In Vitro Cell Dev Biol Anim 2015; 52:314-326. [DOI: 10.1007/s11626-015-9982-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Accepted: 11/16/2015] [Indexed: 12/24/2022]
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Gonadotropin-releasing hormone stimulates biliary proliferation by paracrine/autocrine mechanisms. THE AMERICAN JOURNAL OF PATHOLOGY 2015; 185:1061-72. [PMID: 25794706 PMCID: PMC4380841 DOI: 10.1016/j.ajpath.2014.12.004] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 10/29/2014] [Revised: 12/16/2014] [Accepted: 12/30/2014] [Indexed: 11/22/2022]
Abstract
During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases.
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Chen YH, Chen HL, Chien CS, Wu SH, Ho YT, Yu CH, Chang MH. Contribution of Mature Hepatocytes to Biliary Regeneration in Rats with Acute and Chronic Biliary Injury. PLoS One 2015; 10:e0134327. [PMID: 26308208 PMCID: PMC4550468 DOI: 10.1371/journal.pone.0134327] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2014] [Accepted: 07/08/2015] [Indexed: 12/14/2022] Open
Abstract
Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4’-methylene-dianiline (DAPM) and D-galactosamine (DAPM+D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM+D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1β (HNF-1β) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation.
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Affiliation(s)
- Ya-Hui Chen
- Department of Pediatrics, Taipei Tzu-Chi Hospital, The Buddhist Tzuchi Medical Foundation, Taipei, Taiwan
- Department of Pediatrics, Buddhist Tzu-Chi University College of Medicine, Hualien, Taiwan
- Graduate Institute of Clinical Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
| | - Hui-Ling Chen
- Hepatitis Research Center, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
| | - Chin-Sung Chien
- Graduate Institute of Clinical Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
| | - Shang-Hsin Wu
- Graduate Institute of Clinical Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
| | - Yi-Tian Ho
- Hepatitis Research Center, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
| | - Chun-Hsien Yu
- Department of Pediatrics, Taipei Tzu-Chi Hospital, The Buddhist Tzuchi Medical Foundation, Taipei, Taiwan
- Department of Pediatrics, Buddhist Tzu-Chi University College of Medicine, Hualien, Taiwan
- * E-mail: (MHC); (CHY)
| | - Mei-Hwei Chang
- Graduate Institute of Clinical Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
- Hepatitis Research Center, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
- Department of Pediatrics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
- * E-mail: (MHC); (CHY)
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Zúñiga-García V, Chávez-López MDG, Quintanar-Jurado V, Gabiño-López NB, Hernández-Gallegos E, Soriano-Rosas J, Pérez-Carreón JI, Camacho J. Differential Expression of Ion Channels and Transporters During Hepatocellular Carcinoma Development. Dig Dis Sci 2015; 60:2373-83. [PMID: 25842354 DOI: 10.1007/s10620-015-3633-9] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/04/2014] [Accepted: 03/13/2015] [Indexed: 12/09/2022]
Abstract
BACKGROUND Ion channels and transporters are potential markers and therapeutic targets for several cancers. However, their expression during hepatocellular carcinoma (HCC) development remains unclear. AIM To investigate the mRNA expression of Na(+), K(+) and Ca(2+) channels and ABC transporters during rat HCC development, as well as Abcc3 protein in human liver biopsies. METHODS Wistar rats were treated with diethylnitrosamine (DEN) and developed both cirrhosis (12 weeks of treatment) and either pre-neoplastic lesions (16 weeks of treatment) or multinodular HCC (16 weeks of treatment plus 2 weeks DEN-free). The mRNA expression of 12 ion channels and two ABC transporters was studied using real-time RT-PCR. Tumor-containing or tumor-free liver sections were isolated by laser-capture microdissection. Abcc3 protein expression was studied by immunohistochemistry in healthy, cirrhotic and HCC human biopsies. RESULTS We observed expression changes in seven genes. Kcna3, Kcnn4, Kcnrg and Kcnj11 potassium channel mRNA expression reached peak values at the end of DEN treatment, while Scn2a1 sodium channel, Trpc6 calcium channel and Abcc3 transporter mRNA expression reached their highest levels in the presence of HCC (18 weeks). Whereas Kcnn4 and Scn2a1 channel expression was similar in non-tumor and tumor tissue, the Abcc3 transporter and Kcna3 potassium channels were preferentially overexpressed in the tumor sections. We observed differential Abcc3 protein subcellular localization and expression in human samples. CONCLUSIONS The ion channel/transporter expression profile observed suggests that these genes are potential early markers or therapeutic targets of HCC. The differential localization of Abcc3 may be useful in the diagnosis of cirrhosis and HCC.
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Affiliation(s)
- Violeta Zúñiga-García
- Department of Pharmacology, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Avenida Instituto Politécnico Nacional 2508, C.P. 07360, Mexico City, Mexico,
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Flores-Téllez TNJ, Lopez TV, Vásquez Garzón VR, Villa-Treviño S. Co-Expression of Ezrin-CLIC5-Podocalyxin Is Associated with Migration and Invasiveness in Hepatocellular Carcinoma. PLoS One 2015; 10:e0131605. [PMID: 26135398 PMCID: PMC4489913 DOI: 10.1371/journal.pone.0131605] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2015] [Accepted: 06/03/2015] [Indexed: 11/18/2022] Open
Abstract
Background and Aim Prognostic markers are important for predicting the progression and staging of hepatocellular carcinoma (HCC). Ezrin (EZR) and Podocalyxin (PODXL) are proteins associated with invasion, migration and poor prognosis in various types of cancer. Recently, it has been observed that chloride intracellular channel 5 (CLIC5) forms a complex with EZR and PODXL and that it is required for podocyte structure and function. In this study, we evaluated the overexpression of EZR, PODXL and CLIC5 in HCC. Methods The modified resistant hepatocyte model (MRHR), human biopsies and HCC cell lines (HepG2, Huh7 and SNU387) were used in this study. Gene and protein expression levels were evaluated in the MRHR by qRT-PCR, Western blot and immunohistochemistry analyses, and protein expression in the human biopsies was evaluated by immunohistochemistry. Protein expression in the HCC cell lines was evaluated by immunofluorescence and Western blot, also the migration and invasive abilities of Huh7 cells were evaluated using shRNA-mediated inhibition. Results Our results indicated that these genes and proteins were overexpressed in HCC. Moreover, when the expression of CLIC5 and PODXL was inhibited in Huh7 cells, we observed decreased migration and invasion. Conclusion This study suggested that EZR, CLIC5 and PODXL could be biological markers to predict the prognosis of HCC and that these proteins participate in migration and invasion processes.
