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Sukrithan V, Perez K, Pandit-Taskar N, Jimenez C. Management of metastatic pheochromocytomas and paragangliomas: when and what. Curr Probl Cancer 2024; 51:101116. [PMID: 39024846 DOI: 10.1016/j.currproblcancer.2024.101116] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Accepted: 05/22/2024] [Indexed: 07/20/2024]
Abstract
Recently, the treatment landscape for metastatic pheochromocytomas and paragangliomas (MPPGL) has seen both progress and setbacks. We provide an up-to-date review of the multimodality management of MPPGL and discuss novel opportunities and current challenges in the treatment landscape. Given the unique clinical presentation of MPPGL, we discuss the management of hormone-related clinical sequelae and traditional modalities of therapy. Advances in the understanding of the molecular biology of these diverse tumors have enabled novel strategies such as augmenting DNA damage by targeted delivery of radionuclides such as 131I and 177Lu, abrogating tumor angiogenesis, hypoxia resistance, and DNA damage repair. Despite progress, we address the significant challenges still faced by patients and researchers engaged in efforts to improve outcomes in these rare cancers.
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Affiliation(s)
- Vineeth Sukrithan
- Division of Medical Oncology, Department of Internal Medicine, The Ohio State University Comprehensive Cancer Center, Columbus, OH, United States.
| | - Kimberly Perez
- Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, United States
| | - Neeta Pandit-Taskar
- Molecular Imaging and Therapy Service, Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, United States
| | - Camilo Jimenez
- Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
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Haddad Y, Charousova M, Zivotska H, Splichal Z, Merlos Rodrigo MA, Michalkova H, Krizkova S, Tesarova B, Richtera L, Vitek P, Stokowa-Soltys K, Hynek D, Milosavljevic V, Rex S, Heger Z. Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells. J Nanobiotechnology 2020; 18:95. [PMID: 32660596 PMCID: PMC7359476 DOI: 10.1186/s12951-020-00654-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Accepted: 07/07/2020] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. RESULTS The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% β-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53. CONCLUSION This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.
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Affiliation(s)
- Yazan Haddad
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Marketa Charousova
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Hana Zivotska
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Zbynek Splichal
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Miguel Angel Merlos Rodrigo
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Hana Michalkova
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Sona Krizkova
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Barbora Tesarova
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Lukas Richtera
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Petr Vitek
- Global Change Research Institute of the Czech Academy of Sciences, Belidla 986/4a, 603 00, Brno, Czechia
| | - Kamila Stokowa-Soltys
- Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383, Wrocław, Poland
| | - David Hynek
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Vedran Milosavljevic
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia
| | - Simona Rex
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia.
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia.
| | - Zbynek Heger
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czechia.
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czechia.
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Kortylewicz ZP, Coulter DW, Han G, Baranowska-Kortylewicz J. Norepinephrine-Transporter-Targeted and DNA-Co-Targeted Theranostic Guanidines. J Med Chem 2020; 63:2051-2073. [PMID: 31268317 DOI: 10.1021/acs.jmedchem.9b00437] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
High risk neuroblastoma often recurs, even with aggressive treatments. Clinical evidence suggests that proliferative activities are predictive of poor outcomes. This report describes syntheses, characterization, and biological properties of theranostic guanidines that target norepinephrine transporter and undergo intracellular processing, and subsequently their catabolites are efficiently incorporated into DNA of proliferating neuroblastoma cells. Radioactive guanidines are synthesized from 5-radioiodo-2'-deoxyuridine, a molecular radiotherapy platform with clinically proven minimal toxicities and DNA-targeting properties. The transport of radioactive guanidines into neuroblastoma cells is active as indicated by the competitive suppression of cellular uptake by meta-iodobenzylguanidine. The rate of intracellular processing and DNA uptake is influenced by the agent's catabolic stability and cell population doubling times. The radiotoxicity is directly proportional to DNA uptake and duration of exposure. Biodistribution of 5-[125I]iodo-3'-O-(ε-guanidinohexanoyl)-2'-deoxyuridine in a mouse neuroblastoma model shows significant tumor retention of radioactivity. Neuroblastoma xenografts regress in response to the clinically achievable doses of this agent.
