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Brack E, Wachtel M, Wolf A, Kaech A, Ziegler U, Schäfer BW. Fenretinide induces a new form of dynamin-dependent cell death in pediatric sarcoma. Cell Death Differ 2020; 27:2500-2516. [PMID: 32144381 DOI: 10.1038/s41418-020-0518-z] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2019] [Revised: 02/18/2020] [Accepted: 02/18/2020] [Indexed: 12/17/2022] Open
Abstract
Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by specific chromosomal translocations mostly encoding the oncogenic transcription factor PAX3-FOXO1 and therefore also referred to as fusion-positive RMS (FP-RMS). Previously, we have identified fenretinide (retinoic acid p-hydroxyanilide) to affect PAX3-FOXO1 expression levels as well as FP-RMS cell viability. Here, we characterize the mode of action of fenretinide in more detail. First, we demonstrate that fenretinide-induced generation of reactive oxygen species (ROS) depends on complex II of the mitochondrial respiratory chain, since ROS scavenging as well as complexing of iron completely abolished cell death. Second, we co-treated cells with a range of pharmacological inhibitors of specific cell death pathways including z-vad (apoptosis), necrostatin-1 (necroptosis), 3-methyladenine (3-MA) (autophagy), and ferrostatin-1 (ferroptosis) together with fenretinide. Surprisingly, none of these inhibitors was able to prevent cell death. Also genetic depletion of key players in the apoptotic and necroptotic pathway (BAK, BAX, and RIPK1) confirmed the pharmacological data. Interestingly however, electron microscopy of fenretinide-treated cells revealed an excessive accumulation of cytoplasmic vacuoles, which were distinct from autophagosomes. Further flow cytometry and fluorescence microscopy experiments suggested a hyperstimulation of macropinocytosis, leading to an accumulation of enlarged early and late endosomes. Surprisingly, pharmacological inhibition as well as genetic depletion of large dynamin GTPases completely abolished fenretinide-induced vesicle formation and subsequent cell death, suggesting a new form of dynamin-dependent programmed cell death. Taken together, our data identify a new form of cell death mediated through the production of ROS by fenretinide treatment, highlighting the value of this compound for treatment of sarcoma patients including FP-RMS.
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Affiliation(s)
- Eva Brack
- Department of Oncology and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Marco Wachtel
- Department of Oncology and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Anja Wolf
- Department of Oncology and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Andres Kaech
- Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland
| | - Urs Ziegler
- Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland
| | - Beat W Schäfer
- Department of Oncology and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland.
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2
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Lopez-Barcons L, Maurer BJ, Kang MH, Reynolds CP. P450 inhibitor ketoconazole increased the intratumor drug levels and antitumor activity of fenretinide in human neuroblastoma xenograft models. Int J Cancer 2017; 141:405-413. [PMID: 28340497 DOI: 10.1002/ijc.30706] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Revised: 02/22/2017] [Accepted: 03/13/2017] [Indexed: 01/02/2023]
Abstract
We previously reported that concurrent ketoconazole, an oral anti-fungal agent and P450 enzyme inhibitor, increased plasma levels of the cytotoxic retinoid, fenretinide (4-HPR) in mice. We have now determined the effects of concurrent ketoconazole on 4-HPR cytotoxic dose-response in four neuroblastoma (NB) cell lines in vitro and on 4-HPR activity against two cell line-derived, subcutaneous NB xenografts (CDX) and three patient-derived NB xenografts (PDX). Cytotoxicity in vitro was assessed by DIMSCAN assay. Xenografted animals were treated with 4-HPR/LXS (240 mg/kg/day) + ketoconazole (38 mg/kg/day) in divided oral doses in cycles of five continuous days a week. In one model, intratumoral levels of 4-HPR and metabolites were assessed by HPLC assay, and in two models intratumoral apoptosis was assessed by TUNEL assay, on Day 5 of the first cycle. Antitumor activity was assessed by Kaplan-Meier event-free survival (EFS). The in vitro cytotoxicity of 4-HPR was not affected by ketoconazole (p ≥ 0.06). Ketoconazole increased intratumoral levels of 4-HPR (p = 0.02), of the active 4-oxo-4-HPR metabolite (p = 0.04), and intratumoral apoptosis (p ≤ 0.0006), compared to 4-HPR/LXS-alone. Concurrent ketoconazole increased EFS in both CDX models compared to 4-HPR/LXS-alone (p ≤ 0.008). 4-HPR + ketoconazole also increased EFS in PDX models compared to controls (p ≤ 0.03). Thus, concurrent ketoconazole decreased 4-HPR metabolism with resultant increases of plasma and intratumoral drug levels and antitumor effects in neuroblastoma murine xenografts. These results support the clinical testing of concurrent ketoconazole and oral fenretinide in neuroblastoma.
