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M G MS, Chikhale R, Nanaware PP, Dalvi S, Venkatraman P. A druggable pocket on PSMD10 Gankyrin that can accommodate an interface peptide and doxorubicin. Eur J Pharmacol 2022; 915:174718. [PMID: 34953804 DOI: 10.1016/j.ejphar.2021.174718] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Revised: 12/09/2021] [Accepted: 12/20/2021] [Indexed: 01/08/2023]
Abstract
BACKGROUND PSMD10Gankyrin, a proteasomal chaperone is also an oncoprotein. Overexpression of PSMD10Gankyrin is associated with poor prognosis and survival in many cancers. Therefore, PSMD10Gankyrin is a sought-after drug target in many hard-to-treat cancers. However, its surface appears flat and undruggable. Here, we build on our earlier discovery of a common hot spot region that defined the interface of multiple interacting partners of PSMD10Gankyrin to expose vulnerable spots for a peptide and a small molecule inhibitor. METHODS High throughput virtual screening was used to screen compounds against PSMD10Gankyrin. Interaction of PSMD10Gankyrin with the drug or protein (CLIC1) or peptide was studied using any one or more of these techniques; Microscale Thermophoresis, limited trypsinolysis, SPR and ITC. Cytotoxic effect of doxorubicin was evaluated using MTT assay. RESULTS We identified doxorubicin as the first-generation small molecule inhibitor of PSMD10Gankyrin. K116 and to a lesser extent R41 on PSMD10Gankyrin contribute to the bulk of binding energy for the peptide EEVD, CLIC1 and doxorubicin. We further demonstrate that PSMD10Gankyrin is an intended target for doxorubicin in cells. GENERAL SIGNIFICANCE Drug design against protein interactions in general and PSMD10Gankyrin in particular, remains a challenge. We provide consolidated biophysical evidence for the use of a shared interface motif EEVD as a possible inhibitor of interaction network in cancers driven by PSMD10Gankyrin. We identify a chemical scaffold for designing novel inhibitors of PSMD10Gankyrin. These findings will impact the field of protein interactions in the context of disease biology/drug discovery.
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Affiliation(s)
- Mukund Sudharsan M G
- Protein Interaction Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 41210, India; Homi Bhabha National Institute, 2nd Floor, BARC Training School Complex, Anushaktinagar, Mumbai, Maharashtra, 400094, India
| | - Rupesh Chikhale
- Protein Interaction Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 41210, India
| | - Padma P Nanaware
- Protein Interaction Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 41210, India
| | - Somavally Dalvi
- Protein Interaction Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 41210, India
| | - Prasanna Venkatraman
- Protein Interaction Laboratory for Structural and Functional Biology, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai, Maharashtra, 41210, India; Homi Bhabha National Institute, 2nd Floor, BARC Training School Complex, Anushaktinagar, Mumbai, Maharashtra, 400094, India.
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Romualdo GR, Leroy K, Costa CJS, Prata GB, Vanderborght B, da Silva TC, Barbisan LF, Andraus W, Devisscher L, Câmara NOS, Vinken M, Cogliati B. In Vivo and In Vitro Models of Hepatocellular Carcinoma: Current Strategies for Translational Modeling. Cancers (Basel) 2021; 13:5583. [PMID: 34771745 PMCID: PMC8582701 DOI: 10.3390/cancers13215583] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Revised: 11/02/2021] [Accepted: 11/04/2021] [Indexed: 12/24/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related death globally. HCC is a complex multistep disease and usually emerges in the setting of chronic liver diseases. The molecular pathogenesis of HCC varies according to the etiology, mainly caused by chronic hepatitis B and C virus infections, chronic alcohol consumption, aflatoxin-contaminated food, and non-alcoholic fatty liver disease associated with metabolic syndrome or diabetes mellitus. The establishment of HCC models has become essential for both basic and translational research to improve our understanding of the pathophysiology and unravel new molecular drivers of this disease. The ideal model should recapitulate key events observed during hepatocarcinogenesis and HCC progression in view of establishing effective diagnostic and therapeutic strategies to be translated into clinical practice. Despite considerable efforts currently devoted to liver cancer research, only a few anti-HCC drugs are available, and patient prognosis and survival are still poor. The present paper provides a state-of-the-art overview of in vivo and in vitro models used for translational modeling of HCC with a specific focus on their key molecular hallmarks.
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Affiliation(s)
- Guilherme Ribeiro Romualdo
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
- Department of Structural and Functional Biology, Biosciences Institute, São Paulo State University (UNESP), Botucatu 18618-689, Brazil; (G.B.P.); (L.F.B.)
- Department of Pathology, Botucatu Medical School, São Paulo State University (UNESP), Botucatu 18618-687, Brazil
| | - Kaat Leroy
- Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (K.L.); (M.V.)
| | - Cícero Júlio Silva Costa
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
| | - Gabriel Bacil Prata
- Department of Structural and Functional Biology, Biosciences Institute, São Paulo State University (UNESP), Botucatu 18618-689, Brazil; (G.B.P.); (L.F.B.)
- Department of Pathology, Botucatu Medical School, São Paulo State University (UNESP), Botucatu 18618-687, Brazil
| | - Bart Vanderborght
- Gut-Liver Immunopharmacology Unit, Basic and Applied Medical Sciences, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium;
- Hepatology Research Unit, Internal Medicine and Paediatrics, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium;
| | - Tereza Cristina da Silva
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
| | - Luís Fernando Barbisan
- Department of Structural and Functional Biology, Biosciences Institute, São Paulo State University (UNESP), Botucatu 18618-689, Brazil; (G.B.P.); (L.F.B.)
| | - Wellington Andraus
- Department of Gastroenterology, Clinics Hospital, School of Medicine, University of São Paulo (HC-FMUSP), São Paulo 05403-000, Brazil;
| | - Lindsey Devisscher
- Hepatology Research Unit, Internal Medicine and Paediatrics, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium;
| | - Niels Olsen Saraiva Câmara
- Department of Immunology, Institute of Biomedical Sciences IV, University of São Paulo (USP), São Paulo 05508-000, Brazil;
| | - Mathieu Vinken
- Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (K.L.); (M.V.)
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
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Ranasinghe KNK, Premarathna AD, Mahakapuge TAN, Wijesundera KK, Ambagaspitiya AT, Jayasooriya AP, Kularatne SAM, Rajapakse RPVJ. In vivo anticancer effects of Momordica charantia seed fat on hepatocellular carcinoma in a rat model. J Ayurveda Integr Med 2021; 12:435-442. [PMID: 34275705 PMCID: PMC8377176 DOI: 10.1016/j.jaim.2021.03.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2020] [Revised: 02/26/2021] [Accepted: 03/01/2021] [Indexed: 11/21/2022] Open
Abstract
BACKGROUND Momordica charantia or bitter melon is a well-known vegetable with a number of therapeutic actions in Ayurvedic medicine. Alpha-eleostearic acid, a conjugated trienoic fatty acid present in bitter melon is proven to have anticancer properties. Crude seed oil from local bitter melon varieties could be an effective and economical anticancer therapy. OBJECTIVE(S) The study was conducted to evaluate the anticancer effect of the crude oil from the seeds of Matale green variety of bitter melon on a hepatocellular carcinoma-induced rat model. MATERIALS AND METHODS Hepatocellular carcinoma (HCC) was experimentally induced in Wistar rats. Crude seed oil of Matale green bitter melon (MGBM) was supplemented to one treatment group in concurrence with carcinoma induction and to another treatment group after the development of carcinoma. After 168 days, gross morphological, histopathological, biochemical, hematological and gene-expression analysis of treated and control groups were performed. RESULTS Oral supplementation of MGBM seed oil showed a statistically significant reduction (p < 0.05) in the average number, diameter and area of hepatic dysplastic nodules and a reduction in the size of histopathological neoplastic lesions in both treatment groups compared to the non-treated control group. The expression of tumor suppressor gene p53 and anti-apoptotic gene Bcl-2 were significantly increased while the expression of apoptotic gene caspase 3 was significantly reduced in the treatment group when MGBM supplementation was in concurrence with carcinogenesis (p < 0.05). CONCLUSION Crude seed oil from the MGBM has anticancer effects against experimentally induced HCC in Wistar rats, specially when supplemented in concurrence with carcinoma induction.
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Affiliation(s)
- K N K Ranasinghe
- Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
| | - A D Premarathna
- Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
| | - T A N Mahakapuge
- Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
| | - K K Wijesundera
- Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
| | - A T Ambagaspitiya
- Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
| | - A P Jayasooriya
- Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
| | - S A M Kularatne
- Department of Medicine, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka
| | - R P V J Rajapakse
- Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka.
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Fujita J, Sakurai T. The Oncoprotein Gankyrin/PSMD10 as a Target of Cancer Therapy. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1164:63-71. [PMID: 31576540 DOI: 10.1007/978-3-030-22254-3_5] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Gankyrin (also called PSMD10, p28, or p28GANK) is a crucial oncoprotein that is upregulated in various cancers and assumed to play pivotal roles in the initiation and progression of tumors. Although the in vitro function of gankyrin is relatively well characterized, its role in vivo remains to be elucidated. We have investigated the function of gankyrin in vivo by producing mice with liver parenchymal cell-specific gankyrin ablation (Alb-Cre;gankyrinf/f) and gankyrin deletion both in liver parenchymal and in non-parenchymal cells (Mx1-Cre;gankyrinf/f). Gankyrin deficiency both in non-parenchymal cells and parenchymal cells, but not in parenchymal cells alone, reduced STAT3 activity, interleukin-6 production, and cancer stem cell marker expression, leading to attenuated tumorigenic potential in the diethylnitrosamine hepatocarcinogenesis model. Essentially similar results were obtained by analyzing mice with intestinal epithelial cell-specific gankyrin ablation (Villin-Cre;Gankyrinf/f) and gankyrin deletion both in myeloid and epithelial cells (Mx1-Cre;Gankyrinf/f) in the colitis-associated cancer model. Clinically, gankyrin expression in the tumor microenvironment was negatively correlated with progression-free survival in patients undergoing treatment with Sorafenib for hepatocellular carcinomas. These findings indicate important roles played by gankyrin in non-parenchymal cells as well as parenchymal cells in the pathogenesis of liver cancers and colorectal cancers, and suggest that by acting both on cancer cells and on the tumor microenvironment, anti-gankyrin agents would be promising as therapeutic and preventive strategies against various cancers, and that an in vitro cell culture models that incorporate the effects of non-parenchymal cells and gankyrin would be useful for the study of human cell transformation.