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Affiliation(s)
- Zbigniew P Kortylewicz
- Department of Radiation Oncology, J. Bruce Henriksen Cancer Research Laboratories, University of Nebraska Medical Center, Omaha, Nebraska 68132-6850, United States
| | - Donald W Coulter
- Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska 68132-2168, United States
| | - Guang Han
- Department of Radiation Oncology, J. Bruce Henriksen Cancer Research Laboratories, University of Nebraska Medical Center, Omaha, Nebraska 68132-6850, United States.,Department of Radiation Oncology, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China
| | - Janina Baranowska-Kortylewicz
- Department of Radiation Oncology, J. Bruce Henriksen Cancer Research Laboratories, University of Nebraska Medical Center, Omaha, Nebraska 68132-6850, United States
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Pandit-Taskar N, Modak S. Norepinephrine Transporter as a Target for Imaging and Therapy. J Nucl Med 2017; 58:39S-53S. [PMID: 28864611 DOI: 10.2967/jnumed.116.186833] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2017] [Accepted: 07/19/2017] [Indexed: 01/01/2023] Open
Abstract
The norepinephrine transporter (NET) is essential for norepinephrine uptake at the synaptic terminals and adrenal chromaffin cells. In neuroendocrine tumors, NET can be targeted for imaging as well as therapy. One of the most widely used theranostic agents targeting NET is metaiodobenzylguanidine (MIBG), a guanethidine analog of norepinephrine. 123I/131I-MIBG theranostics have been applied in the clinical evaluation and management of neuroendocrine tumors, especially in neuroblastoma, paraganglioma, and pheochromocytoma. 123I-MIBG imaging is a mainstay in the evaluation of neuroblastoma, and 131I-MIBG has been used for the treatment of relapsed high-risk neuroblastoma for several years, however, the outcome remains suboptimal. 131I-MIBG has essentially been only palliative in paraganglioma/pheochromocytoma patients. Various techniques of improving therapeutic outcomes, such as dosimetric estimations, high-dose therapies, multiple fractionated administration and combination therapy with radiation sensitizers, chemotherapy, and other radionuclide therapies, are being evaluated. PET tracers targeting NET appear promising and may be more convenient options for the imaging and assessment after treatment. Here, we present an overview of NET as a target for theranostics; review its current role in some neuroendocrine tumors, such as neuroblastoma, paraganglioma/pheochromocytoma, and carcinoids; and discuss approaches to improving targeting and theranostic outcomes.
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Affiliation(s)
| | - Shakeel Modak
- Memorial Sloan Kettering Cancer Center, New York, New York
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Grozinsky-Glasberg S, Shimon I, Rubinfeld H. The role of cell lines in the study of neuroendocrine tumors. Neuroendocrinology 2012; 96:173-87. [PMID: 22538498 DOI: 10.1159/000338793] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/20/2010] [Accepted: 04/09/2012] [Indexed: 12/12/2022]
Abstract
Cell lines originating from neuroendocrine tumors (NETs) represent useful experimental models to assess the control of synthesis and release of different hormones and hormone-like peptides, to evaluate the mechanisms of action of these agents in target tissues at the cellular and subcellular levels, and to study cell proliferation and tumor development, as well as the effect of different drugs on these complex processes. To date, the understanding of NET biology (with regard to their mechanisms of hormone secretion, cell proliferation and metastatic spread) has been hampered by the lack of appropriate animal models or cell lines for their study. In the present review, we aim to summarize the recent in vitro/in vivo data regarding cell lines derived from NETs which are most frequently employed in experimental neuroendocrinology.
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Affiliation(s)
- Simona Grozinsky-Glasberg
- Neuroendocrine Tumor Unit, Endocrinology and Metabolism Service, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
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Shah GM, Kandan-Kulangara F, Montoni A, Shah RG, Brind'amour J, Vodenicharov MD, Affar EB. Approaches to detect PARP-1 activation in vivo, in situ, and in vitro. Methods Mol Biol 2011; 780:3-34. [PMID: 21870251 DOI: 10.1007/978-1-61779-270-0_1] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
An accurate and sensitive detection of catalytic activation of poly(ADP-ribose) polymerase-1 (PARP-1) is required to be performed in a wide variety of samples because this activity plays a role in various cellular responses to DNA damage ranging from DNA repair to cell death, as well as in housekeeping functions, such as transcription. Since PARP-1 gene is expressed constitutively, its activation cannot be surmised from increased expression of its mRNA or protein, but by demonstrating the consequences of its catalytic -reaction which results in consumption of the substrate nicotinamide adenine dinucleotide (NAD(+)) and formation of three products, namely, polymer of ADP-ribose (pADPr or PAR), nicotinamide, and protons. Here, we describe various approaches commonly used in our laboratory for detection of PARP-1 activation in vivo (cells, tissues, and tumors), in situ, and in vitro via assessment of formation of pADPr, depletion of the substrate NAD, or formation of protons resulting in rapid and reversible intracellular acidification. It is important to note that although some other members of the PARP family can carry out the same catalytic reaction, many of these assays largely reflect PARP-1 activation in a vast majority of the experimental circumstances and more specifically in DNA damage responses. However, if required, PARP-1-specific action should be confirmed by use of PARP-1 knockout or RNAi-mediated knockdown approaches.
