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Maines LW, Keller SN, Smith RA, Schrecengost RS, Smith CD. Opaganib Downregulates N-Myc Expression and Suppresses In Vitro and In Vivo Growth of Neuroblastoma Cells. Cancers (Basel) 2024; 16:1779. [PMID: 38730731 PMCID: PMC11082966 DOI: 10.3390/cancers16091779] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Revised: 04/25/2024] [Accepted: 04/29/2024] [Indexed: 05/13/2024] Open
Abstract
Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody.
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Affiliation(s)
| | | | | | | | - Charles D. Smith
- Apogee Biotechnology Corporation, 1214 Research Blvd, Suite 2015, Hummelstown, PA 17036, USA
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2
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"Dicing and Splicing" Sphingosine Kinase and Relevance to Cancer. Int J Mol Sci 2017; 18:ijms18091891. [PMID: 28869494 PMCID: PMC5618540 DOI: 10.3390/ijms18091891] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2017] [Revised: 08/29/2017] [Accepted: 08/29/2017] [Indexed: 02/06/2023] Open
Abstract
Sphingosine kinase (SphK) is a lipid enzyme that maintains cellular lipid homeostasis. Two SphK isozymes, SphK1 and SphK2, are expressed from different chromosomes and several variant isoforms are expressed from each of the isozymes, allowing for the multi-faceted biological diversity of SphK activity. Historically, SphK1 is mainly associated with oncogenicity, however in reality, both SphK1 and SphK2 isozymes possess oncogenic properties and are recognized therapeutic targets. The absence of mutations of SphK in various cancer types has led to the theory that cancer cells develop a dependency on SphK signaling (hyper-SphK signaling) or “non-oncogenic addiction”. Here we discuss additional theories of SphK cellular mislocation and aberrant “dicing and splicing” as contributors to cancer cell biology and as key determinants of the success or failure of SphK/S1P (sphingosine 1 phosphate) based therapeutics.
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3
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Dong Y, Li M, Wang S, Dong Y, Zhao H, Dai Z. Xingshentongqiao decoction mediates proliferation, apoptosis, orexin-A receptor and orexin-B receptor messenger ribonucleic acid expression and represses mitogen-activated protein kinase signaling. Chin Med J (Engl) 2015; 128:98-104. [PMID: 25563321 PMCID: PMC4837828 DOI: 10.4103/0366-6999.147826] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
BACKGROUND Hypocretin (HCRT) signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ) is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R]) expression. The signaling pathways involved in these processes were also assessed. METHODS The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK) pathway activation was performed to further assess the signaling mechanism of XSTQ. RESULTS XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk) 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK). CONCLUSIONS XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.
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Affiliation(s)
| | | | - Shaojie Wang
- Department of Traditional Chinese Medicine, Peking University People's Hospital, Beijing 100044, China
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4
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Abstract
The topic of ceramidases has experienced an enormous boost during the last few years. Ceramidases catalyze the degradation of ceramide to sphingosine and fatty acids. Ceramide is not only the central hub of sphingolipid biosynthesis and degradation, it is also a key molecule in sphingolipid signaling, promoting differentiation or apoptosis. Acid ceramidase inhibition sensitizes certain types of cancer to chemo- and radio-therapy and this is suggestive of a role of acid ceramidase inhibitors as chemo-sensitizers which can act synergistically with chemo-therapeutic drugs. In this review, we summarize the development of ceramide analogues as first-generation ceramidase inhibitors together with data on their activity in cells and disease models. Furthermore, we describe the recent developments that have led to highly potent second-generation ceramidase inhibitors that act at nanomolar concentrations. In the third part, various assays of ceramidases are described and their relevance for accurately measuring ceramidase activities and for the development of novel inhibitors is highlighted. Besides potential clinical implications, the recent improvements in ceramidase inhibition and assaying may help to better understand the mechanisms of ceramide biology.
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Affiliation(s)
- Essa M Saied
- Humboldt Universität zu Berlin, Institute for Chemistry, Berlin, Germany; Suez Canal University, Chemistry Department, Faculty of Science, Ismailia, Egypt
| | - Christoph Arenz
- Humboldt Universität zu Berlin, Institute for Chemistry, Berlin, Germany.