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Affiliation(s)
- Jun Fujita
- Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
| | - Toshiharu Sakurai
- Department of Gastroenterology and Hepatology, Faculty of Medicine, Kindai University, Osaka, Japan
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Taheri T, Jamialahmadi K, Khadijeh F. Unexpected Lower Expression of Oncoprotein Gankyrin in Drug Resistant ABCG2 Overexpressing Breast Cancer Cell Lines. Asian Pac J Cancer Prev 2017; 18:3413-3418. [PMID: 29286612 PMCID: PMC5980903 DOI: 10.22034/apjcp.2017.18.12.3413] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Background: Development of a multidrug resistance (MDR) phenotype to chemotherapy remains a major barrier in
the treatment of cancer. Gankyrin (p28, p28GANK or PSMD10) is an oncoprotein overexpressed in different carcinoma
cell lines. The aim of this study was to compare Gankyrin expression level in MDR cells (MCF-7/ADR and MCF-7/
MX) and non-MDR counterparts (MCF-7). Methods: Gankyrin, MDR1 (also known as ABCB1; the ATP-binding
cassette sub-family B member 1) and ABCG2 (also known as BCRP; the human breast cancer resistance protein)
mRNA levels were analyzed by real-time RT-PCR. Western blot analysis was used to detect the protein expression
levels of Gankyrin. Results: The PCR results showed that the expression of Gankyrin was significantly lower in the
ABCG2 overexpressing cell line MCF-7/MX than in non-resistanct MCF-7 cells. In contrast, there were no significant
differences in mRNA expression of Gankyrin in the MDR1 overexpressing cell line MCF-7/ADR in comparison with
MCF-7 cells. Similarly, Western blot analysis confirmed lower expression of Gankyrin protein in the MCF-7/MX cell
line (26% compared to controls) but not in MCF-7/ADR cells. Conclusion: These findings showed that there may be
a relation between down-regulation of Gankyrin and overexpression of ABCG2 but without any clear relationship with
MDR1 expression in breast cancer cell lines.
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Affiliation(s)
- Taheri Taheri
- Department of Biochemistry, Faculty of Science, Payam Noor University of Mashhad, Mashhad, Iran.,Department of Stem Cells and Developmental Biology, Royan Institute for Stem cell Biology and Technology, ACECR, Tehran, Iran.
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Huang SJ, Cheng CL, Chen JR, Gong HY, Liu W, Wu JL. Inducible liver-specific overexpression of gankyrin in zebrafish results in spontaneous intrahepatic cholangiocarcinoma and hepatocellular carcinoma formation. Biochem Biophys Res Commun 2017; 490:1052-1058. [PMID: 28668389 DOI: 10.1016/j.bbrc.2017.06.164] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2017] [Accepted: 06/27/2017] [Indexed: 12/30/2022]
Abstract
Liver cancer is the second leading cause of death worldwide. As such, establishing animal models of the disease is important for both basic and translational studies that move toward developing new therapies. Gankyrin is a critical oncoprotein in the genetic control of liver pathology. In order to evaluate the oncogenic role of gankyrin without cancer cell inoculation and drug treatment, we overexpressed gankyrin under the control of the fabp10a promoter. A Tet-Off system was used to drive expression in hepatocytes. At seven to twelve months of age, gankyrin transgenic fish spontaneously incurred persistent hepatocyte damage, steatosis, cholestasis, cholangitis, fibrosis and hepatic tumors. The tumors were both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). ICC is the second most frequent primary liver cancer in human patients and the first to develop in this tumor model. We further investigated the role of complement C3, a central molecule of the complement system, and found the expression levels of both in mRNA and protein are decreased during tumorigenesis. Together, these findings suggest that gankyrin can promote malignant transformation of liver cells in the context of persistent liver injury. This transformation may be related to compensatory proliferation and the inflammatory microenvironment. The observed decrease in complement C3 may allow transforming cells to escape coordinated induction of the immune response. Herein, we demonstrate an excellent zebrafish model for liver cancers that will be useful for studying the molecular mechanisms of tumorgenesis.
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Affiliation(s)
- Shin-Jie Huang
- Institute of Fisheries Science, National Taiwan University, Taipei 106, Taiwan
| | - Chih-Lun Cheng
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan
| | - Jim-Ray Chen
- Department of Pathology, Chang Gung Memorial Hospital, Keelung 204, Taiwan; College of Medicine, Chang Gung Univeristy, Taoyuan 333, Taiwan
| | - Hong-Yi Gong
- Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan
| | - Wangta Liu
- Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan; Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 807, Taiwan
| | - Jen-Leih Wu
- Institute of Fisheries Science, National Taiwan University, Taipei 106, Taiwan; Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan; College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan; Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan.
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Pei T, Li Y, Wang J, Wang H, Liang Y, Shi H, Sun B, Yin D, Sun J, Song R, Pan S, Sun Y, Jiang H, Zheng T, Liu L. YAP is a critical oncogene in human cholangiocarcinoma. Oncotarget 2016; 6:17206-20. [PMID: 26015398 PMCID: PMC4627302 DOI: 10.18632/oncotarget.4043] [Citation(s) in RCA: 111] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2015] [Accepted: 04/30/2015] [Indexed: 12/15/2022] Open
Abstract
Yes-associated protein (YAP), a transcriptional co-activator, has important regulatory roles in cell signaling and is dysregulated in a number of cancers. However, the role of YAP in cholangiocarcinoma (CCA) progression remains unclear. Here, we demonstrated that YAP was overexpressed in CCA cells and human specimens. High levels of nuclear YAP (nYAP) correlated with histological differentiation, TNM stage, metastasis and poor prognosis in CCA. Silencing YAP increased tumor sensitivity to chemotherapy and inhibited CCA tumorigenesis and metastasis both in vivo and in vitro. YAP overexpression in vivo and in vitro promoted CCA tumorigenesis and metastasis. Additionally, we found that YAP induced epithelial-mesenchymal transition (EMT) and formed a regulatory circuit with miR-29c, IGF1, AKT and gankyrin to promote the progression of CCA. Results of CCA tissue microarray showed positive correlations between nYAP and gankyrin or p-AKT expression. Combination of nYAP and gankyrin or p-AKT exhibited improved prognostic accuracy for CCA patients. In conclusion, YAP promotes carcinogenesis and metastasis by up-regulating gankyrin through activation of the AKT pathway.
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Affiliation(s)
- Tiemin Pei
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Yuejin Li
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Jiabei Wang
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Huanlai Wang
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China.,Department of General Surgery, Qiqihaer City Hospital of Traditional Chinese Medicine, Qiqihaer, China
| | - Yingjian Liang
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Huawen Shi
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Boshi Sun
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Dalong Yin
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Jing Sun
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Ruipeng Song
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Shangha Pan
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Yu Sun
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Hongchi Jiang
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Tongsen Zheng
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
| | - Lianxin Liu
- Key Laboratory of Hepatosplenic Surgery, Ministry of Education, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
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Zhao X, Fu J, Xu A, Yu L, Zhu J, Dai R, Su B, Luo T, Li N, Qin W, Wang B, Jiang J, Li S, Chen Y, Wang H. Gankyrin drives malignant transformation of chronic liver damage-mediated fibrosis via the Rac1/JNK pathway. Cell Death Dis 2015; 6:e1751. [PMID: 25950481 PMCID: PMC4669699 DOI: 10.1038/cddis.2015.120] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2014] [Revised: 03/17/2015] [Accepted: 03/24/2015] [Indexed: 02/07/2023]
Abstract
Hepatocarcinogenesis is a complex process involving chronic liver injury, inflammation, unregulated wound healing, subsequent fibrosis and carcinogenesis. To decipher the molecular mechanism underlying transition from chronic liver injury to dysplasia, we investigated the oncogenic role of gankyrin (PSMD10 or p28GANK) during malignant transformation in a transgenic mouse model. Here, we find that gankyrin increased in patients with cirrhosis. In addition to more severe liver fibrosis and tumorigenesis after DEN plus CCl4 treatment, hepatocyte-specific gankyrin-overexpressing mice (gankyrinhep) exhibited malignant transformation from liver fibrosis to tumors even under single CCl4 administration, whereas wild-type mice merely experienced fibrosis. Consistently, enhanced hepatic injury, severe inflammation and strengthened compensatory proliferation occurred in gankyrinhep mice during CCl4 performance. This correlated with augmented expressions of cell cycle-related genes and abnormal activation of Rac1/c-jun N-terminal kinase (JNK). Pharmacological inhibition of the Rac1/JNK pathway attenuated hepatic fibrosis and prevented CCl4-induced carcinogenesis in gankyrinhep mice. Together, these findings suggest that gankyrin promotes liver fibrosis/cirrhosis progression into hepatocarcinoma relying on a persistent liver injury and inflammatory microenvironment. Blockade of Rac1/JNK activation impeded gankyrin-mediated hepatocytic malignant transformation, indicating the combined inhibition of gankyrin and Rac1/JNK as a potential prevention mechanism for cirrhosis transition.