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Affiliation(s)
- Girish M Shah
- Laboratory for Skin Cancer Research, CHUL (CHUQ) Hospital Research Centre of Laval University, Laval University, Québec, Canada
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Binderup T, Knigge U, Mellon Mogensen A, Palnaes Hansen C, Kjaer A. Quantitative gene expression of somatostatin receptors and noradrenaline transporter underlying scintigraphic results in patients with neuroendocrine tumors. Neuroendocrinology 2008; 87:223-32. [PMID: 18196892 DOI: 10.1159/000113128] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2007] [Accepted: 11/20/2007] [Indexed: 11/19/2022]
Abstract
AIM To measure, by a quantitative approach, the gene expression underlying the results of somatostatin receptor (sst) scintigraphy ((111)In-DTPA-octreotide) and noradrenaline transporter (NAT) scintigraphy ((123)I-MIBG) in patients with neuroendocrine (NE) tumors. METHODS The gene expression of somatostatin receptors 1-5 (sst) and NAT was measured quantitatively by real-time PCR in a group of patients with NE tumors (n = 14) and compared to a group of patients with colorectal adenocarcinomas (n = 15). If available, scintigraphic results were compared with gene expression results (9 octreotide and 3 MIBG scintigraphies). RESULTS The sst(2) was upregulated in 13 of 14 patients (93%) with NE tumors, and the absolute level of gene expression was highest for sst(2). Gene expression alterations of NAT and the other sst subtypes were more variable. Gene expression of sst(2) was in all cases in agreement with positive octreotide scintigraphies. In 2 of 3 cases where MIBG scintigraphy was positive, NAT was also upregulated. Sst(2) was generally downregulated in the colorectal tumor group with the gene expression of the other receptors being more heterogeneous. CONCLUSIONS In general, changes in gene expression of sst(2) corresponded with scintigraphic results. Our data support that sst(2) is the best target for visualization of NE tumors, whereas NAT is only a useful target in a subpopulation of NE tumors. Comparison of scintigraphic results with quantitative gene expression may be used to achieve a better understanding of the link between them, which in turn could aid in planning and development of noninvasive molecular imaging of key molecular processes.
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Affiliation(s)
- Tina Binderup
- Cluster for Molecular Imaging, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark.
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8
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Hopfner M, Sutter AP, Huether A, Baradari V, Scherubl H. Tyrosine kinase of insulin-like growth factor receptor as target for novel treatment and prevention strategies of colorectal cancer. World J Gastroenterol 2006; 12:5635-43. [PMID: 17007015 PMCID: PMC4088163 DOI: 10.3748/wjg.v12.i35.5635] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the antineoplastic potency of the novel insulin-like growth factor 1 receptor (IGF-1R) tyrosine kinase inhibitor (TKI) NVP-AEW541 in cell lines and primary cell cultures of human colorectal cancer (CRC).
METHODS: Cells of primary colorectal carcinomas were from 8 patients. Immunostaining and crystal violet staining were used for analysis of growth factor receptor protein expression and detection of cell number changes, respectively. Cytotoxicity was determined by measuring the release of the cytoplasmic enzyme lactate dehydrogenase (LDH). The proportion of apoptotic cells was determined by quantifying the percentage of sub-G1 (hypodiploid) cells. Cell cycle status reflected by the DNA content of the nuclei was detected by flow cytometry.
RESULTS: NVP-AEW541 dose-dependently inhibited the proliferation of colorectal carcinoma cell lines and primary cell cultures by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by caspase-3 activation and nuclear degradation. Cell cycle was arrested at the G1/S checkpoint. The NVP-AEW541-mediated cell cycle-related signaling involved the inactivation of Akt and extracellular signal-regulated kinase (ERK) 1/2, the upregulation of the cyclin-dependent kinase inhibitors p21Waf1/CIP1 and p27Kip1, and the downregulation of the cell cycle promoter cyclin D1. Moreover, BAX was upregulated during NVP-AEW541-induced apoptosis, whereas Bcl-2 was downregulated. Measurement of LDH release showed that the antineoplastic effect of NVP-AEW541 was not due to general cytotoxicity of the compound. However, augmented antineoplastic effects were observed in combination treatments of NVP-AEW541 with either 5-FU, or the EGFR-antibody cetuximab, or the HMG-CoA-reductase inhibitor fluvastatin.
CONCLUSION: IGF-1R-TK inhibition is a promising novel approach for either mono- or combination treatment strategies of colorectal carcinoma and even for CRC chemoprevention.