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Affiliation(s)
- Lluis Lopez-Barcons
- Cancer Center and Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center School of Medicine, Lubbock, TX, 79430
| | - Barry J Maurer
- Cancer Center and Departments of Cell Biology and Biochemistry, Pediatrics and Internal Medicine, Texas Tech University Health Sciences Center School of Medicine, Lubbock, TX, 79430
| | - Min H Kang
- Cancer Center and Departments of Cell Biology and Biochemistry, Internal Medicine, and Pharmacology/Neuroscience, Texas Tech University Health Sciences Center School of Medicine, Lubbock, TX, 79430
| | - C Patrick Reynolds
- Cancer Center and Departments of Cell Biology and Biochemistry, Pediatrics and Internal Medicine, Texas Tech University Health Sciences Center School of Medicine, Lubbock, TX, 79430
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Yan W, Du J, Du Y, Pu H, Liu S, He J, Zhang J, Hou J. Fenretinide targets the side population in myeloma cell line NCI-H929 and potentiates the efficacy of antimyeloma with bortezomib and dexamethasone regimen. Leuk Res 2016; 51:32-40. [PMID: 27821288 DOI: 10.1016/j.leukres.2016.10.010] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2015] [Revised: 09/01/2016] [Accepted: 10/19/2016] [Indexed: 01/02/2023]
Abstract
Side population (SP) cells, a subset of enriched tumor initiating cells, have been demonstrated to have stem cell-like properties in multiple myeloma (MM) by us as well as other previous studies. A lack of agents targeting tumor initiating cells, however, represents a challenge in the treatment of MM. Previously, fenretinide, a well-tolerated vitamin A derivative, has been shown to exert effect on leukemic stem cells, but its actions against myeloma stem-like cells are still unknown. In this study, the effects of fenretinide on myeloma stem-like cells characteristic was comprehensively examined in SP and non-SP (MP) cells of NCI-H929 cell sorted by flow cytometry-based on Hoechst 33342 stain. We find that fenretinide is capable of eradicating MM SP and MP cells, but not normal bone marrow mononuclear cells (BMMCs) at physiologically achievable concentrations. Fenretinide alone exerted a selective cytotoxic effect on MM SP cells, as well as in combination with bortezomib and dexamethasone. In particular, SP cells were highly sensitive to fenretinide, and in combination with bortezomib and dexamethasone in colony formation and apoptosis assays. Accordingly, the apparent fenretinide-induced-apoptosis was linked to the rapid generation of reactive oxygen species (ROS). Therefore, we propose that fenretinide is a potent agent that targets tumor initiating cells and may be a promising therapeutic agent in MM treatment.
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Affiliation(s)
- Wenqing Yan
- Department of Hematology, The Myeloma & Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China
| | - Juan Du
- Department of Hematology, The Myeloma & Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China
| | - Yanzhi Du
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, China
| | - Honglei Pu
- Department of Hematology, The Myeloma & Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China
| | - Shuyan Liu
- Department of Hematology, The Myeloma & Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China
| | - Jie He
- Department of Hematology, The Myeloma & Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China
| | - Ji Zhang
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, China
| | - Jian Hou
- Department of Hematology, The Myeloma & Lymphoma Center, Changzheng Hospital, The Second Military Medical University, Shanghai, China.
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4
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Albarran L, Lopez JJ, Woodard GE, Salido GM, Rosado JA. Store-operated Ca2+ Entry-associated Regulatory factor (SARAF) Plays an Important Role in the Regulation of Arachidonate-regulated Ca2+ (ARC) Channels. J Biol Chem 2016; 291:6982-8. [PMID: 26817842 DOI: 10.1074/jbc.m115.704940] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Indexed: 01/15/2023] Open
Abstract
The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.