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Affiliation(s)
- X Zhao
- 1] Model Animal Research Center and MOE Key Laboratory of Model Animal for Disease Study, Nanjing University, Nanjing 210061, China [2] International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - J Fu
- 1] International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China [2] National Center for Liver Cancer, Shanghai 200438, China
| | - A Xu
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - L Yu
- 1] International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China [2] National Center for Liver Cancer, Shanghai 200438, China
| | - J Zhu
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - R Dai
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - B Su
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - T Luo
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - N Li
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - W Qin
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - B Wang
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - J Jiang
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China
| | - S Li
- MOE Key Laboratory of Bioinformatics and Bioinformatics Division, TNLIST, Department of Antomation, Tsinghua University, Beijing 100084, China
| | - Y Chen
- 1] International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China [2] National Center for Liver Cancer, Shanghai 200438, China
| | - H Wang
- 1] Model Animal Research Center and MOE Key Laboratory of Model Animal for Disease Study, Nanjing University, Nanjing 210061, China [2] International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai 200438, China [3] National Center for Liver Cancer, Shanghai 200438, China [4] State Key Laboratory of Oncogenes and Related Genes, Cancer Institute of Renji Hospital, Shanghai Jiaotong University, Shanghai 200032, China
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Zhao JA, Peng L, Geng CZ, Liu YP, Wang X, Yang HC, Wang SJ. Preventive effect of hydrazinocurcumin on carcinogenesis of diethylnitrosamine-induced hepatocarcinoma in male SD rats. Asian Pac J Cancer Prev 2014; 15:2115-21. [PMID: 24716943 DOI: 10.7314/apjcp.2014.15.5.2115] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
The purpose of the present study was to evaluate the preventive effects of hydrazinocurcumin (HZC) on diethylnitrosamine (DEN)-induced hepatocarcinogenesis in a male Sprague Dawley (SD) rat model. One hundred and twenty male SD rats used in this study were divided into six groups. Those receiving DEN with curcumin (CUR) or HZC were studied compared with the DEN-alone group. The study demonstrated that DEN induced severe histological and immunohistochemical changes in liver tissues, significantly increasing the levels of liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and total bilirubin level (TBL)). The hepatocarcinoma incidences were 100.0%, 36.7% and 20.0% in the DEN-alone, DEN-CUR and DEN-HZC groups, respectively. Although macroscopic and microscopic features suggested that both CUR and HZC were effective in inhibiting DEN- induced hepatocarcinogenesis, HZC was exerted a stronger influence. Immunohistochemical analysis with PCNA demonstrated significantly differences among the groups (all P < 0.05). Taken together, the results suggested application of CUR and HZC could prevent the occurrence of carcinogenesis and HZC may be a more potent compound for prevention of DEN-induced hepatocarcinogenesis in rats than CUR.
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Affiliation(s)
- Ji-An Zhao
- Department of Hepatobiliary Surgery, The First Affilliated Hospital, Hebei Medical University, Shijiazhuang, China E-mail :
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Jing H, Zhang G, Meng L, Meng Q, Mo H, Tai Y. Gradually elevated expression of Gankyrin during human hepatocarcinogenesis and its clinicopathological significance. Sci Rep 2014; 4:5503. [PMID: 24999092 PMCID: PMC4083285 DOI: 10.1038/srep05503] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Accepted: 06/02/2014] [Indexed: 02/03/2023] Open
Abstract
Gankyrin is an important oncoprotein that is overexpressed in human hepatocellular carcinoma (HCC). However, the gradual alteration of Gankyrin in successive stages during human HCC development and the mechanism of Gankyrin-mediated hepatocarcinogenesis remain largely unknown. In this study, we evaluated the pattern and level of Gankyrin protein expression using immunohistochemistry in various liver tissues, including normal liver, chronic hepatitis, cirrhosis, adenomatous hyperplasia (AH), and HCC tissues, to analyze its clinicopathological significance. Furthermore, we stably transfected the shRNA-Gan vector, which targets human Gankyrin, into HepG2 cells to assess the role of Gankyrin in cell proliferation and tumorigenicity. The expression level of Gankyrin in the cytoplasm, nucleus, and whole cell was gradually elevated during consecutive stages of hepatocarcinogenesis. The nuclear Gankyrin level in AH was significantly higher than that in normal liver, chronic hepatitis, and cirrhotic tissues. The cytoplasmic, nuclear, and total cellular Gankyrin expression levels in HCC were significantly correlated with capsular invasion and intrahepatic metastasis. Silencing Gankyrin expression using shRNA-Gan repressed tumor cell proliferation, tumorigenicity, migration, and invasion in vitro. Our findings demonstrate that Gankyrin is aberrantly expressed beginning at the initiation stage and plays an important role in the initiation, promotion, and progression of hepatocarcinogenesis.
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Affiliation(s)
- Hongbiao Jing
- 1] Department of Pathology, the General Hospital, Jinan Military Command, Jinan, China [2]
| | - Guoming Zhang
- 1] Department of Cardiology, the General Hospital, Jinan Military Command, Jinan, China [2]
| | - Lingsheng Meng
- Laboratory Department, the Sixth Hospital of Jinan, Zhangqiu, China
| | - Qingda Meng
- Laboratory Department, the Sixth Hospital of Jinan, Zhangqiu, China
| | - Haiying Mo
- Department of Pathology, Zaozhuang Hospital, Zaozhuang Mining Group, Zaozhuang, China
| | - Yanhong Tai
- Department of Pathology, the General Hospital, Jinan Military Command, Jinan, China
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Reed C, Hutcheson J, Mayhew CN, Witkiewicz AK, Knudsen ES. RB tumor suppressive function in response to xenobiotic hepatocarcinogens. THE AMERICAN JOURNAL OF PATHOLOGY 2014; 184:1853-9. [PMID: 24726645 DOI: 10.1016/j.ajpath.2014.02.025] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/12/2013] [Revised: 01/27/2014] [Accepted: 02/04/2014] [Indexed: 12/30/2022]
Abstract
Diverse etiologic events are associated with the development of hepatocellular carcinoma. During hepatocarcinogenesis, genetic events likely occur that subsequently cooperate with long-term exposures to further drive the progression of hepatocellular carcinoma. In this study, the frequent loss of the retinoblastoma (RB) tumor suppressor in hepatocellular carcinoma was modeled in response to diverse hepatic stresses. Loss of RB did not significantly affect the response to a steatotic stress as driven by a methionine- and choline-deficient diet. In addition, RB status did not significantly influence the response to peroxisome proliferators that can drive hepatomegaly and tumor development in rodents. However, RB loss exhibited a highly significant effect on the response to the xenobiotic1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene. Loss of RB yielded a unique proliferative response to this agent, which was distinct from both regenerative stresses and genotoxic carcinogens. Long-term exposure to 1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene yielded profound tumor development in RB-deficient livers that was principally absent in RB-sufficient tissue. These data demonstrate the context specificity of RB and the key role RB plays in the suppression of hepatocellular carcinoma driven by xenobiotic stress.
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Affiliation(s)
- Christopher Reed
- Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Jack Hutcheson
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas
| | | | - Agnieszka K Witkiewicz
- Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania; Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas
| | - Erik S Knudsen
- Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania; Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas.
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Song X, Wang J, Zheng T, Song R, Liang Y, Bhatta N, Yin D, Pan S, Liu J, Jiang H, Liu L. LBH589 Inhibits proliferation and metastasis of hepatocellular carcinoma via inhibition of gankyrin/STAT3/Akt pathway. Mol Cancer 2013; 12:114. [PMID: 24093956 PMCID: PMC3853770 DOI: 10.1186/1476-4598-12-114] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2013] [Accepted: 10/01/2013] [Indexed: 01/01/2023] Open
Abstract
Background Gankyrin has shown to be overexpressed in human liver cancers and plays a complex role in hepatocarcinogenesis. Panobinostat (LBH589), a new hydroxamic acid-derived histone deacetylase inhibitor has shown promising anticancer effects recently. Here, we investigated the potential of LBH589 as a form of treatment for hepatocellular carcinoma (HCC). Methods Gankyrin plasmid was transfected into HCC cells, and the cells were selected for more than 4 weeks by incubation with G418 for overexpression clones. The therapeutic effects of LBH589 were evaluated in vitro and in vivo. Cell proliferation, apoptosis, cell cycle, invasive potential, and epithelial-mesenchy-mal transition (EMT) were examined. Results LBH589 significantly inhibited HCC growth and metastasis in vitro and in vivo. Western blotting analysis indicated that LBH589 could decrease the expression of gankyrin and subsequently reduced serine-phosphorylated Akt and tyrosine-phosphorylated STAT3 expression although the total Akt and STAT3 were unaffected. LBH589 inhibited metastasis in vitro via down-regulation of N-cadherin, vimentin, TWIST1, VEGF and up-regulation of E-cadherin. LBH589 also induced apoptosis and G1 phase arrest in HCC cell lines. Ectopic expression of gankyrin attenuated the effects of LBH589, which indicates that gankyrin might play an important role in LBH589 mediated anticancer effects. Lastly, in vivo study indicated that LBH589 inhibited tumor growth and metastasis, without discernable adverse effects comparing to control group, with abrogating gankyrin/STAT3/Akt pathway. Conclusions Our results suggested that LBH589 could inhibit HCC growth and metastasis through down-regulating gankyrin/STAT3/Akt pathway. LBH589 may present itself as a novel therapeutic strategy for HCC.
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Affiliation(s)
- Xuan Song
- Department of Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Key Laboratory of Hepatosplenic Surgery, Ministry of Education, 150001 Harbin, Heilongjiang Province, China.