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Affiliation(s)
- Teresita N. J. Flores-Téllez
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN No. 2508 Col. San Pedro Zacatenco, México 14, CP 07360, México, Distrito Federal
| | - Tania V. Lopez
- Instituto Nacional De Medicina Genómica (INMEGEN), Periférico Sur 4809, Arenal Tepepan, Tlalpan, 14610 Ciudad de México, Distrito Federal
- * E-mail: (TVL); (SVT)
| | - Verónica Rocío Vásquez Garzón
- Facultad de Medicina y Cirugía, Universidad Benito Juárez de Oaxaca. Av Universidad S/N, Col. 5 Señores. C.P. 68120, México, Oaxaca
| | - Saúl Villa-Treviño
- Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN No. 2508 Col. San Pedro Zacatenco, México 14, CP 07360, México, Distrito Federal
- * E-mail: (TVL); (SVT)
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Afroze SH, Munshi MK, Martínez AK, Uddin M, Gergely M, Szynkarski C, Guerrier M, Nizamutdinov D, Dostal D, Glaser S. Activation of the renin-angiotensin system stimulates biliary hyperplasia during cholestasis induced by extrahepatic bile duct ligation. Am J Physiol Gastrointest Liver Physiol 2015; 308:G691-701. [PMID: 25678505 PMCID: PMC4398845 DOI: 10.1152/ajpgi.00116.2014] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2014] [Accepted: 01/08/2015] [Indexed: 01/31/2023]
Abstract
Cholangiocyte proliferation is regulated in a coordinated fashion by many neuroendocrine factors through autocrine and paracrine mechanisms. The renin-angiotensin system (RAS) is known to play a role in the activation of hepatic stellate cells and blocking the RAS attenuates hepatic fibrosis. We investigated the role of the RAS during extrahepatic cholestasis induced by bile duct ligation (BDL). In this study, we used normal and BDL rats that were treated with control, angiotensin II (ANG II), or losartan for 2 wk. In vitro studies were performed in a primary rat cholangiocyte cell line (NRIC). The expression of renin, angiotensin-converting enzyme, angiotensinogen, and angiotensin receptor type 1 was evaluated by immunohistochemistry (IHC), real-time PCR, and FACs and found to be increased in BDL compared with normal rat. The levels of ANG II were evaluated by ELISA and found to be increased in serum and conditioned media of cholangiocytes from BDL compared with normal rats. Treatment with ANG II increased biliary mass and proliferation in both normal and BDL rats. Losartan attenuated BDL-induced biliary proliferation. In vitro, ANG II stimulated NRIC proliferation via increased intracellular cAMP levels and activation of the PKA/ERK/CREB intracellular signaling pathway. ANG II stimulated a significant increase in Sirius red staining and IHC for fibronectin that was blocked by angiotensin receptor blockade. In vitro, ANG II stimulated the gene expression of collagen 1A1, fibronectin 1, and IL-6. These results indicate that cholangiocytes express a local RAS and that ANG II plays an important role in regulating biliary proliferation and fibrosis during extraheptic cholestasis.
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Affiliation(s)
- Syeda H. Afroze
- 2Scott & White Digestive Disease Research Center, Temple, Texas; and
| | | | - Allyson K. Martínez
- 3Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
| | - Mohammad Uddin
- 3Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
| | - Maté Gergely
- 3Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
| | - Claudia Szynkarski
- 3Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
| | - Micheleine Guerrier
- 3Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
| | - Damir Nizamutdinov
- 3Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
| | - David Dostal
- 3Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
| | - Shannon Glaser
- Central Texas Veterans Health Care System, Temple, Texas; Scott & White Digestive Disease Research Center, Temple, Texas; and Department of Internal Medicine, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas
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Roach DR, Garrett WM, Welch G, Caperna TJ, Talbot NC, Shapiro EM. Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation. PLoS One 2015; 10:e0123282. [PMID: 25856627 PMCID: PMC4391930 DOI: 10.1371/journal.pone.0123282] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Accepted: 02/26/2015] [Indexed: 01/19/2023] Open
Abstract
Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP) and stem cell-derived hepatocytes (PICM-19FF). The magnetic particle is a micron-sized iron oxide particle (MPIO) that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.
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Affiliation(s)
- Dwayne R. Roach
- Molecular and Cellular Imaging Laboratory, Department of Radiology, Michigan State University, East Lansing, Michigan, United States of America
- Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, Beltsville Agricultural Research Center, United States Department of Agriculture, Beltsville, Maryland, United States of America
| | - Wesley M. Garrett
- Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, Beltsville Agricultural Research Center, United States Department of Agriculture, Beltsville, Maryland, United States of America
| | - Glenn Welch
- Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, Beltsville Agricultural Research Center, United States Department of Agriculture, Beltsville, Maryland, United States of America
| | - Thomas J. Caperna
- Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, Beltsville Agricultural Research Center, United States Department of Agriculture, Beltsville, Maryland, United States of America
| | - Neil C. Talbot
- Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, Beltsville Agricultural Research Center, United States Department of Agriculture, Beltsville, Maryland, United States of America
| | - Erik M. Shapiro
- Molecular and Cellular Imaging Laboratory, Department of Radiology, Michigan State University, East Lansing, Michigan, United States of America
- * E-mail:
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de Guadalupe Chávez-López M, Pérez-Carreón JI, Zuñiga-García V, Díaz-Chávez J, Herrera LA, Caro-Sánchez CH, Acuña-Macías I, Gariglio P, Hernández-Gallegos E, Chiliquinga AJ, Camacho J. Astemizole-based anticancer therapy for hepatocellular carcinoma (HCC), and Eag1 channels as potential early-stage markers of HCC. Tumour Biol 2015; 36:6149-58. [PMID: 25783527 DOI: 10.1007/s13277-015-3299-0] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2014] [Accepted: 03/01/2015] [Indexed: 12/22/2022] Open
Abstract
Hepatocellular carcinoma (HCC) has very poor prognosis. Astemizole has gained great interest as a potential anticancer drug because it targets several proteins involved in cancer including the Eag1 (ether à-go-go-1) potassium channel that is overexpressed in human HCC. Eag1 channels are regulated by cancer etiological factors and have been proposed as early tumor markers. Here, we found that HepG2 and HuH-7 HCC cells displayed Eag1 messenger RNA (mRNA) and protein expression, determined by real-time RT-PCR and immunochemistry, respectively. Astemizole inhibited human HCC cell proliferation (assessed by metabolic activity assay) and induced apoptosis (studied with flow cytometry) in both cell lines. The subcellular Eag1 protein localization was modified by astemizole in the HepG2 cells. The treatment with astemizole prevented diethylnitrosamine (DEN)-induced rat HCC development in vivo (followed by studying γ-glutamyl transpeptidase (GGT) activity). The Eag1 mRNA and protein levels were increased in most DEN-treated groups but decreased after astemizole treatment. GGT activity was decreased by astemizole. The Eag1 protein was detected in cirrhotic and dysplastic rat livers. Astemizole might have clinical utility for HCC prevention and treatment, and Eag1 channels may be potential early HCC biomarkers. These data provide significant basis to include astemizole in HCC clinical trials.