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Affiliation(s)
- Letizia Albarran
- From the Department of Physiology (Cellular Physiology Research Group), University of Extremadura, 10003 Caceres, Spain and
| | - Jose J Lopez
- From the Department of Physiology (Cellular Physiology Research Group), University of Extremadura, 10003 Caceres, Spain and
| | - Geoffrey E Woodard
- Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland
| | - Gines M Salido
- From the Department of Physiology (Cellular Physiology Research Group), University of Extremadura, 10003 Caceres, Spain and
| | - Juan A Rosado
- From the Department of Physiology (Cellular Physiology Research Group), University of Extremadura, 10003 Caceres, Spain and
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5
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Ivanov I, Kuhn H, Heydeck D. Structural and functional biology of arachidonic acid 15-lipoxygenase-1 (ALOX15). Gene 2015; 573:1-32. [PMID: 26216303 PMCID: PMC6728142 DOI: 10.1016/j.gene.2015.07.073] [Citation(s) in RCA: 153] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2015] [Revised: 06/26/2015] [Accepted: 07/21/2015] [Indexed: 12/14/2022]
Abstract
Lipoxygenases (LOX) form a family of lipid peroxidizing enzymes, which have been implicated in a number of physiological processes and in the pathogenesis of inflammatory, hyperproliferative and neurodegenerative diseases. They occur in two of the three domains of terrestrial life (bacteria, eucarya) and the human genome involves six functional LOX genes, which encode for six different LOX isoforms. One of these isoforms is ALOX15, which has first been described in rabbits in 1974 as enzyme capable of oxidizing membrane phospholipids during the maturational breakdown of mitochondria in immature red blood cells. During the following decades ALOX15 has extensively been characterized and its biological functions have been studied in a number of cellular in vitro systems as well as in various whole animal disease models. This review is aimed at summarizing the current knowledge on the protein-chemical, molecular biological and enzymatic properties of ALOX15 in various species (human, mouse, rabbit, rat) as well as its implication in cellular physiology and in the pathogenesis of various diseases.
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Affiliation(s)
- Igor Ivanov
- Institute of Biochemistry, Charité - University Medicine Berlin, Charitéplatz 1, CCO-Building, Virchowweg 6, D-10117 Berlin, Germany
| | - Hartmut Kuhn
- Institute of Biochemistry, Charité - University Medicine Berlin, Charitéplatz 1, CCO-Building, Virchowweg 6, D-10117 Berlin, Germany.
| | - Dagmar Heydeck
- Institute of Biochemistry, Charité - University Medicine Berlin, Charitéplatz 1, CCO-Building, Virchowweg 6, D-10117 Berlin, Germany
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Yang YP, Lotta L, Beutner G, Li X, Schor NF. Induction of Expression of p75 Neurotrophin Receptor Intracellular Domain Does Not Induce Expression or Enhance Activity of Mitochondrial Complex II. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2015; 2016:8752821. [PMID: 26640617 PMCID: PMC4657150 DOI: 10.1155/2016/8752821] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Revised: 06/14/2015] [Accepted: 06/16/2015] [Indexed: 11/23/2022]
Abstract
Fenretinide is a chemotherapeutic agent in clinical trials for the treatment of neuroblastoma, among the most common and most deadly cancers of childhood. Fenretinide induces apoptosis in neuroblastoma cells through accumulation of mitochondrial reactive oxygen species released from Complex II. The neurotrophin receptor, p75NTR, potentiates this effect. The signaling activity of p75NTR is dependent upon its cleavage to its intracellular domain, p75ICD, trafficking of p75ICD to the nucleus, and functioning of p75ICD as a transcription factor. Mitochondrial Complex II comprises 4 subunits, all of which are encoded by nuclear DNA. We therefore hypothesized that the fenretinide-potentiating effects of p75NTR are the result of transcriptional enrichment of Complex II by p75ICD. However, the present studies demonstrate that neither induced expression of p75ICD or its active fragments nor overexpression of p75NTR results in altered expression or activity of Complex II.