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5
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Gao Y, Gao F, Chen K, Tian ML, Zhao DL. Sphingosine kinase 1 as an anticancer therapeutic target. DRUG DESIGN DEVELOPMENT AND THERAPY 2015; 9:3239-45. [PMID: 26150697 PMCID: PMC4484649 DOI: 10.2147/dddt.s83288] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The development of chemotherapeutic resistance is a major challenge in oncology. Elevated sphingosine kinase 1 (SK1) levels is predictive of a poor prognosis, and SK1 overexpression may confer resistance to chemotherapeutics. The SK/sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor (S1PR) signaling pathway has been implicated in the progression of various cancers and in chemotherapeutic drug resistance. Therefore, SK1 may represent an important target for cancer therapy. Targeting the SK/S1P/S1PR signaling pathway may be an effective anticancer therapeutic strategy, particularly in the context of overcoming drug resistance. This review summarizes our current understanding of the role of SK/S1P/S1PR signaling in cancer and development of SK1 inhibitors.
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Affiliation(s)
- Ying Gao
- Department of Radiotherapy Oncology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, People's Republic of China
| | - Fei Gao
- Department of Neurology, First Affiliated Hospital of Xi'an Medical University, Xi'an, People's Republic of China
| | - Kan Chen
- School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, People's Republic of China
| | - Mei-li Tian
- Department of Radiotherapy Oncology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, People's Republic of China
| | - Dong-li Zhao
- Department of Radiotherapy Oncology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, People's Republic of China
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Muñoz-Sáez E, de Munck García E, Arahuetes Portero RM, Vicente F, Ortiz-López FJ, Cantizani J, Gómez Miguel B. Neuroprotective role of sphingosine-1-phosphate in L-BMAA treated neuroblastoma cells (SH-SY5Y). Neurosci Lett 2015; 593:83-9. [PMID: 25769802 DOI: 10.1016/j.neulet.2015.03.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2014] [Revised: 02/14/2015] [Accepted: 03/06/2015] [Indexed: 10/23/2022]
Abstract
Sphingosine-1-phosphate (S1P) is a bioactive lipid which regulates proliferation, cell migration, survival and differentiation by specific receptors activation. We studied its effects on L-BMAA treated neuroblastoma cells (SH-SY5Y), an amino acid that can trigger neurodegenerative diseases such as amyotrophic lateral sclerosis/Parkinson dementia complex (ALS/PDC). We found that S1P protects from necrosis and prevents the GSK3 increasing as long as the PI3K/AKT pathway is active. Moreover, GSK3 inhibition protects against neuronal death caused by L-BMAA.
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Affiliation(s)
- Emma Muñoz-Sáez
- Departamento de Bioquímica y Biología Molecular-I, Universidad Complutense de Madrid, 28040-Madrid, Spain.
| | | | | | - Francisca Vicente
- Fundación MEDINA, Centro Excelencia Investigación Medicamentos Innovadores en Andalucía, Parque Tecnológico Ciencias de la Salud, 18016-Armilla-Granada, Spain
| | - Francisco Javier Ortiz-López
- Fundación MEDINA, Centro Excelencia Investigación Medicamentos Innovadores en Andalucía, Parque Tecnológico Ciencias de la Salud, 18016-Armilla-Granada, Spain
| | - Juan Cantizani
- Fundación MEDINA, Centro Excelencia Investigación Medicamentos Innovadores en Andalucía, Parque Tecnológico Ciencias de la Salud, 18016-Armilla-Granada, Spain
| | - Begoña Gómez Miguel
- Departamento de Bioquímica y Biología Molecular-I, Universidad Complutense de Madrid, 28040-Madrid, Spain
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7
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Zhu W, Wang X, Zhou Y, Wang H. C2-ceramide induces cell death and protective autophagy in head and neck squamous cell carcinoma cells. Int J Mol Sci 2014; 15:3336-55. [PMID: 24566153 PMCID: PMC3958915 DOI: 10.3390/ijms15023336] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2013] [Revised: 01/20/2014] [Accepted: 02/11/2014] [Indexed: 12/19/2022] Open
Abstract
Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others. The aim of this study was to evaluate the anti-tumor efficacy of C2-ceramide (C2-Cer; N-acetyl-d-sphingosine) by investigating cell death and autophagy in head and neck squamous cell carcinoma (HNSCC) cells. C2-Cer showed concentration-dependent cytotoxicity in HN4 and HN30 cell lines. It simultaneously induced caspase-3-independent apoptosis and programmed necrosis. C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy. An autophagy inhibitor enhanced C2-Cer-mediated cytotoxicity, while a programmed-necrosis inhibitor produced the opposite effect. Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process. These results suggested that C2-Cer exerts anti-tumor effects by inducing programmed apoptosis and necrosis in HNSCC, and these cytotoxic effects are enhanced by an autophagy inhibitor.