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MESH Headings
- Adenocarcinoma/drug therapy
- Adenocarcinoma/metabolism
- Adenocarcinoma/pathology
- Adenocarcinoma/prevention & control
- Antibodies, Monoclonal/therapeutic use
- Antibodies, Monoclonal, Humanized
- Antineoplastic Agents/therapeutic use
- Antineoplastic Combined Chemotherapy Protocols/therapeutic use
- Apoptosis/drug effects
- Cell Cycle/drug effects
- Cell Line, Tumor
- Cetuximab
- Colorectal Neoplasms/drug therapy
- Colorectal Neoplasms/metabolism
- Colorectal Neoplasms/pathology
- Colorectal Neoplasms/prevention & control
- Cytotoxins/therapeutic use
- Dose-Response Relationship, Drug
- Fatty Acids, Monounsaturated/therapeutic use
- Fluvastatin
- Gene Expression Regulation, Neoplastic/drug effects
- Humans
- Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use
- Indoles/therapeutic use
- L-Lactate Dehydrogenase/genetics
- L-Lactate Dehydrogenase/metabolism
- Pyrimidines/pharmacology
- Pyrimidines/therapeutic use
- Pyrroles/pharmacology
- Pyrroles/therapeutic use
- Receptor Protein-Tyrosine Kinases/antagonists & inhibitors
- Receptor Protein-Tyrosine Kinases/drug effects
- Receptor Protein-Tyrosine Kinases/genetics
- Receptor Protein-Tyrosine Kinases/metabolism
- Receptor, IGF Type 1/antagonists & inhibitors
- Receptor, IGF Type 1/drug effects
- Receptor, IGF Type 1/genetics
- Receptor, IGF Type 1/metabolism
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Affiliation(s)
- Michael Hopfner
- Klinik fur Gastroenterologie und Gastrointestinale Onkologie, Vivantes-Klinikum Am Urban, Dieffenbachstr. 1, Berlin 10967, Germany
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Abstract
The norepinephrine transporter (NET) terminates noradrenergic signalling by rapid re-uptake of neuronally released norepinephrine (NE) into presynaptic terminals. NET exerts a fine regulated control over NE-mediated behavioural and physiological effects including mood, depression, feeding behaviour, cognition, regulation of blood pressure and heart rate. NET is a target of several drugs which are therapeutically used in the treatment or diagnosis of disorders among which depression, attention-deficit hyperactivity disorder and feeding disturbances are the most common. Individual genetic variations in the gene encoding the human NET (hNET), located at chromosome 16q12.2, may contribute to the pathogenesis of those diseases. An increasing number of studies concerning the identification of single nucleotide polymorphisms in the hNET gene and their potential association with disease as well as the functional investigation of naturally occurring or induced amino acid variations in hNET have contributed to a better understanding of NET function, regulation and genetic contribution to disorders. This review will reflect the current knowledge in the field of NET from its initial discovery until now.
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Affiliation(s)
- H Bönisch
- Department of Pharmacology and Toxicology, University of Bonn, Reuterstr. 2b, 53115 Bonn, Germany.
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Sutter AP, Höpfner M, Huether A, Maaser K, Scherübl H. Targeting the epidermal growth factor receptor by erlotinib (Tarceva) for the treatment of esophageal cancer. Int J Cancer 2006; 118:1814-22. [PMID: 16217753 DOI: 10.1002/ijc.21512] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Because of very poor 5-year survival new therapeutic approaches are mandatory. Erlotinib (Tarceva), an inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), potently suppresses the growth of various tumors but its effect on esophageal carcinoma, known to express EGFR, remains unexplored. We therefore studied the antineoplastic potency of erlotinib in human esophageal cancer cells. Erlotinib induced growth inhibition of the human esophageal squamous cell carcinoma (ESCC) cell lines Kyse-30, Kyse-70 and Kyse-140, and the esophageal adenocarcinoma cell line OE-33, as well as of primary cell cultures of human esophageal cancers. Combining erlotinib with the EGFR-receptor antibody cetuximab, the insulin-like growth factor receptor tyrosine kinase inhibitor tyrphostin AG1024, or the 3-hydroxy-3-methylglutaryl coenzyme. A reductase (HMG-CoAR) inhibitor fluvastatin resulted in additive or even synergistic antiproliferative effects. Erlotinib induced cell cycle arrest at the G1/S checkpoint. The erlotinib-mediated signaling involved the inactivation of EGFR-TK and ERK1/2, the upregulation of the cyclin-dependent kinase inhibitors p21(Waf1/CIP1) and p27(Kip1), and the downregulation of the cell cycle promoter cyclin D1. However, erlotinib did not induce immediate cytotoxicity or apoptosis in esophageal cancer cells. The inhibition of EGFR-TK by erlotinib appears to be a promising novel approach for innovative treatment strategies of esophageal cancer, as it powerfully induced growth inhibition and cell cycle arrest in human esophageal cancer cells and enhanced the antineoplastic effects of other targeted agents.