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Affiliation(s)
- Yaoli Pu Yang
- Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
| | - Louis Lotta
- Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
| | - Gisela Beutner
- Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
| | - Xingguo Li
- Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
| | - Nina F. Schor
- Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
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7
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Kuhn H, Banthiya S, van Leyen K. Mammalian lipoxygenases and their biological relevance. Biochim Biophys Acta Mol Cell Biol Lipids 2014; 1851:308-30. [PMID: 25316652 DOI: 10.1016/j.bbalip.2014.10.002] [Citation(s) in RCA: 438] [Impact Index Per Article: 39.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2014] [Revised: 09/30/2014] [Accepted: 10/03/2014] [Indexed: 02/07/2023]
Abstract
Lipoxygenases (LOXs) form a heterogeneous class of lipid peroxidizing enzymes, which have been implicated not only in cell proliferation and differentiation but also in the pathogenesis of various diseases with major public health relevance. As other fatty acid dioxygenases LOXs oxidize polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which are further transformed to bioactive lipid mediators (eicosanoids and related substances). On the other hand, lipoxygenases are key players in the regulation of the cellular redox homeostasis, which is an important element in gene expression regulation. Although the first mammalian lipoxygenases were discovered 40 years ago and although the enzymes have been well characterized with respect to their structural and functional properties the biological roles of the different lipoxygenase isoforms are not completely understood. This review is aimed at summarizing the current knowledge on the physiological roles of different mammalian LOX-isoforms and their patho-physiological function in inflammatory, metabolic, hyperproliferative, neurodegenerative and infectious disorders. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".
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Affiliation(s)
- Hartmut Kuhn
- Institute of Biochemistry, University Medicine Berlin - Charite, Chariteplatz 1, CCO-Building, Virchowweg 6, D-10117 Berlin, Germany.
| | - Swathi Banthiya
- Institute of Biochemistry, University Medicine Berlin - Charite, Chariteplatz 1, CCO-Building, Virchowweg 6, D-10117 Berlin, Germany
| | - Klaus van Leyen
- Neuroprotection Research Laboratory, Department of Radiology, Massachusetts Genrel Hospital and Harvard Medical School, Charlestown, MA, USA
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Jang I, Park S, Cho JW, Yigitkanli K, van Leyen K, Roth J. Genetic ablation and short-duration inhibition of lipoxygenase results in increased macroautophagy. Exp Cell Res 2013; 321:276-87. [PMID: 24291223 DOI: 10.1016/j.yexcr.2013.11.017] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2013] [Revised: 11/09/2013] [Accepted: 11/19/2013] [Indexed: 12/24/2022]
Abstract
12/15-lipoxygenase (12/15-LOX) is involved in organelle homeostasis by degrading mitochondria in maturing red blood cells and by eliminating excess peroxisomes in liver. Furthermore, 12/15-LOX contributes to diseases by exacerbating oxidative stress-related injury, notably in stroke. Nonetheless, it is unclear what the consequences are of abolishing 12/15-LOX activity. Mice in which the alox15 gene has been ablated do not show an obvious phenotype, and LOX enzyme inhibition is not overtly detrimental. We show here that liver histology is also unremarkable. However, electron microscopy demonstrated that 12/15-LOX knockout surprisingly leads to increased macroautophagy in the liver. Not only macroautophagy but also mitophagy and pexophagy were increased in hepatocytes, which otherwise showed unaltered fine structure and organelle morphology. These findings were substantiated by immunofluorescence showing significantly increased number of LC3 puncta and by Western blotting demonstrating a significant increase for LC3-II protein in both liver and brain homogenates of 12/15-LOX knockout mice. Inhibition of 12/15-LOX activity by treatment with four structurally different inhibitors had similar effects in cultured HepG2 hepatoma cells and SH-SY5Y neuroblastoma cells with significantly increased autophagy discernable already after 2 hours. Hence, our study reveals a link between ablation or inhibition of 12/15-LOX and stimulation of macroautophagy. The enhanced macroautophagy may be related to the known tissue-protective effects of LOX ablation or inhibition under various diseased conditions caused by oxidative stress and ischemia. This could provide an important cleaning mechanism of cells and tissues to prevent accumulation of damaged mitochondria and other cellular components.