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Affiliation(s)
- Wenyuan Zhu
- Department of Oral and Maxillofacial Surgery, the First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China.
| | - Xinhua Wang
- Department of Oral Implantology, the Affiliated Hospital of Stomatology, College of Medicine, Zhejiang University, Hangzhou 310003, China.
| | - Yi Zhou
- Department of Oral Implantology, the Affiliated Hospital of Stomatology, College of Medicine, Zhejiang University, Hangzhou 310003, China.
| | - Huiming Wang
- Department of Oral and Maxillofacial Surgery, the First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China.
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8
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Abstract
The role of sphingolipids as bioactive signaling molecules that can regulate cell fate decisions puts them at center stage for cancer treatment and prevention. While ceramide and sphingosine have been established as antigrowth molecules, sphingosine-1-phosphate (S1P) offers a progrowth message to cells. The enzymes responsible for maintaining the balance between these "stop" or "go" signals are the sphingosine kinases (SK), SK1 and SK2. While the relative contribution of SK2 is still being elucidated and may involve an intranuclear role, a substantial amount of evidence suggests that regulation of sphingolipid levels by SK1 is an important component of carcinogenesis. Here, we review the literature regarding the role of SK1 as an oncogene that can function to enhance cancer cell viability and promote tumor growth and metastasis; highlighting the importance of developing specific SK1 inhibitors to supplement current cancer therapies.
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Affiliation(s)
- Linda A Heffernan-Stroud
- Molecular and Cellular Biology and Pathobiology Program, Medical University of South Carolina, Charleston, SC, USA
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9
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Bini F, Frati A, Garcia-Gil M, Battistini C, Granado M, Martinesi M, Mainardi M, Vannini E, Luzzati F, Caleo M, Peretto P, Gomez-Muñoz A, Meacci E. New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase. Neuropharmacology 2012; 63:524-37. [DOI: 10.1016/j.neuropharm.2012.04.026] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2012] [Revised: 04/06/2012] [Accepted: 04/21/2012] [Indexed: 01/12/2023]
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10
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Nakamura H, Tada E, Makiyama T, Yasufuku K, Murayama T. Role of cytosolic phospholipase A2α in cell rounding and cytotoxicity induced by ceramide-1-phosphate via ceramide kinase. Arch Biochem Biophys 2011; 512:45-51. [DOI: 10.1016/j.abb.2011.05.006] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2011] [Revised: 05/12/2011] [Accepted: 05/15/2011] [Indexed: 11/26/2022]
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11
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Makiyama T, Nagasaka N, Houjyo Y, Yamaura E, Nakamura H, Koide Y, Nishida A, Murayama T. Newly synthetic ceramide-1-phosphate analogs; their uptake, intracellular localization, and roles as an inhibitor of cytosolic phospholipase A2α and inducer of cell toxicity. Biochem Pharmacol 2010; 80:1396-406. [DOI: 10.1016/j.bcp.2010.07.028] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2010] [Revised: 07/17/2010] [Accepted: 07/23/2010] [Indexed: 01/21/2023]
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12
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Abstract
BACKGROUND The sphingolipids ceramide and sphingosine 1-phosphate (S1P) are key regulators of cell death and proliferation. The subtle balance between their intracellular levels is governed mainly by sphingosine kinase-1, which produces the pro-survival S1P. Sphingosine kinase-1 is an oncogene; is overexpressed in many tumors; protects cancer cells from apoptosis in vitro and in vivo; and its activity is decreased by anticancer therapies. Hence, sphingosine kinase-1 appears to be a target of interest for therapeutic manipulation. OBJECTIVE This review considers recent developments regarding the involvement of sphingosine kinase-1 as a therapeutic target for cancer, and describes the pharmacological tools currently available. RESULTS/CONCLUSION The studies described provide strong evidence that strategies to kill cancer cells via sphingosine kinase-1 inhibition are valid and could have a favorable therapeutic index.