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Affiliation(s)
- María de Guadalupe Chávez-López
- Department of Pharmacology, Centro de Investigación y de Estudios Avanzados del I.P.N., Avenida Instituto Politécnico Nacional 2508, Mexico City, 07360, Mexico
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Johnson C, Hargrove L, Graf A, Kennedy L, Hodges K, Harris R, Francis T, Ueno Y, Francis H. Histamine restores biliary mass following carbon tetrachloride-induced damage in a cholestatic rat model. Dig Liver Dis 2015; 47:211-7. [PMID: 25575430 DOI: 10.1016/j.dld.2014.12.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2014] [Revised: 12/01/2014] [Accepted: 12/06/2014] [Indexed: 12/11/2022]
Abstract
BACKGROUND Bile duct ligation coupled with carbon tetrachloride induces apoptosis of large but not small cholangiocytes. Histidine decarboxylase regulates histamine synthesis. We have shown that: (i) cholangiocytes express histidine decarboxylase and secrete histamine and (ii) histamine stimulates biliary growth. AIMS To demonstrate that histidine decarboxylase/histamine regulates cholangiocyte homeostasis after carbon tetrachloride treatment. METHODS In vivo, normal and bile duct ligated rats were treated with saline or histamine (0.5mg/kg body weight) and given carbon tetrachloride by gavage 2 days before sacrifice. Serum, liver blocks and large cholangiocytes were obtained. Histidine decarboxylase, bile duct mass and proliferation were measured in liver sections and in cholangiocytes. Apoptosis was measured by immunohistochemistry and gene expression. Histamine levels were evaluated in serum. In vitro, large cholangiocytes were treated with carbon tetrachloride in the absence/presence of histamine before evaluating proliferation. RESULTS After bile duct ligation there was enhanced ductal mass, histidine decarboxylase expression and serum histamine levels. Carbon tetrachloride treatment enhanced biliary apoptosis, and decreased histidine decarboxylase and serum histamine levels and biliary proliferation, changes that were restored by histamine. In vitro, cholangiocytes treated with carbon tetrachloride had a lower proliferative capacity that was reversed when cells were pre-treated with histamine. CONCLUSION Histidine decarboxylase may be a key regulator of cholangiocyte homeostasis during biliary injury.
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Affiliation(s)
| | - Laura Hargrove
- Scott & White Digestive Disease Research Center, BaylorScott and White, USA
| | - Allyson Graf
- Scott & White Digestive Disease Research Center, BaylorScott and White, USA
| | - Lindsey Kennedy
- Scott & White Digestive Disease Research Center, BaylorScott and White, USA
| | - Kyle Hodges
- Scott & White Digestive Disease Research Center, BaylorScott and White, USA
| | - Rachel Harris
- Scott & White Digestive Disease Research Center, BaylorScott and White, USA
| | - Taylor Francis
- Scott & White Digestive Disease Research Center, BaylorScott and White, USA
| | - Yoshiyuki Ueno
- Department of Gastroenterology, Yamagata University Faculty of Medicine, Japan; CREST, Japan Science and Technology Agency, Japan
| | - Heather Francis
- Research, Central Texas Veteran's Health Care System, USA; Scott & White Digestive Disease Research Center, BaylorScott and White, USA; Medicine, Texas A&M University HSC, Temple, TX, USA.
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Vásquez-Garzón VR, Beltrán-Ramírez O, Salcido-Neyoy ME, Cervante-Anaya N, Villa-Treviño S. Analysis of gene expression profiles as a tool to uncover tumor markers of liver cancer progression in a rat model. Biomed Rep 2014; 3:167-172. [PMID: 25798242 DOI: 10.3892/br.2014.411] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Accepted: 12/10/2014] [Indexed: 11/05/2022] Open
Abstract
Establishing a transcriptomic profile of human hepatocellular liver cancer (HCC) progression is a complex undertaking. A rat model of HCC was employed to develop a transcriptomic profile. Using three interventions, preneoplastic lesions appeared after 30 days and they progressed to HCC by 9 months. Preneoplastic and cancer lesions were characterized for transcriptomic analysis, and RNA from total liver homogenates was obtained at 1, 7, 11 and 16 days after the initiation treatment. RNA from dissected persistent preneoplastic lesions, adjacent tissue or cancer tissue was used for 30 days, and 5, 9, 12 and 18 months. The GeneChip® Rat Exon 1.0 ST arrays, Partek software and an Affymetrix console were employed for these analyses. LGALS3BP was differentially expressed at each time point, from the initial period, through the preneoplastic evolution period and until the end of cancer progression period. Twelve differentially expressed genes common to the preneoplastic evolution and to the cancer progression period were detected, which included ABCC3. Validation of the microarrays was confirmed by reverse transcription-quantitative polymerase chain reaction of six genes, including LGALS3BP and ABCC3. Of note, the proteins of these two genes are associated with the multidrug response complex, and evasion of immune surveillance and negative regulation of T cell proliferation. This model is useful for identifying candidate genes, and to validate them with regards to determining their relevance in rat HCC progression.