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Affiliation(s)
- Andreas P Sutter
- Medical Clinic I (Gastroenterology/Infectious Diseases/Rheumatology), Campus Benjamin Franklin, Charité -- Universitätsmedizin Berlin, Berlin, Germany
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Höpfner M, Huether A, Sutter AP, Baradari V, Schuppan D, Scherübl H. Blockade of IGF-1 receptor tyrosine kinase has antineoplastic effects in hepatocellular carcinoma cells. Biochem Pharmacol 2006; 71:1435-48. [PMID: 16530734 DOI: 10.1016/j.bcp.2006.02.006] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2005] [Revised: 01/26/2006] [Accepted: 02/03/2006] [Indexed: 12/14/2022]
Abstract
UNLABELLED Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are mandatory. Most HCCs express insulin-like growth factors and their receptors (IGF-R). As IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for innovative treatment strategies of HCC. Here we studied the antineoplastic effects of inhibiting IGF-1R signaling in HCC cells by the novel IGF-1R tyrosine kinase inhibitor NVP-AEW541. METHODS AND RESULTS NVP-AEW541 induced a time- and dose-dependent growth inhibition in the human hepatoblastoma and hepatocellular carcinoma cell lines SK-Hep-1, Hep-3B, Hep-G2 and Huh-7. Measurement of LDH-release showed that the antineoplastic effect of NVP-AEW541 was not due to cytotoxicity. Instead NVP-AEW541 induced apoptosis as evidenced by both caspase-3 and -8 activation as well as by apoptosis-specific morphological and mitochondrial changes. In addition, nuclear degradation was monitored by DNA-laddering. NVP-AEW541-treatment suppressed the expression of the antiapoptotic proteins Bcl-2 and survivin, while the expression of the proapoptotic protein BAX was stimulated in a dose-dependent manner. Moreover, NVP-AEW541 arrested the cell cycle at the G1/S checkpoint. When NVP-AEW541 was combined with cytotoxic chemotherapy or with a specific epidermal growth factor receptor antibody additive antiproliferative effects were observed. INTERPRETATION Inhibition of IGF-1R tyrosine kinase (IGF-1R-TK) by NVP-AEW541 induces growth inhibition, apoptosis and cell cycle arrest in human HCC cell lines without accompanying cytotoxicity. Thus, IGF-1R-TK inhibition may be a promising novel treatment approach in HCC.
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Affiliation(s)
- Michael Höpfner
- Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, 12200 Berlin, Germany
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12
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Sutter AP, Maaser K, Höpfner M, Huether A, Schuppan D, Scherübl H. Cell cycle arrest and apoptosis induction in hepatocellular carcinoma cells by HMG-CoA reductase inhibitors. Synergistic antiproliferative action with ligands of the peripheral benzodiazepine receptor. J Hepatol 2005; 43:808-16. [PMID: 16083991 DOI: 10.1016/j.jhep.2005.04.010] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2004] [Revised: 03/03/2005] [Accepted: 04/04/2005] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer deaths worldwide. Inhibitors of cholesterol biosynthesis ('statins') have been proposed as promising adjunctive anticancer agents to treat HCC, but their mode of action is yet poorly characterized. We additionally investigated the potential benefit of a combination of peripheral benzodiazepine receptor (PBR) ligands and statins. METHODS We analyzed the growth inhibitory effects of PBR ligands, statins, and their combination in two human HCC cell lines. Moreover, we investigated the regulation of cellular cholesterol levels and the expression of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoAR), the target of statins. RESULTS Statins inhibited the proliferation of HCC cells by inducing apoptosis and G1/S cell cycle arrest. Statin-induced apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase activation and nuclear degradation. Furthermore, activation of ERK1/2 was downregulated while p38MAPK was activated. Synergistic growth inhibition was obtained by the combination of the PBR ligand FGIN-1-27 with statins. PBR ligands induced a decrease of HMG-CoAR expression. This downregulation may be responsible for the enhanced sensitivity of HCC cells to statins. CONCLUSIONS Our data shed light on the signaling cascades mediating statin-induced growth inhibition of HCC cells. Moreover, PBR ligands sensitized HCC cells to statins, suggesting a new strategy to treat HCC.