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Affiliation(s)
- Olivier Cuvillier
- Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex 4, France.
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13
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Schmalzbauer R, Eigenbrod S, Winoto-Morbach S, Xiang W, Schtze S, Bertsch U, Kretzschmar HA. Evidence for an association of prion protein and sphingolipid-mediated signaling. J Neurochem 2008; 106:1459-70. [DOI: 10.1111/j.1471-4159.2008.05498.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Kim HJ, Oh JE, Kim SW, Chun YJ, Kim MY. Ceramide induces p38 MAPK-dependent apoptosis and Bax translocation via inhibition of Akt in HL-60 cells. Cancer Lett 2007; 260:88-95. [PMID: 18054155 DOI: 10.1016/j.canlet.2007.10.030] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2007] [Revised: 10/16/2007] [Accepted: 10/17/2007] [Indexed: 11/27/2022]
Abstract
Ceramide induces apoptosis through caspase activation, cytochrome c release, and Bax translocation in HL-60 cells. However, the upstream signal transduction pathways that induce Bax translocation during ceramide-mediated apoptosis have not been well defined yet. In this study, the activation of p38 mitogen-activated protein kinase (MAPK) was found to be critical for the induction of apoptosis and subcellular redistribution of Bax. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a dominant-negative p38 MAPK attenuated DNA fragmentation, caspase-3 activation, and Bax translocation in response to ceramide. Overexpression of Akt also led to suppression of Bax translocation to mitochondria during ceramide-induced apoptosis in HL-60 cells. We also provide evidence for cross-talk between p38 MAPK and Akt pathways. Expression of myr-Akt or inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 had no effect on p38 MAPK activation by ceramide as assessed by phosphorylation, while inhibition of p38 MAPK by a pharmacological inhibitor or a dominant-negative p38 inhibited Akt dephosphorylation in response to ceramide, suggesting that ceramide-induced p38 MAPK activation negatively regulates the Akt pathway.
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Affiliation(s)
- Hae Jong Kim
- Division of Biochemistry, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
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15
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Abstract
Sphingolipid metabolites play critical functions in the regulation of a number of fundamental biological processes including cancer. Whereas ceramide and sphingosine mediate and trigger apoptosis or cell growth arrest, sphingosine 1-phosphate promotes proliferation and cell survival. The delicate equilibrium between the intracellular levels of each of these sphingolipids is controlled by the enzymes that either produce or degrade these metabolites. Sphingosine kinase-1 is a crucial regulator of this two-pan balance, because it produces the prosurvival sphingosine 1-phosphate, and reduces the content of both ceramide and sphingosine, the proapoptotic sphingolipids. Sphingosine kinase-1 controls the levels of sphingolipids having opposite effects on cell survival/death, its gene was found to be of oncogenic nature, its mRNA is overexpressed in many solid tumors, its overexpression protects cells from apoptosis and its activity is decreased during anticancer treatments. Therefore, sphingosine kinase-1 appears to be a target of interest for therapeutic manipulation via its pharmacological inhibition. Strategies to kill tumor cells by increasing their ceramide and/or sphingosine content while blocking sphingosine 1-phosphate generation should have a favorable therapeutic index.
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Affiliation(s)
- Olivier Cuvillier
- Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, 205 route de Narbonne, 31077 Toulouse, France.
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Wang J, Lv XW, Shi JP, Hu XS. Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells. World J Gastroenterol 2007; 13:1129-34. [PMID: 17373752 PMCID: PMC4146880 DOI: 10.3748/wjg.v13.i7.1129] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [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 effect of ceramide on the cell cycle in human hepatocarcinoma Bel7402 cells. Possible molecular mechanisms were explored.
METHODS: [3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, plasmid transfection, reporter assay, FACS and Western blotting analyses were employed to investigate the effect and the related molecular mechanisms of C2-ceramide on the cell cycle of Bel7402 cells.
RESULTS: C2-ceramide was found to inhibit the growth of Bel7402 cells by inducing cell cycle arrest. During the process, the expression of p21 protein increased, while that of cyclinD1, phospho-ERK1/2 and c-myc decreased. Furthermore, the level of CDK7 was downregulated, while the transcriptional activity of PPARγ was upregulated. Addition of GW9662, which is a PPARγ specific antagonist, could reserve the modulation action on CDK7.