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Zohny SF, Mahmoud AH, Borai IH, Bayoumi FS, Eissa E. Chemopreventive and therapeutic efficacy of Salsola inermis extract against N-nitrosodiethylamine-initiated and phenobarbital-promoted hepatocellular carcinogenesis in Wistar rats. ACTA ACUST UNITED AC 2013. [DOI: 10.1016/j.biomag.2013.08.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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14
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Kasai H, Kawai K, Song MF, Li YS, Hattori T, Matsuda T. Analyses of 8-Methyldeoxyadenosine and 8-Methyldeoxyguanosine as Markers of Free Radical-mediated DNA Methylation in Mouse. Genes Environ 2013. [DOI: 10.3123/jemsge.35.5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
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Kim YH, Kim JH, Choi YW, Lim SK, Yim H, Kang SY, Chung YS, Lee GY, Park TJ. Gankyrin is frequently overexpressed in breast cancer and is associated with ErbB2 expression. Exp Mol Pathol 2012; 94:360-5. [PMID: 23276718 DOI: 10.1016/j.yexmp.2012.12.002] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2012] [Accepted: 12/19/2012] [Indexed: 01/26/2023]
Abstract
Gankyrin is a subunit of the 26S proteasome, and has been known to degrade p53 and retinoblastoma protein and promote the tumorigenicity and metastasis in some malignancies. However, the role of gankyrin in breast cancer has not been explored. In this study, we investigated the expression of gankyrin in breast cancer and evaluated its effect on breast cancer. Representative cancer tissues including normal breasts from 60 patients with breast cancer were stained immunohistochemically for gankyrin, estrogen receptor, progesterone receptor, and ErbB2. We evaluated the relationship between gankyrin expression and clinicopathologic parameters or prognostic markers. We also attempted to clarify the mechanism of gankyrin involved in breast carcinogenesis by using MCF7 breast cancer cells. Gankyrin was weakly expressed in normal breast epithelial cells, however, tumor regions of 37/60 (61.7%) cases showed an overexpression of gankyrin. Gankyrin overexpression was associated with extensive intraductal carcinoma (p=0.014) and ErbB2 positivity (p=0.031) in invasive ductal carcinoma. In MCF7 breast cancer cells, downregulation of gankyrin was associated with a reduction of cell proliferation and tumorigenicity. In conclusion, gankyrin was identified in normal breasts and overexpressed in invasive breast cancers. The overexpression of gankyrin was associated with extensive intraductal carcinoma and ErbB2 expression in breast cancer.
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Affiliation(s)
- Yeong Hwa Kim
- Department of Biochemistry and Molecular Biology, Brain Korea 21 Division of Cell Transformation and Restoration, Ajou University, School of Medicine, Suwon 443-721, Republic of Korea
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Pogribny IP, Beland FA. DNA methylome alterations in chemical carcinogenesis. Cancer Lett 2012; 334:39-45. [PMID: 23010082 DOI: 10.1016/j.canlet.2012.09.010] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2012] [Revised: 08/28/2012] [Accepted: 09/14/2012] [Indexed: 01/30/2023]
Abstract
Carcinogenesis, a complex multifactorial process of the transformation of normal cells into malignant cells, is characterized by many biologically significant and interdependent alterations triggered by the mutational and/or non-mutational (i.e., epigenetic) events. One of these events, specific to all types of cancer, is alterations in DNA methylation. This review summarizes the current knowledge of the role of DNA methylation changes induced by various genotoxic chemicals (carcinogenic agents that interact with DNA) and non-genotoxic carcinogens (chemicals causing tumor by mechanisms other than directly damaging DNA) in the lung, colorectal, liver, and hematologic carcinogenesis. It also emphasizes the potential role for epigenetic changes to serve as markers for carcinogen exposure and carcinogen risk assessment.
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Affiliation(s)
- Igor P Pogribny
- Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.
| | - Frederick A Beland
- Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.
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Molecular markers as a prognostic system for hepatocellular carcinoma. J Adv Res 2011. [DOI: 10.1016/j.jare.2011.02.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
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Li J, Poi MJ, Tsai MD. Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer. Biochemistry 2011; 50:5566-82. [PMID: 21619050 PMCID: PMC3127263 DOI: 10.1021/bi200642e] [Citation(s) in RCA: 236] [Impact Index Per Article: 16.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
P16(INK4A) (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly because of the prevalence of genetic inactivation of the p16(INK4A) (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that an elevated level of expression (upregulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications for the structure-function relationship of P16 and for human cancers.
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Affiliation(s)
- Junan Li
- Division of Environmental Health Sciences, College of Public Health, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, Ohio 43210, USA.
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Overexpression of gankyrin induces liver steatosis in zebrafish (Danio rerio). Biochim Biophys Acta Mol Cell Biol Lipids 2011; 1811:536-48. [PMID: 21722753 DOI: 10.1016/j.bbalip.2011.06.011] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2010] [Revised: 05/24/2011] [Accepted: 06/09/2011] [Indexed: 02/08/2023]
Abstract
Gankyrin is a small ankyrin-repeat protein that previous research has confirmed to be overexpressed in hepatocellular carcinoma (HCC). Although relevant literature has reported on gankyrin functions in cellular proliferation and tumorigenesis, the exact role of gankyrin is poorly understood in animal model systems. This study analyzed hepatic lipid accumulation in gankyrin transgenic (GK) zebrafish. Bromodeoxyuridine (BrdU)-positive cells were predominantly increased in the liver bud of GK larvae, indicating that gankyrin functionally promoted cell proliferation at the larval stage in GK fish. However, over 90% of the viable GK adults showed an increased lipid content, leading in turn to liver steatosis. Liver histology and oil red O staining also indicated the accumulation of fatty droplets in GK fish, consistent with the specific pathological features of severe steatosis. Molecular analysis revealed that gankyrin overexpression induced hepatic steatosis and modulated the expression profiles of four hepatic microRNAs, miR-16, miR-27b, miR-122, and miR-126, and 22 genes involved in lipid metabolism. Moreover, significantly increased hepatic cell apoptosis resulted in liver damage in GK adults, leading to liver failure and death after approximately 10months. This study is the first to report gankyrin as a potential link between microRNAs and liver steatosis in zebrafish.
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Wang G, Rong J, Zhou Z, Duo J. A novel gene P28GANK confers multidrug resistance by modulating the expression of MDR-1, Bcl-2, and bax in osteosarcoma cells. Mol Biol 2010. [DOI: 10.1134/s0026893310060063] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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Iakova P, Timchenko L, Timchenko NA. Intracellular signaling and hepatocellular carcinoma. Semin Cancer Biol 2010; 21:28-34. [PMID: 20850540 DOI: 10.1016/j.semcancer.2010.09.001] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2010] [Revised: 09/05/2010] [Accepted: 09/09/2010] [Indexed: 12/13/2022]
Abstract
Liver cancer is the fifth most common cancer and the third most common cause of cancer related death in the world. The recent development of new techniques for the investigations of global change in the gene expression, signaling pathways and wide genome binding has provided novel information for the mechanisms underlying liver cancer progression. Although these studies identified gene expression signatures in hepatocellular carcinoma, the early steps of the development of hepatocellular carcinomas (HCC) are not well understood. The development of HCC is a multistep process which includes the progressive alterations of gene expression leading to the increased proliferation and to liver cancer. This review summarizes recent progress in the identification of the key steps of the development of HCC with the focus on early events of carcinogenesis and on the role of translational and epigenetic alterations in the development of HCC. Quiescent stage of the liver is supported by several tumor suppressor proteins including p53, Rb and C/EBPα. Studies with chemical models of liver carcinogenesis and with human HCC have shown that the elevation of gankyrin is responsible for the elimination of these three proteins at early steps of carcinogenesis. Later stages of progression of the liver cancer are associated with alterations in many signaling pathways including translation which leads to epigenetic silencing/activation of many genes. Particularly, recent reports suggest a critical role of histone deacetylase 1, HDAC1, in the development of HCC through the interactions with transcription factors such as C/EBP family proteins.
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Affiliation(s)
- Polina Iakova
- Department of Pathology and Immunology and Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, United States
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Abstract
Gankyrin, a newly defined oncoprotein also known as PSMD10 and P28, functions as a dual-negative regulator of the two most prominent tumor suppressor pathways, the CDK/pRb and HDM2/P53 pathways. Its aberrant expression has been prevalently found in human hepatocellular carcinomas (HCC) and esophagus squamous cell carcinomas (ESCC), indicative of the potential of gankyrin as a rational diagnostic and therapeutic target in cancers. Here, we review the unique structural features and functional diversity of gankyrin, and discuss its implication in cancer diagnostics and therapeutics from the perspective of chemical biology.
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Affiliation(s)
- Junan Li
- Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH 43210, USA.,Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
| | - Yi Guo
- Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA
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Kim SY, Hur W, Choi JE, Kim D, Wang JS, Yoon HY, Piao LS, Yoon SK. Functional characterization of human oncoprotein gankyrin in Zebrafish. Exp Mol Med 2009; 41:8-16. [PMID: 19287195 DOI: 10.3858/emm.2009.41.1.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2- hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.
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Affiliation(s)
- So Yeon Kim
- Department of Internal Medicine and WHO Collaborating Center of Viral Hepatitis, The Catholic University of Korea, Seoul 137-701, Korea
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Ni Y, Wang H, Chen F, Li J, DeKeyzer F, Feng Y, Yu J, Bosmans H, Marchal G. Tumor models and specific contrast agents for small animal imaging in oncology. Methods 2009; 48:125-38. [PMID: 19328231 DOI: 10.1016/j.ymeth.2009.03.014] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2009] [Accepted: 03/11/2009] [Indexed: 02/08/2023] Open
Abstract
Despite the widespread use of various imaging modalities in clinical and experimental oncology without or with combined application of commercially available nonspecific contrast agents (CAs), development of tissue- or organ- or disease-specific CAs has been a continuing effort for pursuing ever-improved sensitivity, specificity, and applicability. This is particularly true with magnetic resonance imaging (MRI) due to its intrinsic superb spatial/temporal/contrast resolutions and adequate detectability for tiny amount of substances. In this context, research using small animal tumor models has played an indispensible role in preclinical exploration of tissue specific CAs. Emphasizing more on methodological and practical aspects, this article aims to share our cumulated experiences on how to create tumor models for evaluation and development of new tissue specific MRI CAs and how to apply such models in imaging-based research studies. With the results that are repeatedly confirmed by later clinical applications in cancer patients, some of our early preclinical studies have contributed to the designs of subsequent clinical trials on the new CAs, some studies have predicted new utilities of these CAs; and other studies have led to the discoveries of new tissue- or disease-specific CAs with novel diagnostic or even therapeutic potentials. Among commonly adopted tumor models, the chemically induced and surgically implanted nodules in the liver prove very useful to simulate primary and metastatic intrahepatic tumors, respectively in clinical patients. The methods to create tumor models have eased procedures and yielded high success rates. The specific properties of the new CAs could be outshined by intraindividual comparison to the commercial CAs as nonspecific controls. Meticulous imaging-microangiography-histology matching techniques guaranteed colocalization of the lesion on in vivo MRI and postmortem tissue specimen, hence correct imaging interpretation and longstanding conclusions. As exemplified in the real study cases, the present experimental set-up proves applicable in small animals for imaging-based oncological investigations, and may provide a platform for the currently booming molecular imaging in a multimodality environment.