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Affiliation(s)
- Insook Jang
- Department of Integrated OMICS for Biomedical Science, WCU Program of Graduate School, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, South Korea
| | - Sujin Park
- Department of Integrated OMICS for Biomedical Science, WCU Program of Graduate School, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, South Korea
| | - Jin Won Cho
- Department of Integrated OMICS for Biomedical Science, WCU Program of Graduate School, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, South Korea
| | - Kazim Yigitkanli
- Neuroprotection Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
| | - Klaus van Leyen
- Neuroprotection Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
| | - Jürgen Roth
- Department of Integrated OMICS for Biomedical Science, WCU Program of Graduate School, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, South Korea.
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Ganeshan VR, Schor NF. p75 neurotrophin receptor and fenretinide-induced signaling in neuroblastoma. Cancer Chemother Pharmacol 2013; 73:271-9. [PMID: 24253178 DOI: 10.1007/s00280-013-2355-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Accepted: 11/08/2013] [Indexed: 10/26/2022]
Abstract
PURPOSE Neuroblastoma is the most common extracranial solid tumor of childhood. The retinoic acid analogue, fenretinide (4-hydroxyphenyl retinamide; 4-HPR), induces apoptosis in neuroblastoma cells in vitro and is currently in clinical trials for children with refractory neuroblastoma. We have previously shown that expression of the p75 neurotrophin receptor (p75NTR) enhances apoptosis induction and mitochondrial accumulation of reactive oxygen species by 4-HPR in neuroblastoma cells. We now examine the signaling events that underlie this effect. METHODS Systematic examination of pro- and anti-apoptotic signaling effectors was performed by Western blot. Specific inhibitors of JNK phosphorylation and scavengers of mitochondrial reactive oxygen species were used to demonstrate the roles of these phenomena in the enhancement of fenretinide efficacy. RESULTS The present studies demonstrate that enhancement of 4-HPR-induced apoptosis by p75NTR is dependent upon p38MAPK phosphorylation, JNK phosphorylation, caspase 3 activation, Akt cleavage, and decreased Akt phosphorylation. In addition, treatment with 4-HPR results in upregulation of MKK4 and MEKK1, and phosphorylation of MKK3/6. Efforts to enhance the efficacy of 4-HPR and to identify those tumors most likely to respond to it might exploit these effectors of 4-HPR-induced apoptosis. CONCLUSIONS Pharmacological agents that enhance MKK4 or MEKK1 expression or JNK expression or phosphorylation may enhance efficacy of 4-HPR in neuroblastomas that do not express high levels of p75NTR.
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Affiliation(s)
- Veena R Ganeshan
- Center for Neural Development and Disease, University of Rochester Medical Center, Rochester, NY, 14642, USA
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10
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Ovarian and breast cancer spheres are similar in transcriptomic features and sensitive to fenretinide. BIOMED RESEARCH INTERNATIONAL 2013; 2013:510905. [PMID: 24222909 PMCID: PMC3816214 DOI: 10.1155/2013/510905] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/17/2013] [Revised: 08/16/2013] [Accepted: 08/18/2013] [Indexed: 01/06/2023]
Abstract
Cancer stem cells (CSCs) are resistant to chemotherapy and are ability to regenerate cancer cell populations, thus attracting much attention in cancer research. In this report, we first demonstrated that sphere cells from ovarian cancer cell line A2780 shared many features of CSCs, such as resistance to cisplatin and able to initiate tumors in an efficient manner. Then, we conducted cDNA microarray analysis on spheres from ovarian A2780 cells, and from breast MCF7 and SUM159 cells, and found that molecular pathways underlying spheres from these cancer cell lines were similar to a large extent, suggesting that similar mechanisms are involved in the genesis of CSCs in both ovarian and breast cancer types. In addition, we showed that spheres from these cancer types were highly sensitive to fenretinide, a stimulus of oxidative stress-mediated apoptosis in cancer cells. Thus, our results not only provide important insights into mechanisms underlying CSCs in ovarian and breast cancer, but also lead to the development of more sophisticated protocols of cancer therapy in near future.