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Affiliation(s)
- Andreas P Sutter
- Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Berlin, Germany
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Höpfner M, Sutter AP, Huether A, Schuppan D, Zeitz M, Scherübl H. Targeting the epidermal growth factor receptor by gefitinib for treatment of hepatocellular carcinoma. J Hepatol 2004; 41:1008-16. [PMID: 15582135 DOI: 10.1016/j.jhep.2004.08.024] [Citation(s) in RCA: 134] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2004] [Revised: 07/19/2004] [Accepted: 08/17/2004] [Indexed: 12/19/2022]
Abstract
BACKGROUND/AIMS Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are mandatory. Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), potently suppresses the growth of various tumors, but its effect on HCC remains unexplored. We therefore studied the antineoplastic potency of gefitinib in human HCC cells. RESULTS Gefitinib induced a time- and dose-dependent growth inhibition of the human HCC cell lines Huh-7 and HepG2. Gefitinib-treatment induced both mitochondria-dependent and -independent apoptosis. Changes in mitochondrial membrane potential and caspase-8 activation, followed by caspase-3 activation and nuclear degradation, were detected. Moreover, gefitinib induced cell cycle arrest at the G1/S checkpoint and decreased the phosphorylation of mitogen-activated protein kinase ERK1/2. Finally, gefitinib suppressed the expression of antiapoptotic Bcl-2 and Bcl-X(L), further rendering HCC cells prone to apoptosis. CONCLUSIONS Our data demonstrate that the inhibition of EGFR-TK by gefitinib induced growth inhibition, apoptosis and cell cycle arrest in human HCC cells. Thus, EGFR-TK inhibition appears to be a promising novel approach for future treatment strategies of HCC.
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Affiliation(s)
- Michael Höpfner
- Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, Hindenburgdamm 30, 12200 Berlin, Germany
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Sutter AP, Maaser K, Grabowski P, Bradacs G, Vormbrock K, Höpfner M, Krahn A, Heine B, Stein H, Somasundaram R, Schuppan D, Zeitz M, Scherübl H. Peripheral benzodiazepine receptor ligands induce apoptosis and cell cycle arrest in human hepatocellular carcinoma cells and enhance chemosensitivity to paclitaxel, docetaxel, doxorubicin and the Bcl-2 inhibitor HA14-1. J Hepatol 2004; 41:799-807. [PMID: 15519653 DOI: 10.1016/j.jhep.2004.07.015] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2004] [Revised: 06/21/2004] [Accepted: 07/12/2004] [Indexed: 12/12/2022]
Abstract
BACKGROUND/AIMS Hepatocellular carcinoma (HCC) is one of the most common causes of cancer deaths worldwide. Thus, novel therapies are urgently needed. A promising approach is the use of peripheral benzodiazepine receptor (PBR) ligands which inhibit the proliferation of various tumors. METHODS PBR expression both in human HCC cell lines and in tumor specimens of HCC patients was analyzed by RT-PCR and immunostaining. To evaluate PBR ligands for the treatment of HCC, we tested their effects on human HCC cells. RESULTS PBR was localized to the mitochondria both of HCC cell lines and tumor tissues of HCC patients. In contrast, normal liver did not express PBR. PBR ligands inhibited the proliferation of HCC cell lines by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase-3 activation and nuclear degradation. Furthermore, pro-apoptotic Bax was overexpressed while anti-apoptotic Bcl-2 and Bcl-X(L) were suppressed. Cell cycle was arrested both at the G1/S- and G2/M-checkpoints. Synergistic anti-neoplastic effects were obtained by a combination of PBR ligands with cytostatic drugs (paclitaxel, docetaxel, doxorubicin), or with an experimental Bcl-2 inhibitor. CONCLUSIONS This is the first report on the induction of apoptosis and cell cycle arrest by PBR ligands in HCC cells. Moreover, PBR ligands sensitized HCC cells to taxans and doxorubicin.