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Affiliation(s)
- Verónica R Vásquez-Garzón
- Department of Cell Biology, Center for Research and Advanced Studies (CINVESTAV-IPN), México, DF 07360, México
| | | | - Martha E Salcido-Neyoy
- National Cancer Institute, Colonia Sección XVI Delegación Tlalpan, México, DF 14080, México
| | - Nancy Cervante-Anaya
- Department of Cell Biology, Center for Research and Advanced Studies (CINVESTAV-IPN), México, DF 07360, México
| | - Saúl Villa-Treviño
- Department of Cell Biology, Center for Research and Advanced Studies (CINVESTAV-IPN), México, DF 07360, México
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Kennedy LL, Hargrove LA, Graf AB, Francis TC, Hodges KM, Nguyen QP, Ueno Y, Greene JF, Meng F, Huynh VD, Francis HL. Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents. J Transl Med 2014; 94:1406-18. [PMID: 25365204 DOI: 10.1038/labinvest.2014.129] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2014] [Accepted: 07/07/2014] [Indexed: 02/07/2023] Open
Abstract
Cholangiopathies are characterized by dysregulation of the balance between biliary growth and loss. We have shown that histamine (HA) stimulates biliary growth via autocrine mechanisms. To evaluate the paracrine effects of mast cell (MC) stabilization on biliary proliferation, sham or BDL rats were treated by IP-implanted osmotic pumps filled with saline or cromolyn sodium (24 mg/kg BW/day (inhibits MC histamine release)) for 1 week. Serum, liver blocks and cholangiocytes were collected. Histidine decarboxylase (HDC) expression was measured using real-time PCR in cholangiocytes. Intrahepatic bile duct mass (IBDM) was evaluated by IHC for CK-19. MC number was determined using toluidine blue staining and correlated to IBDM. Proliferation was evaluated by PCNA expression in liver sections and purified cholangiocytes. We assessed apoptosis using real-time PCR and IHC for BAX. Expression of MC stem factor receptor, c-kit, and the proteases chymase and tryptase were measured by real-time PCR. HA levels were measured in serum by EIA. In vitro, MCs and cholangiocytes were treated with 0.1% BSA (basal) or cromolyn (25 μM) for up to 48 h prior to assessing HDC expression, HA levels and chymase and tryptase expression. Supernatants from MCs treated with or without cromolyn were added to cholangiocytes before measuring (i) proliferation by MTT assays, (ii) HDC gene expression by real-time PCR and (iii) HA release by EIA. In vivo, cromolyn treatment decreased BDL-induced: (i) IBDM, MC number, and biliary proliferation; (ii) HDC and MC marker expression; and (iii) HA levels. Cromolyn treatment increased cholangiocyte apoptosis. In vitro, cromolyn decreased HA release and chymase and tryptase expression in MCs but not in cholangiocytes. Cromolyn-treated MC supernatants decreased biliary proliferation and HA release. These studies provide evidence that MC histamine is key to biliary proliferation and may be a therapeutic target for the treatment of cholangiopathies.
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Affiliation(s)
- Lindsey L Kennedy
- Digestive Disease Research Center, Central Texas Veterans Health Care System, Temple, TX, USA
| | - Laura A Hargrove
- Scott and White Digestive Disease Research Center, Scott and White, Temple, TX, USA
| | - Allyson B Graf
- Digestive Disease Research Center, Central Texas Veterans Health Care System, Temple, TX, USA
| | - Taylor C Francis
- Department of Medicine, Texas A&M Health Science Center, Temple, TX, USA
| | - Kyle M Hodges
- Scott and White Digestive Disease Research Center, Scott and White, Temple, TX, USA
| | - Quy P Nguyen
- Digestive Disease Research Center, Central Texas Veterans Health Care System, Temple, TX, USA
| | - Yoshi Ueno
- CREST, Japan Science and Technology Corporation, Tokyo, Japan
| | - John F Greene
- Scott and White Digestive Disease Research Center, Scott and White, Temple, TX, USA
| | - Fanyin Meng
- 1] Digestive Disease Research Center, Central Texas Veterans Health Care System, Temple, TX, USA [2] Scott and White Digestive Disease Research Center, Scott and White, Temple, TX, USA [3] Department of Medicine, Texas A&M Health Science Center, Temple, TX, USA
| | - Victoria D Huynh
- Department of Medicine, Texas A&M Health Science Center, Temple, TX, USA
| | - Heather L Francis
- 1] Digestive Disease Research Center, Central Texas Veterans Health Care System, Temple, TX, USA [2] Scott and White Digestive Disease Research Center, Scott and White, Temple, TX, USA [3] Department of Medicine, Texas A&M Health Science Center, Temple, TX, USA
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Han Y, Onori P, Meng F, DeMorrow S, Venter J, Francis H, Franchitto A, Ray D, Kennedy L, Greene J, Renzi A, Mancinelli R, Gaudio E, Glaser S, Alpini G. Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2014; 307:G894-904. [PMID: 25214401 PMCID: PMC4216989 DOI: 10.1152/ajpgi.00288.2014] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Biliary hyperplasia and liver fibrosis are common features in cholestatic liver disease. Melatonin is synthesized by the pineal gland as well as the liver. Melatonin inhibits biliary hyperplasia of bile duct-ligated (BDL) rats. Since melatonin synthesis (by the enzyme serotonin N-acetyltransferase, AANAT) from the pineal gland increases after dark exposure, we hypothesized that biliary hyperplasia and liver fibrosis are diminished by continuous darkness via increased melatonin synthesis from the pineal gland. Normal or BDL rats (immediately after surgery) were housed with light-dark cycles or complete dark for 1 wk before evaluation of 1) the expression of AANAT in the pineal gland and melatonin levels in pineal gland tissue supernatants and serum; 2) biliary proliferation and intrahepatic bile duct mass, liver histology, and serum chemistry; 3) secretin-stimulated ductal secretion (functional index of biliary growth); 4) collagen deposition, liver fibrosis markers in liver sections, total liver, and cholangiocytes; and 5) expression of clock genes in cholangiocytes. In BDL rats exposed to dark there was 1) enhanced AANAT expression/melatonin secretion in pineal gland and melatonin serum levels; 2) improved liver morphology, serum chemistry and decreased biliary proliferation and secretin-stimulated choleresis; and 4) decreased fibrosis and expression of fibrosis markers in liver sections, total liver and cholangiocytes and reduced biliary expression of the clock genes PER1, BMAL1, CLOCK, and Cry1. Thus prolonged dark exposure may be a beneficial noninvasive therapeutic approach for the management of biliary disorders.