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11
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Bell E, Ponthan F, Whitworth C, Westermann F, Thomas H, Redfern CPF. Cell survival signalling through PPARδ and arachidonic acid metabolites in neuroblastoma. PLoS One 2013; 8:e68859. [PMID: 23874790 PMCID: PMC3706415 DOI: 10.1371/journal.pone.0068859] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2012] [Accepted: 06/06/2013] [Indexed: 12/11/2022] Open
Abstract
Retinoic acid (RA) has paradoxical effects on cancer cells: promoting cell death, differentiation and cell cycle arrest, or cell survival and proliferation. Arachidonic acid (AA) release occurs in response to RA treatment and, therefore, AA and its downstream metabolites may be involved in cell survival signalling. To test this, we inhibited phospholipase A2-mediated AA release, cyclooxygenases and lipoxygenases with small-molecule inhibitors to determine if this would sensitise cells to cell death after RA treatment. The data suggest that, in response to RA, phospholipase A2-mediated release of AA and subsequent metabolism by lipoxygenases is important for cell survival. Evidence from gene expression reporter assays and PPARδ knockdown suggests that lipoxygenase metabolites activate PPARδ. The involvement of PPARδ in cell survival is supported by results of experiments with the PPARδ inhibitor GSK0660 and siRNA-mediated knockdown. Quantitative reverse transcriptase PCR studies demonstrated that inhibition of 5-lipoxygenase after RA treatment resulted in a strong up-regulation of mRNA for PPARδ2, a putative inhibitory PPARδ isoform. Over-expression of PPARδ2 using a tetracycline-inducible system in neuroblastoma cells reduced proliferation and induced cell death. These data provide evidence linking lipoxygenases and PPARδ in a cell survival-signalling mechanism and suggest new drug-development targets for malignant and hyper-proliferative diseases.
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Affiliation(s)
- Emma Bell
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Frida Ponthan
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Claire Whitworth
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Frank Westermann
- Division of Tumor Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Huw Thomas
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Christopher P. F. Redfern
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom
- * E-mail:
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12
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12S-Lipoxygenase is necessary for human vascular smooth muscle cell survival. Exp Cell Res 2013; 319:1586-93. [DOI: 10.1016/j.yexcr.2013.04.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2013] [Revised: 03/31/2013] [Accepted: 04/01/2013] [Indexed: 11/19/2022]
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13
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Sogno I, Venè R, Ferrari N, De Censi A, Imperatori A, Noonan DM, Tosetti F, Albini A. Angioprevention with fenretinide: Targeting angiogenesis in prevention and therapeutic strategies. Crit Rev Oncol Hematol 2010; 75:2-14. [DOI: 10.1016/j.critrevonc.2009.10.007] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2009] [Revised: 10/15/2009] [Accepted: 10/29/2009] [Indexed: 01/01/2023] Open
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Abstract
Oxidative stress has been implicated previously in the regulation of ceramide metabolism. In the present study, its effects on dihydroceramide desaturase were investigated. To stimulate oxidative stress, HEK (human embyronic kidney)-293, MCF7, A549 and SMS-KCNR cells were treated with H2O2, menadione or tert-butylhydroperoxide. In all cell lines, an increase in dihydroceramide was observed upon oxidative stress as measured by LC (liquid chromatography)/MS. In contrast, total ceramide levels were relatively unchanged. Mechanistically, dihydroceramide desaturase activity was measured by an in situ assay and decreased in a time- and dose-dependent fashion. Interestingly, no detectable changes in the protein levels were observed, suggesting that oxidative stress does not induce degradation of dihydroceramide desaturase. In summary, oxidative stress leads to potent inhibition of dihydroceramide desaturase resulting in significant elevation in dihydroceramide levels in vivo.