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Affiliation(s)
- Yicheng Ni
- Section of Radiology, Department of Medical Diagnostic Sciences, Biomedical Sciences Group, University of Leuven, Belgium.
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Li X, Zhang Y, Xiong C, Jin H, Jing B, Zhang Y, Fan D. Overexpression of a new gene P28GANK confers multidrug resistance of gastric cancer cells. Cancer Invest 2009; 27:129-39. [PMID: 19235584 DOI: 10.1080/07357900802189816] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Here we investigated the roles of P28GANK in multidrug resistance of gastric cancer cells and the possible mechanisms. We constructed the siRNA vector of P28GANK and transfected it into human vincristine-resistant gastric adenocarcinoma cell line SGC7901/VCR. Down-regulation of P28GANK could enhance the sensitivity of SGC7901/VCR cells towards anticancer drugs and could decrease the capacity of cells to efflux adriamycin. P28GANK could down-regulate the expression of P-gp, but not affect MRP or GST. In vivo experiment also confirmed our above results. Further study of the biological functions of P28GANK might be helpful for understanding the mechanisms of MDR in gastric cancer.
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Affiliation(s)
- Xiaohua Li
- State Key Laboratory of Cancer Biology, Xijing Hospital, the Fourth Military Medical University, Xi'an, China
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Dual therapeutic effects of interferon-alpha gene therapy in a rat hepatocellular carcinoma model with liver cirrhosis. Mol Ther 2008; 16:1681-1687. [PMID: 18665156 DOI: 10.1038/mt.2008.160] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2008] [Accepted: 07/02/2008] [Indexed: 02/03/2023] Open
Abstract
Human hepatocellular carcinoma (HCC) often arises from a background of liver cirrhosis. Therefore, in order to develop therapeutic strategies for HCC, an animal model bearing multifocal liver tumors accompanied by liver cirrhosis is a preferred experimental setting. In this study, we developed a rapid and reproducible method for generating such a model in rats by weekly administration of diethylnitrosamine (DEN) at doses based on body weight (BW). By adjusting the duration of administration of DEN, the animals could be induced to develop HCC alone, or HCC and liver cirrhosis simultaneously. The latter model was used for evaluating the therapeutic effects of adenoviral delivery of interferon-alpha (IFN-alpha). Our results demonstrated that targeting of IFN-alpha expression to the liver significantly reduced liver tumor volume and ameliorated liver cirrhosis. Mechanistic studies revealed that IFN-alpha gene therapy induced immunomodulatory, antiproliferative, and proapoptotic activities that were effective in the control of tumor growth, and reduced the expressions of transforming growth factor-beta (TGF-beta) and tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to amelioration of liver cirrhosis. These results suggest that IFN-alpha gene therapy is a promising strategy to treat HCC patients who have concomitant liver cirrhosis.
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Umemura A, Itoh Y, Itoh K, Yamaguchi K, Nakajima T, Higashitsuji H, Onoue H, Fukumoto M, Okanoue T, Fujita J. Association of gankyrin protein expression with early clinical stages and insulin-like growth factor-binding protein 5 expression in human hepatocellular carcinoma. Hepatology 2008; 47:493-502. [PMID: 18161051 DOI: 10.1002/hep.22027] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
UNLABELLED Gankyrin (also known as PSMD10) is a liver oncoprotein that interacts with multiple proteins including MDM2 and accelerates degradation of the tumor suppressors p53 and Rb. We produced a monoclonal anti-gankyrin antibody and immunohistochemically assessed the clinicopathological significance of gankyrin overexpression in 43 specimens of human hepatocellular carcinoma (HCC). Specific cytoplasmic staining for gankyrin was observed in 62.8% (27/43) of HCCs, which was significantly associated with low TNM stage (P = 0.004), no capsular invasion (P = 0.018), no portal venous invasion (P = 0.008), and no intrahepatic metastasis (P = 0.012). The cumulative survival rate of patients with gankyrin-positive HCC was significantly higher than that with gankyrin-negative HCC (P = 0.037). p53 and MDM2 were positively stained by antibodies in 30.2% and 23.3%, respectively, of HCCs, but neither was inversely associated with gankyrin expression. In the Huh-7 human HCC cell line, overexpression of gankyrin up-regulated expression of insulin-like growth factor binding protein 5 (IGFBP-5), whereas suppression of gankyrin expression by siRNA down-regulated it. Supression of IGFBP-5 expression inhibited proliferation of Huh-7 cells as well as U-2 OS osteosarcoma cells. In HCC specimens, positive staining for IGFBP-5 was observed by immunohistochemistry in 41.9% (18/43), and the level of expression was significantly correlated with that of gankyrin (rho = 0.629, P < 0.001). CONCLUSION These results suggest that gankyrin plays an oncogenic role(s) mainly at the early stages of human hepatocarcinogenesis, and that IGFBP-5 inducible by gankyrin overexpression may be involved in it.
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Affiliation(s)
- Atsushi Umemura
- Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Muscarella P, Bloomston M, Brewer AR, Mahajan A, Frankel WL, Ellison EC, Farrar WB, Weghorst CM, Li J. Expression of the p16INK4A/Cdkn2a gene is prevalently downregulated in human pheochromocytoma tumor specimens. Gene Expr 2008; 14:207-16. [PMID: 19110720 PMCID: PMC6042001 DOI: 10.3727/105221608786883825] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
A number of hereditary syndromes have been found to be associated with pheochromocytoma development, but there is a paucity of data regarding secondary molecular events, such as downregulation of the p16INK4A/Cdkn2a gene (hereafter p16), contributing to pheochromocytoma tumorigenesis. Using tissue microarray and immunohistochemistry, we evaluated the expression of p16 in 31 pheochromocytoma tumor specimens. Our results showed that the p16 gene was expressed at low level or even not expressed in all but one specimens [30/31 (96.8%)], indicative of the prevalence of p16 downregulation in pheochromocytomas. In contrast, high expression of pl6 was observed in the majority of control "normal" specimens [5/7 (71.6%)]. To further investigate the molecular mechanisms underlying pl6 downregulation in pheochromocytomas, we used quantitative real-time PCR, methylation-specific PCR, and direct DNA sequencing to analyze these specimens for potential genetic alterations of the p16 gene. Deletions and aberrant CpG methylation of pl6 were identified in 9 (29.0%) and 11 (35.5%) specimens, respectively, while one specimen harbored a point mutation, Ala --> Pro at residue 20 of P16, and this mutation led to an eightfold decrease in the CDK4-inhibitory activity of P16. The overall frequency of pl6 genetic alterations is 67.7%. Taken together, our results demonstrate that reduced expression of pl6 is a common event in human pheochromocytomas, and the primary cause for such downregulation is inactivating genetic abnormalities in the p16 gene.
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Affiliation(s)
- Peter Muscarella
- *Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH, USA
- §Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
| | - Mark Bloomston
- *Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH, USA
- §Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
| | - Alexander R. Brewer
- *Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH, USA
| | - Anjali Mahajan
- †Department of Chemistry, The Ohio State University, Columbus, OH, USA
| | - Wendy L. Frankel
- ‡Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH, USA
- §Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
| | - E. Christopher Ellison
- *Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH, USA
- §Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
| | - William B. Farrar
- *Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH, USA
- §Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
| | - Christopher M. Weghorst
- §Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
- ¶Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH, USA
| | - Junan Li
- §Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
- ¶Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH, USA
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Pogribny IP, Rusyn I, Beland FA. Epigenetic aspects of genotoxic and non-genotoxic hepatocarcinogenesis: studies in rodents. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2008; 49:9-15. [PMID: 17879298 PMCID: PMC2705440 DOI: 10.1002/em.20342] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
Hepatocellular carcinoma, which is one of the most prevalent life-threatening human cancers, is showing an increased incidence worldwide. Recent evidence indicates that the development of hepatocellular carcinoma is associated with not only genetic alterations, but also with profound epigenetic changes. This review summarizes the current knowledge about epigenetic alterations during rodent hepatocarcinogenesis, considers the similarities and differences in epigenetic effects of genotoxic and non-genotoxic rodent liver carcinogens, and discusses the possible role of these effects in the causality of liver tumor development.
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Affiliation(s)
- Igor P Pogribny
- Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
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Nitta RT, Smith CL, Kennedy BK. Evidence that proteasome-dependent degradation of the retinoblastoma protein in cells lacking A-type lamins occurs independently of gankyrin and MDM2. PLoS One 2007; 2:e963. [PMID: 17896003 PMCID: PMC1978514 DOI: 10.1371/journal.pone.0000963] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2007] [Accepted: 09/06/2007] [Indexed: 12/11/2022] Open
Abstract
Background A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB) stabilization through unknown mechanism(s). Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna−/− fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna−/− fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16ink4a-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna−/− cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna−/− cells with p14arf. p14arf expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna−/− cells. Conclusions/Significance Our findings suggest that pRB degradation in Lmna−/− cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna−/− fibroblasts. Second, Lmna−/− cells are refractory to p14arf-mediated cell cycle arrest, as was previously shown with p16ink4a. Potential roles of lamin A/C in the suppression of tumorigenesis are discussed.