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Affiliation(s)
- Andreas P Sutter
- Medical Clinic I, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
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Höpfner M, Sutter AP, Huether A, Ahnert-Hilger G, Scherübl H. A novel approach in the treatment of neuroendocrine gastrointestinal tumors: additive antiproliferative effects of interferon-gamma and meta-iodobenzylguanidine. BMC Cancer 2004; 4:23. [PMID: 15154969 PMCID: PMC442128 DOI: 10.1186/1471-2407-4-23] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2003] [Accepted: 05/21/2004] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Therapeutic options to effectively inhibit growth and spread of neuroendocrine gastrointestinal tumors are still limited. As both meta-iodobenzylguanidine (MIBG) and interferon-gamma (IFNgamma) cause antineoplastic effects in neuroendocrine gastrointestinal tumor cells, we investigated the antiproliferative effects of the combination of IFNgamma and non-radiolabeled MIBG in neuroendocrine gut STC-1 and pancreatic carcinoid BON tumor cells. METHODS AND RESULTS IFNgamma receptors were expressed in both models. IFNgamma dose- and time-dependently inhibited the growth of both STC-1 and of BON tumor cells with IC50-values of 95 +/- 15 U/ml and 135 +/- 10 U/ml, respectively. Above 10 U/ml IFNgamma induced apoptosis-specific caspase-3 activity in a time-dependent manner in either cell line and caused a dose-dependent arrest in the S-phase of the cell cycle. Furthermore, IFNgamma induced cytotoxic effects in NE tumor cells. The NE tumor-targeted drug MIBG is selectively taken up via norepinephrine transporters, thereby specifically inhibiting growth in NE tumor cells. Intriguingly, IFNgamma treatment induced an upregulation of norepinephrine transporter expression in neuroendocrine tumors cells, as determined by semi-quantitative RT-PCR. Co-application of sub-IC50 concentrations of IFNgamma and MIBG led to additive growth inhibitory effects, which were mainly due to increased cytotoxicity and S-phase arrest of the cell cycle. CONCLUSION Our data show that IFNgamma exerts antiproliferative effects on neuroendocrine gastrointestinal tumor cells by inducing cell cycle arrest, apoptosis and cytotoxicity. The combination of IFNgamma with the NE tumor-targeted agent MIBG leads to effective growth control at reduced doses of either drug. Thus, the administration of IFNgamma alone and more so, in combination with MIBG, is a promising novel approach in the treatment of neuroendocrine gastrointestinal tumors.
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Affiliation(s)
- Michael Höpfner
- Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Hindenburgdamm 30, 12200 Berlin, Germany
| | - Andreas P Sutter
- Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Hindenburgdamm 30, 12200 Berlin, Germany
| | - Alexander Huether
- Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Hindenburgdamm 30, 12200 Berlin, Germany
| | - Gudrun Ahnert-Hilger
- Charité – Universitätsmedizin Berlin, Campus Mitte, Institut für Anatomie, Philippstrasse 12, 10115 Berlin, Germany
| | - Hans Scherübl
- Charité – Universitätsmedizin Berlin, Campus Mitte, Institut für Anatomie, Philippstrasse 12, 10115 Berlin, Germany
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Sutter AP, Maaser K, Gerst B, Krahn A, Zeitz M, Scherübl H. Enhancement of peripheral benzodiazepine receptor ligand-induced apoptosis and cell cycle arrest of esophageal cancer cells by simultaneous inhibition of MAPK/ERK kinase. Biochem Pharmacol 2004; 67:1701-10. [PMID: 15081869 DOI: 10.1016/j.bcp.2004.01.009] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2003] [Accepted: 01/05/2004] [Indexed: 11/16/2022]
Abstract
Specific ligands of the peripheral benzodiazepine receptor (PBR) activate pro-apoptotic and anti-proliferative signaling pathways. Previously, we found that PBR ligands activated the p38 mitogen-activated protein kinase (MAPK) pathway in esophageal cancer cells, and that the activation of p38MAPK contributed to tumor cell apoptosis and cell cycle arrest. Here, we report that PBR ligands also activate the pro-survival MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in esophageal cancer cells, which might compromise the efficacy of PBR ligands. Hence, a combination treatment of PBR ligands and MEK inhibitors, which are emerging as promising anticancer agents, was pursued to determine whether this treatment could lead to enhanced apoptosis and cell cycle arrest. Using Western blotting we demonstrated a time- and dose-dependent phosphorylation of ERK1/2 in response to PBR ligands. Apoptosis was investigated by assessment of mitochondrial alterations and caspase-3 activity. Cell cycle arrest was measured by flow cytometric analysis of stained isolated nuclei. The inhibition of MEK/ERK with a pharmacologic inhibitor, 2'-amino-3'-methoxyflavone (PD 98059), resulted in a synergistic enhancement of PBR-ligand-induced growth inhibition, apoptosis and cell cycle arrest. Specifity of the pharmacologic inhibitor was confirmed by the use of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 0126), a second MEK/ERK inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 0124), a structural analogue of it which does not display any affinity to MEK. Enhanced pro-apoptotic and anti-proliferative effects were observed both in KYSE-140 esophageal squamous cancer and OE-33 adenocarcinoma cells, suggesting that this effect was not cell-type specific. In addition, the PBR-mediated overexpression of the stress response gene (growth arrest and DNA-damage-inducible gene gadd153) was synergistically enhanced by MEK inhibition. This is the first report of enhanced PBR-ligand-mediated apoptosis and cell cycle arrest by simultaneous MEK inhibition, suggesting a new anticancer strategy.