CONCLUSION: Our results support the hypothesis that cell cycle arrest induced by C2-ceramide may be mediated via accumulation of p21 and reduction of cyclinD1 and CDK7, at least partly, through PPARγ activation. The ERK signaling pathway was involved in this process.
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Affiliation(s)
- Jing Wang
- Research Center for Eco-Environmental Sciences, The Chinese Academy of Sciences, Haidian District, Beijing 100085, China.
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17
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Mitra P, Maceyka M, Payne SG, Lamour N, Milstien S, Chalfant CE, Spiegel S. DNA polymerase beta catalytic efficiency mirrors the Asn279-dCTP H-bonding strength. FEBS Lett 2007; 581:735-40. [PMID: 17274985 DOI: 10.1016/j.febslet.2007.01.041] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2007] [Accepted: 01/17/2007] [Indexed: 11/20/2022]
Abstract
Ternary complexes of wild type or mutant form of human DNA polymerase beta (pol beta) bound to DNA and dCTP substrates were studied by molecular dynamics (MD) simulations. The occurrences of contact configurations (CC) of structurally important atom pairs were sampled along the MD trajectories, and converted into free-energy differences, DeltaG(CC). DeltaG(CC) values were correlated with the experimental binding and catalytic free energies for the wild type pol beta and its Arg183Ala, Tyr271Ala, Asp276Val, Lys280Gly, Arg283Ala, and Glu295Ala mutants. The correlation coefficients show that the strength of the H-bond between dCTP and Asn279 is a strong predictor of the mutation-induced changes in the catalytic efficiency of pol beta. This finding is consistent with the view that enzyme preorganization plays a major role in controlling DNA polymerase specific activity.
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Affiliation(s)
- Poulami Mitra
- Department of Biochemistry and Massey Cancer Center, Virginia Commonwealth University School of Medicine, 2-011 Sanger Hall, 1101 E. Marshall Street, Richmond, VA 23298-0614, USA
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18
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Posse de Chaves EI. Sphingolipids in apoptosis, survival and regeneration in the nervous system. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2006; 1758:1995-2015. [PMID: 17084809 DOI: 10.1016/j.bbamem.2006.09.018] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/14/2006] [Revised: 09/20/2006] [Accepted: 09/21/2006] [Indexed: 12/27/2022]
Abstract
Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.
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Affiliation(s)
- Elena I Posse de Chaves
- Centre for Alzheimer and Neurodegenerative Research, Signal Transduction Research Group and Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
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Kong JY, Klassen SS, Rabkin SW. Ceramide activates a mitochondrial p38 mitogen-activated protein kinase: a potential mechanism for loss of mitochondrial transmembrane potential and apoptosis. Mol Cell Biochem 2006; 278:39-51. [PMID: 16180087 DOI: 10.1007/s11010-005-1979-6] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2004] [Accepted: 02/03/2005] [Indexed: 10/25/2022]
Abstract
This study examined the impact of ceramide, an intracellular mediator of apoptosis, on the mitochondria to test the hypothesis that ceramide utilized p38 MAPK in the mitochondria to alter mitochondrial potential and induce apoptosis. The capacity of ceramide to adversely affect mitochondria was demonstrated by the significant loss of mitochondrial potential (DeltaPsim), indicated by a J-aggregate fluorescent probe, after embryonic chick cardiomyocytes were treated with the cell permeable ceramide analogue C2-ceramide. p38 MAPK was identified in the mitochondrial fraction of the cell and p38 MAPK phosphorylation in this mitochondrial fraction of the cell occurred with ceramide treatment. In addition, SAPK phosphorylation and a decrease in ERK phosphorylation occurred in whole cell lysates after ceramide treatment. The p38 MAPK inhibitor SB 202190 but not the MEK inhibitor PD 98059 significantly inhibited ceramide-induced apoptosis and loss of DeltaPsim. These data suggest that p38 MAPK is present in the mitochondria and its activation by ceramide indicates local signaling more directly coupled to the mitochondrial pathway in apoptosis.