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Affiliation(s)
- Ryan T. Nitta
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
| | - Catherine L. Smith
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
| | - Brian K. Kennedy
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
- * To whom correspondence should be addressed. E-mail:
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Shan YF, Zhou WP, Fu XY, Yan HX, Yang W, Liu SQ, Cao HF, Kang B, Wu MC, Wang HY. The role of p28GANK in rat oval cells activation and proliferation. Liver Int 2006; 26:240-7. [PMID: 16448463 DOI: 10.1111/j.1478-3231.2005.01203.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
BACKGROUND Human gankyrin gene product (p28GANK) is a novel oncogenic protein ubiquitously overexpressed in hepatocellular carcinoma and also plays a role in cell cycle progression in normal hepatocytes and liver regeneration. However, little is known about the physiological role of p28GANK in the liver oval cell activation and proliferation. We investigated the possible involvement of p28GANK in oval cell-mediated liver regeneration and cell cycle progression. METHODS We examined the different p28GANK expression in 2-acetylaminofuorene/partial heptectomy (2-AAF/PH) rats, as a model of oval cell activation, and PH rats by Western blot and immunohistochemistry. Oval cells isolated from 2-AAF/PH rat model were cultured in our study. p28GANK expression was examined in the oval cells after mitogenic stimulation. RESULTS In 2-AAF/PH rats, p28GANK was expressed in the activated oval cells and located in the nucleus. p28GANK protein expression was increased in 2-AFF/PH rats after hepatectomy lasting for 96 h when retinoblastoma maintained hyperphosphorylation status at Ser-795. The isolated oval cells express AFP, OV6, CK19, CD34, CD45, c-kit and albumin. After epidermal growth factor stimulation, p28GANK protein was up-regulated in oval cells from 24 to 72 h, which coincided with increased expression of CyclinD1, CDK4 and decreased of Rb protein. CONCLUSIONS p28GANK expression was increased in oval cell-mediated liver regeneration and oval cells after mitogenic stimulation. Thus, p28GANK may play a role in oval cell-mediated liver regeneration and liver oval cell cycle progression.
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Affiliation(s)
- Yun-Feng Shan
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Shanghai, China
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Sreepriya M, Bali G. Effects of administration of Embelin and Curcumin on lipid peroxidation, hepatic glutathione antioxidant defense and hematopoietic system during N-nitrosodiethylamine/Phenobarbital-induced hepatocarcinogenesis in Wistar rats. Mol Cell Biochem 2006; 284:49-55. [PMID: 16477385 DOI: 10.1007/s11010-005-9012-7] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2005] [Accepted: 09/21/2005] [Indexed: 12/17/2022]
Abstract
The effects of administration of Embelin (EMB) and Curcumin (CUR) on lipid peroxidation, hepatic glutathione antioxidant defense and hematopoietic cells were examined during N-nitrosodiethylamine (DENA-200 mg kg(-1)body wt, single I.P injection) initiated and Phenobarbital (PB-0.05% in drinking water orally for 13 weeks) promoted hepatocarcinogenesis in Wistar strain male albino rats. DENA/PB-induced hepatic damage was manifested by a significant drop in the hepatic glutathione antioxidant defense, increased lipid peroxidation and histological alterations like dysplasia, and atypical cells with abnormal chromatin pattern. Treatment with Curcumin (100 mg kg(-1)body wt) and Embelin (50 mg kg(-1)body wt) prevented the drop in hepatic glutathione antioxidant defense, decreased lipid peroxidation, minimized the histological alterations induced by DENA/PB, but showed toxic effects on the hematopoietic cells. Results indicate the beneficial effects of Embelin and Curcumin against oxidative tissue damage during chemically-induced hepatocarinogenesis in rats.
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Affiliation(s)
- Meenakshisundaram Sreepriya
- Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharati Campus, Bangalore 560 056, Karnataka, India.
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Sreepriya M, Bali G. Chemopreventive effects of embelin and curcumin against N-nitrosodiethylamine/phenobarbital-induced hepatocarcinogenesis in Wistar rats. Fitoterapia 2005; 76:549-55. [PMID: 16009505 DOI: 10.1016/j.fitote.2005.04.014] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2004] [Accepted: 04/27/2005] [Indexed: 11/28/2022]
Abstract
The effects of embelin (50 mg/kg/day), a benzoquinone derivative of Embelia ribes, and the effects of curcumin (100 mg/kg/day), the active principle of Curcuma longa, against N-nitrosodiethylamine (DENA)-initiated and phenobarbital (PB)-promoted hepatocarcinogenesis were studied in Wistar rats. They were able to prevent the induction of hepatic hyper plastic nodules, body weight loss, increase in the levels of hepatic diagnostic markers, and hypoproteinemia induced by DENA/PB treatment. Hence, results of our study suggest the possible chemopreventive effects of embelin (EMB) and curcumin (CUR) against DENA/PB-induced hepatocarcinogenesis in Wistar rats.
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Affiliation(s)
- M Sreepriya
- Department of Microbiology and Biotechnology, Jnana Bharti Campus, Bangalore University, Bangalore 560 056, Karnataka, India.
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Higashitsuji H, Higashitsuji H, Itoh K, Sakurai T, Nagao T, Sumitomo Y, Sumitomo H, Masuda T, Dawson S, Shimada Y, Mayer RJ, Fujita J. The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53. Cancer Cell 2005; 8:75-87. [PMID: 16023600 DOI: 10.1016/j.ccr.2005.06.006] [Citation(s) in RCA: 179] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/21/2005] [Revised: 04/25/2005] [Accepted: 06/01/2005] [Indexed: 02/08/2023]
Abstract
Gankyrin is an ankyrin repeat oncoprotein commonly overexpressed in hepatocellular carcinomas. Gankyrin interacts with the S6 proteasomal ATPase and accelerates the degradation of the tumor suppressor Rb. We show here that gankyrin has an antiapoptotic activity in cells exposed to DNA damaging agents. Downregulation of gankyrin induces apoptosis in cells with wild-type p53. In vitro and in vivo experiments revealed that gankyrin binds to Mdm2, facilitating p53-Mdm2 binding, and increases ubiquitylation and degradation of p53. Gankyrin also enhances Mdm2 autoubiquitylation in the absence of p53. Downregulation of gankyrin reduced amounts of Mdm2 and p53 associated with the 26S proteasome. Thus, gankyrin is a cofactor that increases the activities of Mdm2 on p53 and probably targets polyubiquitylated p53 into the 26S proteasome.
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Affiliation(s)
- Hiroaki Higashitsuji
- Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan
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Li H, Fu X, Chen Y, Hong Y, Tan Y, Cao H, Wu M, Wang H. Use of adenovirus-delivered siRNA to target oncoprotein p28GANK in hepatocellular carcinoma. Gastroenterology 2005; 128:2029-41. [PMID: 15940635 DOI: 10.1053/j.gastro.2005.03.001] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
BACKGROUND & AIMS RNA interference (RNAi) is a powerful tool to silence gene expression. The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells. However, its potential use as a therapeutic tool to target an oncogene specifically remains to be seen. We applied the adenovirus-delivered siRNA (AdSiRNA) to inhibit a hepatocellular carcinoma (HCC) oncogene, p28GANK, in HCC cell lines and investigated its antitumor effects. METHODS The T7-RNA polymerase system was used to screen the specific target site. Double-strand oligonucleotide for transcription of short hairpin RNA was constructed into the adenoviral vector. Four HCC cell lines were infected with the RNAi-containing adenovirus. The RNAi effects on HCC were studied in cultured cells as well as in animal models. RESULTS p28GANK expression was suppressed by up to 80% in HCC cells. Depletion of p28GANK inhibited HCC cell growth and tumorigenesis, enhanced dephosphorylation of RB1, and decreased transcription activity of E2F-1 in HuH-7 cells. Furthermore, depletion of p28GANK induced caspase-8- and caspase-9-mediated apoptosis of HCC cells. Finally, targeting p28GANK by adenovirus injection inhibited the growth of established tumors in nude mice. CONCLUSIONS This study shows that the T7-system screening-based AdSiRNA can be used successfully to silence an oncogene. We proved the therapeutic potential of AdSiRNA on the treatment of HCC by targeting p28GANK. Our results indicate that p28GANK may serve as a novel therapeutic target for treating HCC.
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Affiliation(s)
- Honghai Li
- International Co-operation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Shanghai, Peoples Republic of China
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Yuan C, Li J, Mahajan A, Poi MJ, Byeon IJL, Tsai MD. Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship. Biochemistry 2004; 43:12152-61. [PMID: 15379554 DOI: 10.1021/bi049116o] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.
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Affiliation(s)
- Chunhua Yuan
- Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
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Abstract
To date, dozens of melanoma-associated antigens (MAGEs) have been identified and classified into 2 subgroups, I and II. Subgroup I consists of antigens which expression is generally restricted to tumor or germ cells, also named as cancer/testis (CT) antigen. Proteins and peptides derived from some of these antigens have been utilized in promising clinical trials of immunotherapies for gastrointestinal carcinoma, esophageal carcinoma, pulmonary carcinoma and so on. Various MAGE family members play important physiological and pathological roles during embryogenesis, germ cell genesis, apoptosis, etc. However, little is known regarding the role of MAGE family members in cell activities. It is reasonable to speculate that the genes for subgroup I MAGEs, which play important roles during embryogenesis, could be later deactivated by a genetic mechanism such as methylation. In the case of tumor formation, these genes are reactivated and the resultant proteins may be recognized and attacked by the immune system. Thus, the subgroup I MAGEs may play important roles in the immune surveillance of certain tumor types. Here, we review the classifications of MAGE family genes and what is known of their biological functions.