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Raguénez G, Mühlethaler-Mottet A, Meier R, Duros C, Bénard J, Gross N. Fenretinide-induced caspase-8 activation and apoptosis in an established model of metastatic neuroblastoma. BMC Cancer 2009; 9:97. [PMID: 19331667 PMCID: PMC2670318 DOI: 10.1186/1471-2407-9-97] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2008] [Accepted: 03/30/2009] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Resistance of high-risk metastatic neuroblastoma (HR-NB) to high dose chemotherapy (HD-CT) raises a major therapeutic challenge in pediatric oncology. Patients are treated by maintenance CT. For some patients, an adjuvant retinoid therapy is proposed, such as the synthetic retinoid fenretinide (4-HPR), an apoptotic inducer. Recent studies demonstrated that NB metastasis process is enhanced by the loss of caspase-8 involved in the Integrin-Mediated Death (IMD) process. As the role of caspase-8 appears to be critical in preventing metastasis, we aimed at studying the effect of 4-HPR on caspase-8 expression in metastatic neuroblasts. METHODS We used the human IGR-N-91 MYCN-amplified NB experimental model, able to disseminate in vivo from the primary nude mouse tumor xenograft (PTX) into myocardium (Myoc) and bone marrow (BM) of the animal. NB cell lines, i.e., IGR-N-91 and SH-EP, were treated with various doses of Fenretinide (4-HPR), then cytotoxicity was analyzed by MTS proliferation assay, apoptosis by the propidium staining method, gene or protein expressions by RT-PCR and immunoblotting and caspases activity by colorimetric protease assays. RESULTS The IGR-N-91 parental cells do not express detectable caspase-8. However the PTX cells established from the primary tumor in the mouse, are caspase-8 positive. In contrast, metastatic BM and Myoc cells show a clear down-regulation of the caspase-8 expression. In parallel, the caspases -3, -9, -10, Bcl-2, or Bax expressions were unchanged. Our data show that in BM, compared to PTX cells, 4-HPR up-regulates caspase-8 expression that parallels a higher sensitivity to apoptotic cell death. Stable caspase-8-silenced SH-EP cells appear more resistant to 4-HPR-induced cell death compared to control SH-EP cells. Moreover, 4-HPR synergizes with drugs since apoptosis is restored in VP16- or TRAIL-resistant-BM cells. These results demonstrate that 4-HPR in up-regulating caspase-8 expression, restores and induces apoptotic cell death in metastatic neuroblasts through caspase-8 activation. CONCLUSION This study provides basic clues for using fenretinide in clinical treatment of HR-NB patients. Moreover, since 4-HPR induces cell death in caspase-8 negative NB, it also challenges the concept of including 4-HPR in the induction of CT of these patients.
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Affiliation(s)
- Gilda Raguénez
- Centre National de Recherche Scientifique, Unité Mixte de Recherche 8126, Institut Fédératif de Recherche 54, Institut Gustave Roussy, Villejuif, France.
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16
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Windsor R, Strauss S, Seddon B, Whelan J. Experimental therapies in Ewing's sarcoma. Expert Opin Investig Drugs 2009; 18:143-59. [DOI: 10.1517/13543780802715784] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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Magwere T, Myatt SS, Burchill SA. Manipulation of oxidative stress to induce cell death in Ewing’s sarcoma family of tumours. Eur J Cancer 2008; 44:2276-87. [DOI: 10.1016/j.ejca.2008.06.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2008] [Revised: 05/22/2008] [Accepted: 06/13/2008] [Indexed: 10/21/2022]
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Zaheer A, Sahu SK, Traynelis VC. Inhibitors of EGFR signaling retard cytotoxicity of fenretinide in rat gliosarcoma cells. Neurochem Res 2007; 33:22-6. [PMID: 17577665 DOI: 10.1007/s11064-007-9401-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2007] [Accepted: 05/30/2007] [Indexed: 10/23/2022]
Abstract
PURPOSE Fenretinide, 4-(N-hydroxyphenyl) retinamide, (4-HPR) is a well tolerated analog of alltrans retinoic acid. The gangliosideGM3, is a non-specific inhibitor of EGF receptor autophosphorylation (EGFR-phos). Both compounds were found preferentially cytotoxic to malignant and proliferating cells when compared to non-proliferating normal brain cells. Some of the small molecule inhibitors of EGFR-phos are also known to inhibit growth of brain tumors at relatively non-toxic doses. The purpose of this investigation was to evaluate if 4-HPR and inhibitors of EGFR-phos could be used together in the treatment of brain tumors. METHODS The 9L rat gliosarcoma cells were treated in vitro with 4-HPR either alone or in combination with the non-specific or specific inhibitors of EGFR-phos, GM3 or AG-1478, respectively. The relative viability of the control and treated cells was determined using 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The experimental data were analyzed for statistical significance. RESULTS In contrast to the expected additive/synergistic effect on cell growth inhibition, the sub-toxic and toxic concentrations of 4-HPR protected GM3 treated cells. The viable cells were 3.86 times higher following GM3 plus 4-HPR treatments compared to GM3 treatment alone. Additionally, a specific inhibitor of EGFR-phos signaling, AG-1478 caused a concentration dependent protection of cells from the toxicity of 4-HPR. Our results show counteracting cytotoxic responses of 4-HPR and EGFR-phos inhibitors when used together in 9L rat gliosarcoma cells.