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Affiliation(s)
- Yuyan Han
- 4Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
| | - Paolo Onori
- 7Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, La Sapienza, Rome, Italy
| | - Fanyin Meng
- 1Research, Central Texas Veterans Health Care System, Temple, Texas; ,2Baylor Scott & White Digestive Disease Research Center, Scott & White, Temple, Texas; ,3Operational Funds, Baylor Scott & White, Temple, Texas; ,4Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
| | - Sharon DeMorrow
- 2Baylor Scott & White Digestive Disease Research Center, Scott & White, Temple, Texas; ,4Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
| | - Julie Venter
- 4Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
| | - Heather Francis
- 1Research, Central Texas Veterans Health Care System, Temple, Texas; ,2Baylor Scott & White Digestive Disease Research Center, Scott & White, Temple, Texas; ,3Operational Funds, Baylor Scott & White, Temple, Texas; ,4Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
| | - Antonio Franchitto
- 5Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy; ,7Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, La Sapienza, Rome, Italy
| | - Debolina Ray
- 4Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
| | - Lindsey Kennedy
- 1Research, Central Texas Veterans Health Care System, Temple, Texas;
| | - John Greene
- 6Pathology, Baylor Scott & White, Temple, Texas; and
| | - Anastasia Renzi
- 7Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, La Sapienza, Rome, Italy
| | - Romina Mancinelli
- 7Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, La Sapienza, Rome, Italy
| | - Eugenio Gaudio
- 7Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, La Sapienza, Rome, Italy
| | - Shannon Glaser
- 1Research, Central Texas Veterans Health Care System, Temple, Texas; ,2Baylor Scott & White Digestive Disease Research Center, Scott & White, Temple, Texas; ,4Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
| | - Gianfranco Alpini
- Research, Central Texas Veterans Health Care System, Temple, Texas; Baylor Scott & White Digestive Disease Research Center, Scott & White, Temple, Texas; Department of Medicine, Division Gastroenterology, Texas A&M University Health Science Center, Temple, Texas;
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Pucci A, Franzini M, Matteucci M, Ceragioli S, Marconi M, Ferrari M, Passino C, Basolo F, Emdin M, Paolicchi A. b-Gamma-glutamyltransferase activity in human vulnerable carotid plaques. Atherosclerosis 2014; 237:307-13. [DOI: 10.1016/j.atherosclerosis.2014.09.028] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2014] [Revised: 09/02/2014] [Accepted: 09/19/2014] [Indexed: 11/28/2022]
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Lin SY, Wang YY, Chen WY, Chuang YH, Pan PH, Chen CJ. Beneficial effect of quercetin on cholestatic liver injury. J Nutr Biochem 2014; 25:1183-1195. [PMID: 25108658 DOI: 10.1016/j.jnutbio.2014.06.003] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2013] [Revised: 05/29/2014] [Accepted: 06/04/2014] [Indexed: 12/15/2022]
Abstract
Bile duct obstruction and subsequent cholestasis are associated with hepatocellular injury, cholangiocyte proliferation, stellate cell activation, Kupffer cell activation, oxidative stress, inflammation and fibrosis. Flavonoids have been shown to confer beneficial health effects, including hepatoprotection. However, the molecular mechanism of flavonoid-mediated hepatoprotection is incompletely understood. In this study, we report the protective effect of quercetin on cholestatic liver injury. Cholestasis was produced by bile duct ligation (BDL) in male Sprague-Dawley rats for 3 weeks. Daily oral administration of quercetin was started 1 week before injury and lasted for 4 weeks. In comparison with the control group, the BDL group showed liver injury, as evidenced by histological changes, and elevation in serum biochemicals, ductular reaction, fibrosis, inflammation and oxidative stress. These pathophysiological changes were attenuated by daily quercetin supplementation. Quercetin alleviated BDL-induced transforming growth factor beta-1 (TGF-β1), interleukin-1 beta, connective tissue growth factor and collagen expression. The antifibrotic effect of quercetin was accompanied by reductions in α-smooth muscle actin-positive matrix-producing cells and Smad 2/3 activity critical to the fibrogenic potential of TGF-β1. Quercetin also attenuated BDL-induced oxidative stress, leukocyte accumulation, nuclear factor (NF)-κB activation and proinflammatory cytokine production. Further studies demonstrated an inhibitory effect of quercetin on MyD88 and TGF-β-activated kinase-1 critical for linking toll-like receptor (TLR) and NF-κB. Taken together, the hepatoprotective, anti-inflammatory and antifibrotic effects of quercetin seem to be multifactorial. The beneficial effects of daily quercetin supplementation are associated with antioxidative and anti-inflammatory potential as well as down-regulation of NF-κB and TGF-β/Smad signaling, probably via interference with TLR signaling.
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Affiliation(s)
- Shih-Yi Lin
- Division of Endocrinology and Metabolism, Taichung Veterans General Hospital, Taichung 407, Taiwan; School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
| | - Ya-Yu Wang
- School of Medicine, National Yang-Ming University, Taipei 112, Taiwan; Division of Family Medicine, Taichung Veterans General Hospital, Taichung 407, Taiwan
| | - Wen-Ying Chen
- Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan
| | - Yu-Han Chuang
- Division of Endocrinology and Metabolism, Taichung Veterans General Hospital, Taichung 407, Taiwan
| | - Pin-Ho Pan
- Department of Pediatrics, Tungs' Taichung MetroHarbor Hospital, Taichung 435, Taiwan
| | - Chun-Jung Chen
- Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan; Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan; Center for General Education, Tunghai University, Taichung 407, Taiwan; Department of Nursing, HungKuang University, Taichung 433, Taiwan.
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A medium-term gpt delta rat model as an in vivo system for analysis of renal carcinogenesis and the underlying mode of action. ACTA ACUST UNITED AC 2014; 67:31-9. [PMID: 25446801 DOI: 10.1016/j.etp.2014.09.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2014] [Accepted: 09/26/2014] [Indexed: 01/16/2023]
Abstract
The kidney is a major target site of chemical carcinogenesis. However, a reliable in vivo assay for rapid identification of renal carcinogens has not been established. The purpose of this study was to develop a new medium-term gpt delta rat model (the GNP model) to facilitate identification of renal carcinogens. In this model, we carried out an in vivo mutation assay using unilaterally nephrectomized kidney tissue and a tumor-promoting assay using residual kidney tissue, with diethylnitrosamine (DEN) as the renal tumor initiator. To clarify the optimal time of DEN injection after nephrectomy, time-dependent changes in bromodeoxyuridine-labeling indices in the tubular epithelium of nephrectomized rats were examined. The optimal dose of DEN injection and sufficient duration of subsequent nitrilotriacetic acid treatment were determined for detection of renal preneoplastic lesions. The standard protocol for the GNP model was determined as follows. Six-week-old female gpt delta rats were treated with test chemicals for 4 weeks, followed by a 2-week washout period, and 40 mg/kg DEN was administered intraperitoneally to initiate renal carcinogenesis. Unilateral nephrectomy was performed 48 h before DEN injection, followed by gpt assays using excised kidney tissues. One week after DEN injection, rats were further exposed to test chemicals for 12 weeks, and histopathological analysis of renal preneoplastic lesions was performed as an indicator of tumor-promoting activity in residual kidney tissue. Validation studies using aristolochic acid, potassium dibasic phosphate, phenylbutazone, and d-limonene indicated the reliability of the GNP model for predicting renal carcinogens and the underlying mode of action.