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Affiliation(s)
- Jiang Xiao
- Hepatology Institute, People's Hospital, Peking University, Beijing 100044, China
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Fu X, Tan L, Liu S, Li H, Chen L, Qin J, Wu M, Wang H. A novel diagnostic marker, p28GANK distinguishes hepatocellular carcinoma from potential mimics. J Cancer Res Clin Oncol 2004; 130:514-20. [PMID: 15221469 DOI: 10.1007/s00432-004-0562-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2003] [Accepted: 02/22/2004] [Indexed: 12/15/2022]
Abstract
PURPOSE To investigate the sensitivity, specificity, and spatial distribution of the product of p28 gene (p28(GANK) protein) in human hepatocellular carcinoma (HCC) and nonhepatocellular carcinomas in relation to immunostaining with Cytokeratin 18 (CK18), alpha-fetoprotein (AFP), and Hepatocyte paraffin 1 (HepPar1). METHOD In this retrospective study, formalin-fixed paraffin-embedded tissues from 24 HCCs, five intrahepatic cholangiocarcinomas (ICC), five combined hepatocellular cholangiocarcinomas (C-HCC-CC) and mine metastatic hepatic carcinomas (MHC) were immunostained for p28(GANK) as well as CK18, AFP and HepPar1. Only cases with more intensified staining in carcinoma contrast to the adjacent liver tissues were accepted as positive. RESULT In HCC, p28(GANK) was expressed restrictively in hepatocytes of both para-lesion and carcinoma liver tissues, while absent in the bile duct epithelial cells, Kupffer cells, and other interstitial cells. The positive staining of p28(GANK) was noted in 16 (66.7%) specimens of HCC and three (60.0%) specimens of C-HCC-CC, and no specific lesion staining was found in all tested specimens of ICC and MHC. Sensitivity and specificity for hepatocyte-originated carcinoma were, respectively, 65.5% and 100% for p28(GANK), 79.3% and 85.2% for CK18, 20.7% and 100% for AFP, 79.3% and 92.0% for HepPar1. CONCLUSION The hepatocytic staining for p28(GANK) is clearly useful in differentiating hepatocyte-originated carcinoma from non-HCC. p28(GANK) may be used as an ancillary marker for the diagnosis of HCC.
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Affiliation(s)
- Xiaoyong Fu
- International Co-operation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 225 Changhai Road, 200438 Shanghai, P.R. China
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Sakurai T, Itoh K, Higashitsuji H, Nagao T, Nonoguchi K, Chiba T, Fujita J. A Cleaved Form of MAGE-A4 Binds to Miz-1 and Induces Apoptosis in Human Cells. J Biol Chem 2004; 279:15505-14. [PMID: 14739298 DOI: 10.1074/jbc.m310437200] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Gankyrin, a recently discovered oncoprotein, is a promising target for drug therapy because it is overexpressed in most hepatocellular carcinomas. Since gankyrin interacts with MAGE-A4, we made several MAGE-A4 mutants and assessed their effects on cell growth. We found that the C-terminal 107 amino acids of MAGE-A4 (MAGE-A4DeltaN1) induced p53-dependent and p53-independent apoptosis. MAGE-A4DeltaN1 increased the p53 protein level, but decreased the p21(Cip1) transcript and protein levels. During apoptosis Bcl-xL was down-regulated and mitochondrial integrity was disrupted. A yeast two-hybrid screen identified Miz-1 as a MAGE-A4DeltaN1-binding protein. MAGE-A4DeltaN1 was recruited through association with Miz-1 to the p21(Cip1) promoter and down-regulated transcription of p21(Cip1). In 293T cells and U-2 OS cells, full-length MAGE-A4 was processed to generate a C-terminal fragment of 104 amino acids with activities similar to MAGE-A4DeltaN1. Processing was inhibited with a broad range caspase inhibitor Z-VAD-FMK, but not by site-directed mutagenesis of aspartic acids in MAGE-A4, suggesting an indirect involvement of caspase(s) in the processing. The amount of the processed form was increased by exposure of cells to adriamycin. Transduction with a HIV Tat-MAGE-A4DeltaN1 fusion protein suppressed anchorage-independent growth of gankyrin-overexpressing cells in vitro and in vivo. These results demonstrate that the C-terminal fragment of MAGE-A4 induces apoptosis at least partly by binding to Miz-1, and that the fragment may be exploited as an anticancer agent. Furthermore, the finding that a C-terminal fragment with pro-apoptotic activity is generated from full-length MAGE-A4 after genotoxic stress in human cells suggests a novel function for MAGE-A4.
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MESH Headings
- Amino Acid Chloromethyl Ketones/pharmacology
- Amino Acid Sequence
- Animals
- Antigens, Neoplasm/chemistry
- Antigens, Neoplasm/metabolism
- Apoptosis
- COS Cells
- Carcinoma, Hepatocellular/metabolism
- Caspases/metabolism
- Cell Line
- Cell Line, Tumor
- DNA-Binding Proteins/chemistry
- DNA-Binding Proteins/metabolism
- Down-Regulation
- Doxorubicin/pharmacology
- Enzyme Inhibitors/pharmacology
- Female
- Gene Products, tat/metabolism
- Genes, Reporter
- Humans
- In Situ Nick-End Labeling
- Intracellular Membranes/metabolism
- Kruppel-Like Transcription Factors
- Membrane Potentials
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- Mitochondria/metabolism
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- NIH 3T3 Cells
- Neoplasm Proteins
- Plasmids/metabolism
- Proto-Oncogene Proteins c-bcl-2/metabolism
- Recombinant Fusion Proteins/metabolism
- Sequence Homology, Amino Acid
- Time Factors
- Transcription Factors/chemistry
- Transcription Factors/metabolism
- Transfection
- Tumor Suppressor Protein p53/metabolism
- Two-Hybrid System Techniques
- bcl-X Protein
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Affiliation(s)
- Toshiharu Sakurai
- Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan
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Qin JM, Fu XY, Li SJ, Liu SQ, Zeng JZ, Qiu XH, Wu MC, Wang HY. Gene and protein expressions of p28GANK in rat with liver regeneration. World J Gastroenterol 2003; 9:2523-7. [PMID: 14606089 PMCID: PMC4656533 DOI: 10.3748/wjg.v9.i11.2523] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/22/2002] [Revised: 02/01/2003] [Accepted: 02/11/2003] [Indexed: 02/06/2023] Open
Abstract
AIM To observe the gene and protein expression changes of p28GANK in regenerating liver tissues, and to reveal the biological function of p28GANK on the regulation of liver regeneration. METHODS One hundred and thirty two adult male Sprague-Dawley rats were selected, weighing 200-250 g, and divided randomly into sham operation (SO) group and partial hepatectomy (PH) group. Each group had eleven time points: 0, 2, 6, 12, 24, 30, 48, 72, 120, 168 and 240 h, six rats were in each time point. The rats were undergone 70% PH under methoxyflurane anesthesia by resection of the anterior and left lateral lobes of the liver. SO was conducted by laparotomy plus slight mobilization of the liver without resection. Liver specimens were collected at the indicated time points after PH or SO. The expression level of p28GANK mRNA was determined by Northern blot as well as at protein level via immunohistochemical staining. The expressions of p28GANK mRNA in these tissues were analyzed by imaging analysis system of FLA-2000 FUJIFILM and one way analysis of variance. The protein expressions of p28GANK in these tissues were analyzed with Fromowitz' method and Rank sum test. RESULTS The expression of p28GANK mRNA in the regenerating liver tissues possessed two transcripts, which were 1.5 kb and 1.0 kb. There was a significantly different expression patterns of p28GANK mRNA between SO and PH groups (P<0.01). The expression of p28GANK mRNA increased 2 h after PH, the peak time was 72 h (SO group: 163.83+/-1.4720; PH group: 510.5+/-17.0499, P<0.01). There was a significant difference in the 1.5 kb transcript, which decreased gradually after 72 hours. The protein expression of p28GANK was mainly in the cytoplasm of regenerating hepatocytes, and increased near the central region 24 h after PH, and became strongly positive at 48 h (+++, vs the other time points P<0.05), but decreased 72 h after PH. CONCLUSION The expression of p28GANK mRNA increases in the early stage of rat liver regeneration, the protein expression of p28GANK is mainly in the cytoplasm of regenerating liver cells. It suggests that the gene of p28GANK may be an important regulatory and controlled factor involved in hepatocyte proliferation during liver regeneration.
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Affiliation(s)
- Jian-Min Qin
- International Co-operation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, Shanghai, 200438, China
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Krzywda S, Brzozowski AM, Higashitsuji H, Fujita J, Welchman R, Dawson S, Mayer RJ, Wilkinson AJ. The crystal structure of gankyrin, an oncoprotein found in complexes with cyclin-dependent kinase 4, a 19 S proteasomal ATPase regulator, and the tumor suppressors Rb and p53. J Biol Chem 2003; 279:1541-5. [PMID: 14573599 DOI: 10.1074/jbc.m310265200] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Gankyrin is a 25-kDa hepatocellular carcinoma-associated protein that mediates protein-protein interactions in cell cycle control and protein degradation. It has been reported to form complexes with cyclin-dependent kinase 4, retinoblastoma protein, the S6b ATPase subunit of the 19 S regulator of the 26 S proteasome, and Mdm2, an E3 ubiquitin ligase involved in p53 degradation. It is the first protein described to bind both to the 26 S proteasome and to proteins in other complexes containing cyclin-dependent kinase(s) and p53 ubiquitylating activities, thus providing a mechanism for delivering cell cycle regulating machinery and ubiquitylated substrates to the proteasome for degradation. Gankyrin contains a 33-residue motif known as the ankyrin repeat that occurs five and a half to six times in the sequence. As a step toward understanding gankyrin interactions with its protein partners we have determined its three-dimensional crystal structure to 2.0-A resolution. It reveals that the entire 226-residue gankyrin polypeptide folds into seven ankyrin repeat elements. The ankyrin repeats, consisting of an antiparallel beta-hairpin followed by a perpendicularly oriented helix-loop-helix, pack side-by-side, creating an extended curved structure with a groove running across the long concave surface. Comparison with the structures of other ankyrin repeat proteins suggests that interactions with partner proteins are mediated by residues situated on this concave surface.