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Affiliation(s)
- Jennifer Y Kong
- Department of Medicine (Cardiology), University of British Columbia, Vancouver, BC, Canada
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Saito M, Saito M, Cooper TB, Vadasz C. Ethanol-Induced Changes in the Content of Triglycerides, Ceramides, and Glucosylceramides in Cultured Neurons. Alcohol Clin Exp Res 2005; 29:1374-83. [PMID: 16131844 DOI: 10.1097/01.alc.0000175011.22307.61] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Ethanol induces apoptosis in cultured neurons. To assess the involvement of sphingolipids and neutral lipids in the apoptotic process, ethanol-induced alterations in lipid content and metabolism were examined by using primary cultured rat cerebellar granule neurons (CGNs), human neuroblastoma SK-N-SH cells, and mouse neuroblastoma Neuro2a cells. Ethanol treatment conditions that induced apoptosis in CGNs and SK-N-SH cells but not in Neuro2a cells were used for these experiments. METHODS Cultured neurons were treated with and without 100 mM ethanol for one to three days, and the amounts of cellular sphingolipids [ceramide, glucosylceramide (GlcCer), and sphingomyelin] and neutral lipids [cholesterol, triglyceride (TG), and cholesterol ester (ChE)] were analyzed by high-performance thin-layer chromatography, using a Coomassie brilliant blue staining method. The incorporation of [C] acetate into each lipid fraction was measured in CGNs treated with and without ethanol. Also, the effect of delipidated serum, sterols, myriocin (a serine-palmitoyltransferase inhibitor), and desipramine (an acid sphingomyelinase inhibitor) on ethanol-induced lipid changes was studied by using Neuro2a cells. RESULTS The most prominent change common to CGN, SK-N-SH, and Neuro2a cells was ethanol-induced TG accumulation. Higher incorporation of radioactivity into TG was also observed in ethanol-treated cultures when cellular lipids were metabolically labeled with [C] acetate in CGNs. In addition, ethanol elevated ceramide levels in all these neurons. However, ethanol induced decreases in GlcCer along with the reduction of cell viability in SK-N-SH cells and CGNs, whereas it increased GlcCer in Neuro2a cells that remained viable. Myriocin, which reduced ceramide levels, attenuated ethanol-induced cell death in SK-N-SH cells. Ethanol-induced accumulation of TG was sterol-independent, whereas changes in ceramide and GlcCer were affected in Neuro2a cells by the presence of sterols in the medium. Staurosporine, which induced cell death in SK-N-SH cells, increased levels of TG, ChE, and ceramides and reduced the level of GlcCer. CONCLUSIONS The results showing that ethanol induces accumulation of TG and ceramide in cultured neurons suggest that ethanol enhances lipogenesis and/or reduces fatty acid degradation in neurons, as previously observed in other cell types. Further, ethanol-induced changes in lipid metabolism, specifically those of ceramide and GlcCer, may be related to the ethanol-induced apoptotic pathway.
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Affiliation(s)
- Mariko Saito
- Laboratory of Neurobehavior Genetics and the Division of Analytical Psychopharmacology, The Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA.
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Albi E, Cataldi S, Bartoccini E, Magni MV, Marini F, Mazzoni F, Rainaldi G, Evangelista M, Garcia-Gil M. Nuclear sphingomyelin pathway in serum deprivation-induced apoptosis of embryonic hippocampal cells. J Cell Physiol 2005; 206:189-95. [PMID: 16021626 DOI: 10.1002/jcp.20448] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Sphingomyelin (SM) cycle has been involved in the regulation of proliferation, differentiation, and apoptosis. Increases in ceramide have been found after a larger number of apoptotic stimuli including cytokines, cytotoxic drugs, and environmental stresses. Accumulating evidence suggest that the subcellular localization of ceramide generation is a critical factor in determining the cellular behavior. Since recently enzymes involved in ceramide metabolism such as sphingomyelinase, SM synthase, sphingosine kinase and ceramidase have been found in the nucleus of hepatocyte cells, we have studied first the presence and the physicochemical characteristics of SM metabolism enzymes in nuclei isolated from embryonic hippocampal cells (cell line HN9.10e). The activities of sphingomyelinase and SM-synthase have been assayed and the ceramide production evaluated at different times after serum deprivation in these neurones cultivated in serum-deficient medium. We report that both enzymes are present in the nucleus of embryonic hippocampal cells and differ from those present in the homogenate in optimum pH. After serum deprivation, that induces a time-dependent decrease in cell viability and increase of the cell percentage in G1 phase of the cell cycle, a nuclear sphingomyelinase activation together with SM-synthase inhibition and a consequent increase of nuclear ceramide pool have been demonstrated. No similar enzyme activity modifications in homogenate have been identified. The possible role of nuclear sphingomyelinase/sphingomyelin-synthase balance in serum deprivation-induced apoptosis in the embryonic hippocampal cell is discussed.