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Guerrier M, Attili F, Alpini G, Glaser S. Prolonged administration of secretin to normal rats increases biliary proliferation and secretin-induced ductal secretory activity. Hepatobiliary Surg Nutr 2014; 3:118-25. [PMID: 25019073 DOI: 10.3978/j.issn.2304-3881.2014.04.04] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/01/2014] [Accepted: 04/14/2014] [Indexed: 11/14/2022]
Abstract
BACKGROUND AND AIM Cholangiocyte proliferation is coordinately regulated by a number of gastrointestinal hormones/peptides, some of which display stimulatory effects and some have inhibitory actions on cholangiocyte proliferation. Enhanced biliary proliferation [for example after bile duct ligation (BDL) and partial hepatectomy] is associated with increased expression of secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR) and Cl(-)/HCO3 (-) anion exchanger 2 and secretin-stimulated ductal secretion, whereas loss/damage of bile ducts [for example after acute carbon tetrachloride (CCl4) administration] is associated with reduced secretin-stimulated ductal secretory activity. There is growing information regarding the role of gastrointestinal hormones the regulation of biliary growth. For example, while gastrin, somatostatin and serotonin inhibit bile duct hyperplasia of cholestatic rats by downregulation of cAMP signaling, secretin has been shown to stimulate the proliferation of normal mice by activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signaling. However, no information exists regarding the stimulatory effects of secretin on biliary proliferation of normal rats. Thus, we evaluated the in vivo and in vitro effect of secretin on biliary proliferation, the expression of markers key of ductal secretion and secretin-stimulated ductal secretion. METHODS Normal male rats were treated with saline or secretin (2.5 nmoles/kg BW/day by osmotic minipumps for one week). We evaluated: (I) intrahepatic bile duct mass (IBDM) in liver sections and PCNA expression in purified cholangiocytes; (II) SR and CFTR mRNA expression and secretin-stimulated cAMP levels in purified cholangiocytes; and (III) secretin-stimulated bile and bicarbonate secretion in bile fistula rats. In vitro, normal rat intrahepatic cholangiocyte lines (NRIC) were treated with BSA (basal) or secretin (100 nM) for 24 to 72 hours in the absence/presence of a PKA or a MEK inhibitor before evaluating proliferation by MTS assays. RESULTS Prolonged administration of secretin to normal rats increased IBDM and PCNA expression in purified cholangiocytes compared to saline-treated normal rats. Also, secretin increased the expression of proteins (SR and CFTR) that are key in the regulating ductal secretion and enhanced secretin-stimulated cAMP levels and bile and bicarbonate secretion. In vitro, secretin increased the proliferation of NRIC, increase that was prevented by PKA and MAPK inhibitors. CONCLUSIONS We have demonstrated that secretin stimulates both in vivo and in vitro biliary proliferation and secretin-stimulated ductal secretory activity in normal rats. We suggest that the stimulatory effect of secretin on biliary proliferation and secretion may be important for preventing biliary dysfunction during ductopenic disorders.
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Affiliation(s)
- Micheleine Guerrier
- 1 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX, USA ; 2 University of Rome Sapienza, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, TX, USA ; 4 Scott & White Digestive Disease Research Center, Scott & White, TX, USA
| | - Fabia Attili
- 1 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX, USA ; 2 University of Rome Sapienza, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, TX, USA ; 4 Scott & White Digestive Disease Research Center, Scott & White, TX, USA
| | - Gianfranco Alpini
- 1 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX, USA ; 2 University of Rome Sapienza, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, TX, USA ; 4 Scott & White Digestive Disease Research Center, Scott & White, TX, USA
| | - Shannon Glaser
- 1 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX, USA ; 2 University of Rome Sapienza, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, TX, USA ; 4 Scott & White Digestive Disease Research Center, Scott & White, TX, USA
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Patil PB, Begum S, Joshi M, Kleman MI, Olausson M, Sumitran-Holgersson S. Phenotypic and in vivo functional characterization of immortalized human fetal liver cells. Scand J Gastroenterol 2014; 49:705-14. [PMID: 24730442 PMCID: PMC4059185 DOI: 10.3109/00365521.2013.830328] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.
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Affiliation(s)
- Pradeep B. Patil
- Laboratory of Transplantation and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden,Correspondence: Professor, Suchitra Sumitran-Holgersson, Laboratory of Transplantation Surgery and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg, Sahlgrenska Science Park, Medicinaregatan 8A, S-413 46 Gothenburg, Sweden. +46 0 31 3432100.
| | - Setara Begum
- Laboratory of Transplantation and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden,Correspondence: Professor, Suchitra Sumitran-Holgersson, Laboratory of Transplantation Surgery and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg, Sahlgrenska Science Park, Medicinaregatan 8A, S-413 46 Gothenburg, Sweden. +46 0 31 3432100.
| | - Meghnad Joshi
- Laboratory of Transplantation and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
| | | | - Michael Olausson
- Laboratory of Transplantation and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
| | - Suchitra Sumitran-Holgersson
- Laboratory of Transplantation and Regenerative Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
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Luo M, Yang F, Huang SX, Kuang ZP, Luo XL, Li YD, Wu JN, Xie YA. Two-stage model of chemically induced hepatocellular carcinoma in mouse. Oncol Res 2014; 20:517-28. [PMID: 24063282 DOI: 10.3727/096504013x13747716581336] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
The aim of this study was to develop an efficient and reproducible mouse model for hepatocellular carcinoma (HCC) research and assess the expression of two proto-oncogenes (c-myc and N-ras) and tumor suppressor gene p53 in the carcinogenic process. In this study, we found that diethylnitrosamine initiation with CCl4 and ethanol promotion could induce a short-term, two-stage liver carcinogenesis model in male BALB/c mice, the process of hepatocarcinogenesis including liver damage, liver necrosis/cell death, liver inflammation, liver proliferation, liver hyperplasia, liver steatosis, and liver cirrhosis and hepatocellular nodules, which mimicked the usual sequence of events observed in human HCC. We also identified that the increase in expression of the p53 gene is related to the proliferation of hepatocytes, whereas overexpression of the c-myc and N-ras genes is associated with hepatocarcinogenesis. This animal model may serve as a basis for recapitulating the molecular pathogenesis of HCC seen in humans.