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Affiliation(s)
- Szymon Krzywda
- Structural Biology Laboratory, University of York, York YO10 5YW, United Kingdom
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42
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Lim IK. Spectrum of molecular changes during hepatocarcinogenesis induced by DEN and other chemicals in Fisher 344 male rats [Mechanisms of Ageing and Development 123 (2002) 1665-1680]. Mech Ageing Dev 2003; 124:697-708. [PMID: 12825548 DOI: 10.1016/s0047-6374(03)00010-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
UNLABELLED Unlike other tissues such as breast, colon and renal cell carcinoma, it is not an easy task to single out any representative oncogene or tumor suppressor genes in the development of hepatocellular carcinoma (HCC), which play a pivotal role. To investigate putatively altered main pathways in HCC, F344 male rats were treated with a single injection of N-nitrosodiethylamine (DEN), followed by either twice/week injections of nodularin for 10 weeks or thioacetamide (TAA) in drinking water for 39 weeks. p53 expression was dramatic in both hepatocytes and mesenchymal cells after a single injection of DEN, however, PCR-SSCP assay could not detect any p53 mutation during the development of hepatocellular adenoma (HCA). The data indicate that wtp53 response was mostly for removal of damaged cells during the initiation of carcinogenesis. When treated with DEN-TAA, induction of gankyrin expression during hepatic fibrosis preceded the loss of pRB protein, accompanied with significant expressions of G1phase cyclins and CDKs. Moreover, p16(INK4A) exon 1 was hypermethylated during the development of poorly differentiated HCCs. These changes would result in complete inactivation of the pRB regulatory pathway during hepatocarcinogenesis. Induction of TGF-beta1 expression with loss of its receptor expression occurred rapidly in the altered hepatocytes by DEN-nodularin treatment. CONCLUSION Therefore, escape from TGF-beta1 induced apoptosis and severe degradation of pRB protein during the early stage of carcinogenesis can perform a symphony to proliferate and to transform the altered hepatocytes to tumor cells. Inactivation of p16(INK4A) and p53 genes at the later stage of carcinogenesis would endow HCC with malignancy, which is highly resistant to any therapeutic trials.
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Affiliation(s)
- In Kyoung Lim
- Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Gyunggi-do, Suwon, South Korea.
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Nagao T, Higashitsuji H, Nonoguchi K, Sakurai T, Dawson S, Mayer RJ, Itoh K, Fujita J. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Biol Chem 2003; 278:10668-74. [PMID: 12525503 DOI: 10.1074/jbc.m206104200] [Citation(s) in RCA: 83] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa. Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas. Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein. The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells. The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth. These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins.
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Affiliation(s)
- Toshikazu Nagao
- Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, Japan
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44
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Pogribny IP, James SJ. De novo methylation of the p16INK4A gene in early preneoplastic liver and tumors induced by folate/methyl deficiency in rats. Cancer Lett 2002; 187:69-75. [PMID: 12359353 DOI: 10.1016/s0304-3835(02)00408-1] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Previous studies have established that chronic dietary insufficiency of the lipotropic nutrients choline and methionine with or without chemical initiation is hepatocarcinogenic in the rat and certain mouse strains. In the present study, the folate/methyl-deficient model of multistage hepatocarcinogenesis was used to evaluate progressive in vivo changes in p16 promoter methylation in both preneoplastic and tumor tissues. Previous studies using this model have demonstrated stage-dependent alterations in genome-wide and p53 gene-specific methylation. In the present study, we used highly sensitive methylation specific PCR (MSP) to determine time of appearance of methylated sequences within p16 promoter. In addition, methylation-sensitive single nucleotide primer extension methodology was applied to determine methylation status of the remaining CpG sites within amplified methylated alleles. Using this approach, extensive methylation in p16 promoter was found in 100% of tumors, but the pattern of methylation varied depending on tumor type. The incidence and extent of de novo methylation in the CpG island of the p16 promoter increased with tumor progression. To further explore the evolution of p16 gene hypermethylation, we examined the appearance and progression of site-specific de novo methylation during early preneoplasia. Our data show that site-specific de novo methylation of 5' CpG island of p16 gene precedes tumor development and undergoes dynamic expansion during tumor progression.
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Affiliation(s)
- Igor P Pogribny
- Division of Biochemical Toxicology, Federal Drug Administration, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA.
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45
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Lim IK. Spectrum of molecular changes during hepatocarcinogenesis induced by DEN and other chemicals in Fischer 344 male rats. Mech Ageing Dev 2002; 123:1665-80. [PMID: 12470904 DOI: 10.1016/s0047-6374(02)00087-8] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
UNLABELLED Unlike other tissues such as breast, colon and renal cell carcinoma, it is not an easy task to single out any representative oncogene or tumor suppressor genes in the development of hepatocellular carcinoma (HCC), which play a pivotal role. To investigate putatively altered main pathways in HCC, F344 male rats were treated with a single injection of N-nitrosodiethylamine (DEN), followed by either twice/week injections of nodularin for 10 weeks or thioacetamide (TAA) in drinking water for 39 weeks. p53 expression was dramatic in both hepatocytes and mesenchymal cells after a single injection of DEN, however, PCR-SSCP assay could not detect any p53 mutation during the development of hepatocellular adenoma. The data indicate that wtp53 response was mostly for removal of damaged cells during the initiation of carcinogenesis. When treated with DEN-TAA, induction of gankyrin expression during hepatic fibrosis preceded the loss of pRB protein, accompanied with significant expressions of G1 phase cyclins and CDKs. Moreover, p16(INK4A) exon 1 was hypermethylated during the development of poorly differentiated HCCs. These changes would result in complete inactivation of the pRB regulatory pathway during hepatocarcinogenesis. Induction of TGF-beta1 expression with loss of its receptor expression occurred rapidly in the altered hepatocytes by DEN-nodularin treatment. CONCLUSION Therefore, escape from TGF-beta1 induced apoptosis and severe degradation of pRB protein during the early stage of carcinogenesis can perform a symphony to proliferate and to transform the altered hepatocytes to tumor cells. Inactivation of p16(INK4A) and p53 genes at the later stage of carcinogenesis would endow HCC with malignancy, which is highly resistant to any therapeutic trials.
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Affiliation(s)
- In Kyoung Lim
- Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 442-721, South Korea.
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Abe M, Okochi E, Kuramoto T, Kaneda A, Takato T, Sugimura T, Ushijima T. Cloning of the 5' upstream region of the rat p16 gene and its role in silencing. Jpn J Cancer Res 2002; 93:1100-6. [PMID: 12417039 PMCID: PMC5926886 DOI: 10.1111/j.1349-7006.2002.tb01211.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Hypermethylation of the 5' upstream region (5' region) of the human p16(CDKN2A) (p16) gene is known to cause silencing, which is involved in a wide range of human cancers. For the rat p16 gene, its 5' region has not been cloned, and it is uncertain whether surrogate use of exon 1 alpha is adequate for analysis of p16 silencing. In this study, we observed that methylation analysis of exon 1 alpha gave false positive results in three samples of normal rat mammary epithelia and in two of six primary mammary carcinomas. Therefore, we determined the nucleotide sequence of the 5' region of the rat p16 gene. To confirm that methylation status of the 5' region is correlated with p16 expression, the methylation status was analyzed by bisulfite sequencing and methylation-specific PCR in three samples of normal mammary glands, six samples of mammary carcinomas and four cell lines. The 5' region was demethylated in all of the three normal and six carcinoma samples that fully expressed p16. On the other hand, the 5' region was highly methylated in the 3Y1 cell line, which lacked p16 expression, but without deletion. These results showed that the methylation status of the 5' region was more closely correlated with p16 expression than that of the exon 1 alpha and analysis of the methylation status is useful in examining p16 silencing in various rat tumors.
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Affiliation(s)
- Masanobu Abe
- Carcinogenesis Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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Dawson S, Apcher S, Mee M, Higashitsuji H, Baker R, Uhle S, Dubiel W, Fujita J, Mayer RJ. Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome. J Biol Chem 2002; 277:10893-902. [PMID: 11779854 DOI: 10.1074/jbc.m107313200] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.
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Affiliation(s)
- Simon Dawson
- Laboratory of Intracellular Proteolysis, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.
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Mizesko MC, Grewe C, Grabner A, Miller MS. Alterations at the Ink4a locus in transplacentally induced murine lung tumors. Cancer Lett 2001; 172:59-66. [PMID: 11595130 DOI: 10.1016/s0304-3835(01)00647-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
The malignant phenotype results from multiple genetic alterations, including the activation of oncogenes and inactivation of tumor suppressor genes. Activation of the Ki-ras oncogene has been implicated as an early event in the pathogenesis of lung adenocarcinomas in humans and experimental animal models. Previous studies from this laboratory have shown that, following treatment of pregnant [D2 x B6D2F(1)]F(2) or Balb/c mice with the polycyclic aromatic hydrocarbon, 3-methylcholanthrene (MC), lung tumors from the transplacentally exposed offspring exhibited a high incidence of mutations in the Ki-ras gene. The role of genetic alterations at other oncogenic or tumor suppressor loci that can mediate lung tumor initiation and/or progression have not been well characterized in either human or murine models. Using the transplacental carcinogenesis model, which results in the induction of both lung and liver tumors following in utero exposure to MC, the results of this and our previous studies show that alterations in the Ink4a locus occur in only 15 and 27% of the lung and liver tumors, respectively. Preliminary data also suggests that the type of mutation induced in the Ki-ras gene following the initial exposure to MC may influence lung tumor progression. These results imply that damage to the Ink4a gene is not a frequent pathway to malignant progression in mouse lung and liver tumors following in utero exposure to environmental carcinogens.
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Affiliation(s)
- M C Mizesko
- Department of Cancer Biology, Comprehensive Cancer Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1082, USA
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