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Affiliation(s)
- Elisabetta Albi
- Department of Clinic and Experimental Medicine, Physiopathology, Policlinico Monteluce, Perugia, Italy
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Colombaioni L, Garcia-Gil M. Sphingolipid metabolites in neural signalling and function. ACTA ACUST UNITED AC 2004; 46:328-55. [PMID: 15571774 DOI: 10.1016/j.brainresrev.2004.07.014] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/26/2004] [Indexed: 11/20/2022]
Abstract
Sphingolipid metabolites, such as ceramide, sphingosine, sphingosine-1-phosphate (S1P) and complex sphingolipids (gangliosides), are recognized as molecules capable of regulating a variety of cellular processes. The role of sphingolipid metabolites has been studied mainly in non-neuronal tissues. These studies have underscored their importance as signals transducers, involved in control of proliferation, survival, differentiation and apoptosis. In this review, we will focus on studies performed over the last years in the nervous system, discussing the recent developments and the current perspectives in sphingolipid metabolism and functions.
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Schultz A, Larsson C. Ceramide influences neurite outgrowth and neuroblastoma cell apoptosis regulated by novel protein kinase C isoforms. J Neurochem 2004; 89:1427-35. [PMID: 15189345 DOI: 10.1111/j.1471-4159.2004.02431.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
We have previously seen that protein kinase C (PKC) epsilon induces neurite outgrowth and that PKCdelta and PKCtheta elicit apoptosis in neuroblastoma cells. In this study we investigate the effects of cell-permeable C(2)-ceramide on these events in SK-N-BE(2) neuroblastoma cells. C(2)-ceramide abolishes neurite formation induced by overexpression of PKCepsilon and, in cells overexpressing PKCdelta or PKCtheta, ceramide treatment leads to apoptosis. Exposure to C(2)-ceramide also suppressed neurite outgrowth induced by retinoic acid, but ceramide did not abrogate neurite induction by treatment with the ROCK inhibitor Y-27632, demonstrating that C(2)-ceramide is not a general inhibitor of neurite outgrowth. The neurite-suppressing effect occurs independently of cell-death. Furthermore, C(2)-ceramide relocated PKCepsilon and the isolated regulatory domain of PKCepsilon from the cytosol to the perinuclear region. In contrast, neither the localization of PKCdelta nor of PKCtheta was affected by C(2)-ceramide. Taken together, the data indicate that the neurite-inhibiting effect of C(2)-ceramide treatment may be caused by a re-localization of PKCepsilon and thus identify a functional consequence of ceramide effects on PKCepsilon localization.
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Affiliation(s)
- Anna Schultz
- Molecular Medicine, Lund University, 205-02 Malmö, Sweden
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Ramos B, Lahti JM, Claro E, Jackowski S. Prevalence of necrosis in C2-ceramide-induced cytotoxicity in NB16 neuroblastoma cells. Mol Pharmacol 2003; 64:502-11. [PMID: 12869656 DOI: 10.1124/mol.64.2.502] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The mechanism of cell death triggered by C2-ceramide was investigated using the NB16 neuroblastoma cell line. Treatment of NB16 cells with 20 microM C2-ceramide for 20 h resulted in approximately 75% loss of cell viability, but only 25% of cells were scored as apoptotic based on terminal deoxynucleotidyl transferase nick-end labeling. Ultrastructural analysis revealed early development of necrotic cytoplasmic vacuolization. After 20 h of treatment with C2-ceramide, the majority of cells possessed necrotic morphology with pronounced cytoplasmic vacuolization and without any nuclear changes, although a quarter of the cell population also exhibited clear perinuclear chromatin condensation characteristic of apoptosis. Flow cytometric analysis of cells labeled with both annexin V and propidium iodide showed the rapid accumulation of C2-ceramide-treated cells in the necrotic/late apoptotic fraction. In contrast, cells treated with tumor necrosis factor alpha plus cycloheximide (TNFalpha + CHX) first appeared in the early apoptotic fraction and then accumulated in the necrotic/late apoptotic fraction. Both C2-ceramide and TNFalpha + CHX increased caspase 8- and 3-like activities in cytosolic extracts; however, treatment of cells with the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected NB16 cells from TNFalpha + CHX-induced cell death but did not prevent C2-ceramide cytotoxicity. Although C2-ceramide triggered apoptosis in a fraction of the cells, cell death in the population was primarily caused by necrosis. Thus, C2-ceramide does not faithfully mimic the effects of apoptotic ligands such as TNFalpha, which are thought to be mediated by an accumulation of endogenous ceramide. The inhibition of phosphatidylcholine synthesis is a target for C2-ceramide-mediated cytotoxicity, and this work suggests that other agents that kill cells by inhibiting this pathway may also use a mixture of mechanisms, including necrosis as well as apoptosis.