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Affiliation(s)
- Min Luo
- Biomedical Research Center, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, People's Republic of China
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Renzi A, Mancinelli R, Onori P, Franchitto A, Alpini G, Glaser S, Gaudio E. Inhibition of the liver expression of arylalkylamine N-acetyltransferase increases the expression of angiogenic factors in cholangiocytes. Hepatobiliary Surg Nutr 2014; 3:4-10. [PMID: 24696833 DOI: 10.3978/j.issn.2304-3881.2014.01.02] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2014] [Accepted: 01/24/2014] [Indexed: 12/30/2022]
Abstract
BACKGROUND AND AIMS Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl(-)/HCO3 (-) AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by cholangiocytes. METHODS In this study, we evaluated the effect of local modulation of biliary AANAT expression on the cholangiocytes synthesis of VEGF-A/C. RESULTS The decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in cholangiocyte lines decreased the expression of VEGF-A/C. CONCLUSIONS Modulation of melatonin synthesis may affect the expression of VEGF-A/C by cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions.
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Affiliation(s)
- Anastasia Renzi
- 1 Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy ; 2 Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, 4 Scott & White Digestive Disease Research Center, Scott & White, Academic Operations, Scott & White, 5 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA
| | - Romina Mancinelli
- 1 Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy ; 2 Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, 4 Scott & White Digestive Disease Research Center, Scott & White, Academic Operations, Scott & White, 5 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA
| | - Paolo Onori
- 1 Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy ; 2 Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, 4 Scott & White Digestive Disease Research Center, Scott & White, Academic Operations, Scott & White, 5 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA
| | - Antonio Franchitto
- 1 Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy ; 2 Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, 4 Scott & White Digestive Disease Research Center, Scott & White, Academic Operations, Scott & White, 5 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA
| | - Gianfranco Alpini
- 1 Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy ; 2 Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, 4 Scott & White Digestive Disease Research Center, Scott & White, Academic Operations, Scott & White, 5 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA
| | - Shannon Glaser
- 1 Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy ; 2 Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, 4 Scott & White Digestive Disease Research Center, Scott & White, Academic Operations, Scott & White, 5 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA
| | - Eugenio Gaudio
- 1 Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza, Rome, Italy ; 2 Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy ; 3 Research, Central Texas Veterans Health Care System, 4 Scott & White Digestive Disease Research Center, Scott & White, Academic Operations, Scott & White, 5 Department of Medicine, Division Gastroenterology, Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA
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Torres Mena JE, Sánchez Rodríguez R, Quintanar Jurado V, Mojica Espinosa R, Del Pozo Yauner L, Meléndez Zajgla J, Villa Treviño S, Pérez Carreón JI. Laser capture microdissection after γ-glutamyl transferase histochemistry: An optimization for gene expression analysis. Anal Biochem 2014; 447:126-32. [DOI: 10.1016/j.ab.2013.11.015] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2013] [Revised: 11/08/2013] [Accepted: 11/11/2013] [Indexed: 11/16/2022]
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Ghosh S, Sengupta A, Sharma S, Sonawat HM. Metabolic perturbations of kidney and spleen in murine cerebral malaria: (1)H NMR-based metabolomic study. PLoS One 2013; 8:e73113. [PMID: 24039868 PMCID: PMC3765208 DOI: 10.1371/journal.pone.0073113] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2013] [Accepted: 07/18/2013] [Indexed: 02/02/2023] Open
Abstract
A significant fraction of global population is under the threat of malaria. Majority of annual death is due to the more complicated form of the infection i.e. the cerebral form, also known as Cerebral Malaria (CM). Host parasite interaction is known to cause a cascade of events in various tissues like brain, liver, kidney, and spleen. We have employed (1)H NMR based metabolomics to understand the specific perturbations of various tissues in CM. In our previous paper we have delineated the differences between CM vis-a-vis non-cerebral malaria (NCM) mice in serum, liver and brain. In this paper we focus on their differences of metabolic profile in kidney and spleen as kidney dysfunction and splenomegaly are known to be associated to neurological outcome of the disease. Moreover we have also looked into how the biological compartments (kidney, spleen and serum) interact with each other. The various metabolites involved in such interactions and their correlational aspects across the compartments have been studied in CM, NCM and control mice. The idea was to find out the specific pathways that are altered in CM mice. Our results demonstrate that both the kidney as well as spleen metabolism are differentially perturbed in CM with respect to NCM. The results point out that glutamate levels are decreased in CM mice with respect to NCM mice both in case of spleen and kidney while creatine, myo-inositol and betaine levels are increased in kidney of CM mice with respect to NCM mice. From the analysis of Multiway Principal Component Analysis (MPCA) we see that lipid metabolism and TCA cycle is altered in kidney and spleen.
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Affiliation(s)
- Soumita Ghosh
- Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India
| | - Arjun Sengupta
- Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India
| | - Shobhona Sharma
- Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
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The effect of caffeic acid phenethyl ester analogues in a modified resistant hepatocyte model. Anticancer Drugs 2013; 24:394-405. [PMID: 23388162 DOI: 10.1097/cad.0b013e32835e9743] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
We present a study of the chemoprotective effects of two caffeic acid phenethyl ester (CAPE)-related structures: LQM717 and LQM706. The modified resistant hepatocyte model in rats was used to study the chemoprevention of these CAPE analogues, which are inexpensive and easily obtained. In the liver cancer model used, we detected extensive necrosis and lipid peroxidation after 24 h, many altered hepatic foci, putatively preneoplastic lesions with γ-glutamyl transpeptidase staining after 30 days, and liver tumors at 12 months. We tested the effect of the CAPE analogues on necrosis, lipid peroxidation, proliferation, p65 activation, altered hepatic foci, and tumors. Both compounds exerted protective effects on lipid peroxidation, necrosis, cell proliferation, p65 activation, and preneoplastic lesions. Rats under a carcinogenic protocol showed a 52, 71.74, and 51.6% decrease in the number of preneoplastic nodules when pretreated with CAPE, LQM706, and LQM717, respectively. At 12 months after carcinogenic treatment, eight of eight rats developed liver cancer, whereas in the group of rats that received pretreatment with CAPE, LQM706, or LQM717, 62.5, 83.3, or 42.85%, respectively, had tumors. In conclusion, LQM717 has the potential to enhance chemoprotection activity much better than CAPE by markedly reducing the formation of liver cancers in this model, and this is a compound that is easy to obtain.