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Affiliation(s)
- Andreas P Sutter
- Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Berlin, Germany
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Höpfner M, Sutter AP, Gerst B, Zeitz M, Scherübl H. A novel approach in the treatment of neuroendocrine gastrointestinal tumours. Targeting the epidermal growth factor receptor by gefitinib (ZD1839). Br J Cancer 2003; 89:1766-75. [PMID: 14583782 PMCID: PMC2394425 DOI: 10.1038/sj.bjc.6601346] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Therapeutic options to inhibit the growth and spread of neuroendocrine (NE) gastrointestinal tumours are still limited. Since gefitinib (4-(3-chloro-4-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline), an inhibitor of epidermal growth factor receptor-sensitive tyrosine kinase (EGFR-TK), had been shown to suppress potently the growth of various non-NE tumour entities, we studied the antineoplastic potency of gefitinib in NE gastrointestinal tumour cells. In human insulinoma (CM) cells, in human pancreatic carcinoid (BON) cells and in NE tumour cells of the gut (STC-1), gefitinib induced a time- and dose-dependent growth inhibition by almost 100%. The antiproliferative potency of gefitinib correlated with the proliferation rate of the tumour cells. So the IC50 value of gefitinib was 4.7±0.6 μM in the fast-growing CM cells, still 16.8±0.4 μM in the moderate-growing BON cells, and up to 31.5±2.5 μM in the slow-growing STC-1 cells. Similarly, the induction of apoptosis and cell-cycle arrest by gefitinib differed according to growth characteristics: fast-growing CM cells displayed a strong G0/G1 arrest in response to gefitinib, while no significant cell-cycle alterations were seen in the slow-growing STC-1. Vice versa, the proapoptotic effects of gefitinib, as determined by caspase-3 activation and DNA fragmentation, were most pronounced in the slow-growing STC-1 cells. Using cDNA microarrays, we found extensive changes in the expression of genes involved in the regulation of apoptosis and cell cycle after incubation with gefitinib. Among them, an upregulation of the growth arrest and DNA damage-inducible gene GADD153 was observed. Phosphorylation of ERK1/2, which inhibits GADD153 expression, was reduced in a time-dependent manner. However, no gefitinib-induced activation of the GADD153-inducing p38 mitogen-activated protein kinase was detected. Our data demonstrate that the inhibition of EGFR-TK by gefitinib induces growth inhibition, apoptosis and cell-cycle arrest in NE gastrointestinal tumour cells. Thus, EGFR-TK inhibition appears to be a promising novel approach for the treatment of NE tumour disease.
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Affiliation(s)
- M Höpfner
- Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
| | - A P Sutter
- Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
| | - B Gerst
- Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
| | - M Zeitz
- Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
| | - H Scherübl
- Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
- Medical Clinic I, Gastroenterology/Infectious Diseases/Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail:
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Sutter AP, Maaser K, Barthel B, Scherübl H. Ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in oesophageal cancer cells: involvement of the p38MAPK signalling pathway. Br J Cancer 2003; 89:564-72. [PMID: 12888831 PMCID: PMC2394363 DOI: 10.1038/sj.bjc.6601125] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2003] [Revised: 04/30/2003] [Accepted: 05/07/2003] [Indexed: 12/31/2022] Open
Abstract
Specific ligands of the peripheral benzodiazepine receptor (PBR) are known to induce apoptosis and cell cycle arrest in oesophageal cancer cells. However, the underlying mechanisms are still unknown. Here, we investigated the transcriptional alterations and activation of protein kinases in response to PBR-specific ligands. Using cDNA arrays, we examined the transcriptional effects of the PBR-specific ligand FGIN-1-27 in two oesophageal cancer cell lines, KYSE-140 (squamous cell carcinoma) and OE-33 (adenocarcinoma). In oesophageal cancer cells, FGIN-1-27 induced extensive changes in the expression of genes involved in the regulation of apoptosis and cell cycle. Both in oesophageal cancer cell lines (KYSE-140, OE-33) we observed a strong upregulation of the growth arrest and DNA-damage-inducible genes, gadd45 and gadd153, in response to PBR ligands. gadd genes are known to be induced by p38MAPK activation. Using Western blotting we detected a time- and dose-dependent phosphorylation of p38MAPK, which was found to be functionally involved in gadd induction, apoptosis, and cell cycle arrest. In conclusion, our data indicate that PBR-specific ligands cause apoptosis and cell cycle arrest by activation of the p38MAPK pathway and induction of gadd45 and gadd153.
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Affiliation(s)
- A P Sutter
- Medical Clinic I,
Gastroenterology, Infectious Diseases, Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
| | - K Maaser
- Medical Clinic I,
Gastroenterology, Infectious Diseases, Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
| | - B Barthel
- Medical Clinic I,
Gastroenterology, Infectious Diseases, Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
| | - H Scherübl
- Medical Clinic I,
Gastroenterology, Infectious Diseases, Rheumatology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
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