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Affiliation(s)
- Belen Ramos
- Departamento de Fisiología, Universidad de Extremadura, Cáceres, Spain
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Sietsma H, Dijkhuis AJ, Kamps W, Kok JW. Sphingolipids in neuroblastoma: their role in drug resistance mechanisms. Neurochem Res 2002; 27:665-74. [PMID: 12374201 DOI: 10.1023/a:1020228117739] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Disseminated neuroblastoma usually calls for chemotherapy as the primary approach for treatment. Treatment failure is often attributable to drug resistance. This involves a variety of cellular mechanisms, including increased drug efflux through expression of ATP-binding cassette transporters (e.g., P-glycoprotein) and the inability of tumor cells to activate or propagate the apoptotic response. In recent years it has become apparent that sphingolipid metabolism and the generation of sphingolipid species, such as ceramide, also play a role in drug resistance. This may involve an autonomous mechanism, related to direct effects of sphingolipids on the apoptotic response, but also a subtle interplay between sphingolipids and ATP-binding cassette transporters. Here, we present an overview of the current understanding of the multiple levels at which sphingolipids function in drug resistance, with an emphasis on sphingolipid function in neuroblastoma and how modulation of sphingolipid metabolism may be used as a novel treatment paradigm.
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Affiliation(s)
- Hannie Sietsma
- Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands
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Colombaioni L, Frago LM, Varela-Nieto I, Pesi R, Garcia-Gil M. Serum deprivation increases ceramide levels and induces apoptosis in undifferentiated HN9.10e cells. Neurochem Int 2002; 40:327-36. [PMID: 11792463 DOI: 10.1016/s0197-0186(01)00090-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Sphingolipid metabolites have been involved in the regulation of proliferation, differentiation and apoptosis. While cellular mechanisms of these processes have been extensively analysed in the post-mitotic neurons, little is known about proliferating neuronal precursors. We have taken as a model of neuroblasts the embryonic hippocampal cell line HN9.10e. Apoptosis was induced by serum deprivation and by treatment with N-acetylsphingosine (C2-Cer), a membrane-permeant analogue of the second messenger ceramide. Following C2-Cer addition, cytochrome c was released from mitochondria, [Ca(2+)](i) and caspase-3-like activity increased. Both cytochrome c release and rise of [Ca(2+)](i) occurred before caspase-3 activation and nuclear condensation. The intracellular levels of ceramide peaked at 1h following the serum deprivation. These results indicate that the serum deprivation induces a rise in the intracellular ceramide level, and that increased ceramide concentration leads to calcium dysregulation and release of cytochrome c followed by caspase-3 activation. We show that cytochrome c is released without a loss of mitochondrial transmembrane potential.
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Osawa Y, Banno Y, Nagaki M, Brenner DA, Naiki T, Nozawa Y, Nakashima S, Moriwaki H. TNF-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2001; 167:173-80. [PMID: 11418646 DOI: 10.4049/jimmunol.167.1.173] [Citation(s) in RCA: 136] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.
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Affiliation(s)
- Y Osawa
- First Department of Internal Medicine and Department of Biochemistry, Gifu University School of Medicine, Gifu, Japan
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