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Dittrich KL, Smith R, Maronpot RR. Stability of Gamma-glutamyltranspeptidase (GGT) in Paraffin Embedded Liver Tissue. J Histotechnol 2013. [DOI: 10.1179/his.1990.13.3.185] [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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Kim SE, An SY, Woo DH, Han J, Kim JH, Jang YJ, Son JS, Yang H, Cheon YP, Kim JH. Engraftment potential of spheroid-forming hepatic endoderm derived from human embryonic stem cells. Stem Cells Dev 2013; 22:1818-29. [PMID: 23373441 DOI: 10.1089/scd.2012.0401] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Transplantation and drug discovery programs for liver diseases are hampered by the shortage of donor tissue. While recent studies have shown that hepatic cells can be derived from human embryonic stem cells (hESCs), few cases have shown selective enrichment of hESC-derived hepatocytes and their integration into host liver tissues. Here we demonstrate that the dissociation and reaggregation procedure after an endodermal differentiation of hESC produces spheroids mainly consisted of cells showing hepatic phenotypes in vitro and in vivo. A combined treatment with Wnt3a and bone morphogenic protein 4 efficiently differentiated hESCs into definitive endoderm in an adherent culture. Dissociation followed by reaggregation of these cells in a nonadherent condition lead to the isolation of spheroid-forming cells that preferentially expressed early hepatic markers from the adherent cell population. Further differentiation of these spheroid cells in the presence of the hepatocyte growth factor, oncostatin M, and dexamethasone produced a highly enriched population of cells exhibiting characteristics of early hepatocytes, including glycogen storage, indocyanine green uptake, and synthesis of urea and albumin. Furthermore, we show that grafted spheroid cells express hepatic features and attenuate the serum aspartate aminotransferase level in a model of acute liver injury. These data suggest that hepatic progenitor cells can be enriched by the spheroid formation of differentiating hESCs and that these cells have engraftment potential to replace damaged liver tissues.
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Affiliation(s)
- Sung-Eun Kim
- Division of Biotechnology, Laboratory of Stem Cell Biology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea
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Chen YH, Chang MH, Chien CS, Wu SH, Yu CH, Chen HL. Contribution of mature hepatocytes to small hepatocyte-like progenitor cells in retrorsine-exposed rats with chimeric livers. Hepatology 2013; 57:1215-24. [PMID: 23080021 DOI: 10.1002/hep.26104] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2012] [Accepted: 10/01/2012] [Indexed: 12/25/2022]
Abstract
UNLABELLED The potential lineage relationship between hepatic oval cells, small hepatocyte-like progenitor cells (SHPCs), and hepatocytes in liver regeneration is debated. To test whether mature hepatocytes can give rise to SHPCs, rats with dipeptidyl peptidase IV (DPPIV) chimeric livers, which harbored endogenous DPPIV-deficient hepatocytes and transplanted DPPIV-positive hepatocytes, were subjected to retrorsine treatment followed by partial hepatectomy (PH). DPPIV-positive hepatocytes comprised about half of the DPPIV chimeric liver mass. Tissues from DPPIV chimeric livers after retrorsine/PH treatment showed large numbers of SHPC clusters. None of the SHPC clusters were stained positive for DPPIV in any analyzed samples. Furthermore, serial sections stained for gamma-glutamyl-transpeptidase (GGT, a marker of fetal hepatoblasts) and glucose-6-phosphatase (G6Pase, a marker of mature hepatocytes) showed inverse expression of the two enzymes and a staining pattern consistent with a lineage that begins with GGT(+)/G6Pase(-) to GGT(-)/G6Pase(+) within a single SHPC cluster. Using double immunofluorescence staining for markers specific for hepatic oval cells and hepatocytes in serial sections, oval cell proliferations with CK-19(+)/laminin(+) and OV-6(+)/C/EBP-α(-) were shown to extend from periportal areas into the SPHC clusters, differentiating into hepatic lineage by progressive loss of CK-19/laminin expression and appearance of C/EBP-α expression towards the cluster side. Cells in the epithelial cell adhesion molecule (EpCAM(+)) SHPC clusters showed membranous EpCAM(+)/HNF-4α(+) (hepatocyte nuclear factor-4α) staining and were contiguous to the surrounding cytoplasmic EpCAM(+)/HNF-4α(-) ductular oval cells. Extensive elimination of oval cell response by repeated administration of 4,4'-methylenedianiline (DAPM) to retrorsine-exposed rats impaired the emergence of SHPC clusters. CONCLUSION These findings highly suggest the hepatic oval cells but not mature hepatocytes as the origin of SHPC clusters in retrorsine-exposed rats.
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Affiliation(s)
- Ya-Hui Chen
- Graduate Institute of Clinical Medicine, Department of Pediatrics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
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Talbot NC, Caperna TJ. A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion. Cytotechnology 2013; 66:1-7. [PMID: 23397443 DOI: 10.1007/s10616-013-9541-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2012] [Accepted: 01/21/2013] [Indexed: 01/28/2023] Open
Abstract
A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line's growth was finite and the cells essentially ceased dividing after approximately 20 passages over a 1 year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to 90-98 % confluency after each passage at 2 week intervals, and the cells maintained a high cell density after 2 years and 48 passages in culture (average of 2.6 × 10(6) cells/T25 flask or 1 × 10(5) cells/cm(2)). Morphologically, the PICM-FF cells closely resembled the finite feeder-independent PICM-19 cultures previously reported, and, as before, no spontaneous formation of 3D multicellular ductules occurred in the cells' monolayer. Their bipotent stem cell nature was therefore not evident. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. Two other attributes of fetal hepatocytes, γ-glutamyl transpeptidase activity and serum-protein secretion, were also shown to be maintained by the PICM-19FF cells. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line and this should enhance the experimental usefulness of the cell line, in general, and may also improve its application to toxicological/pharmacological assays and artificial liver devices.
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Affiliation(s)
- Neil C Talbot
- US Department of Agriculture, Beltsville Agricultural Research Center, Animal Biosciences and Biotechnology Laboratory, Bldg. 200, Rm. 13, BARC-East, Beltsville, MD, 20705, USA,
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