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Affiliation(s)
- Ayesha Zaheer
- Department of Neurosurgery, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA
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19
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Abstract
Fenretinide, a synthetic retinoid, has emerged as a promising anticancer agent based on numerous in vitro and animal studies, as well as chemoprevention clinical trials. In vitro observations suggest that the anticancer activity of fenretinide may arise from its ability to induce apoptosis in tumor cells. Diverse signaling molecules including reactive oxygen species, ceramide, and ganglioside GD3 can mediate apoptosis induction by fenretinide in transformed, premalignant, and malignant cells. In many cell types, these signaling intermediates appear to be induced by mechanisms that are independent of retinoic acid receptor activation, and ultimately initiate the intrinsic or mitochondrial-mediated pathway of cell elimination. Numerous investigations conducted during the past 10 years have discovered a great deal about the apoptogenic activity of fenretinide. In this review we explore the mechanisms associated with fenretinide-induced apoptosis and highlight certain mechanistic underpinnings of fenretinide-induced cell death that remain poorly understood and thus warrant further characterization.
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Affiliation(s)
- N Hail
- Department of Clinical Pharmacy, School of Pharmacy, The University of Colorado at Denver and Health Sciences Center, Box C238, 80262, USA.
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20
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Kwon KJ, Jung YS, Lee SH, Moon CH, Baik EJ. Arachidonic acid induces neuronal death through lipoxygenase and cytochrome P450 rather than cyclooxygenase. J Neurosci Res 2005; 81:73-84. [PMID: 15931672 DOI: 10.1002/jnr.20520] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Arachidonic acid (AA) is released from membrane phospholipids during normal and pathologic processes such as neurodegeneration. AA is metabolized via lipoxygenase (LOX)-, cyclooxygenase (COX)-, and cytochrome P450 (CYP450)-catalyzed pathways. We investigated the relative contributions of these pathways in AA-induced neuronal death. Exposure of cultured cortical neurons to AA (50 microM) yielded significantly apoptotic neuronal death, which was attenuated greatly by LOX inhibitors (nordihydroguaiaretic acid, AA861, and baicalein), or CYP450 inhibitors (SKF525A and metyrapone), rather than COX inhibitors (indomethacin and NS398). AA (10 microM)-induced neurotoxicity was prevented by all kinds of inhibitors. Compared, the neurotoxic effects of three pathway metabolites, 12-hydroxyeicosatetraenoic acid (12-HETE), a major LOX metabolite, induced a significant neurotoxicity. AA also produced reactive oxygen species within 30 min, which was reduced by all inhibitors tested, including COX inhibitors, and AA neurotoxicity was abolished by the antioxidant Trolox. AA treatment also depleted glutathione levels; this depletion was reduced by the LOX or CYP450 inhibitors rather than by the COX inhibitors. Taken together, our data suggested that the LOX pathway likely plays a major role in AA-induced neuronal death with the modification of intracellular free radical levels.
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Affiliation(s)
- Kyoung Ja Kwon
- Department of Physiology, Ajou University School of Medicine, Suwon, Korea
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21
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Lovat PE, Corazzari M, Di Sano F, Piacentini M, Redfern CPF. The role of gangliosides in fenretinide-induced apoptosis of neuroblastoma. Cancer Lett 2005; 228:105-10. [PMID: 15907365 DOI: 10.1016/j.canlet.2005.01.044] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2004] [Accepted: 01/12/2005] [Indexed: 10/25/2022]
Abstract
Fenretinide is thought to induce apoptosis via increases in ceramide levels but the mechanisms of ceramide generation and the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. In SH-SY5Y neuroblastoma cells, evidence suggests that acid sphingomyelinase activity is essential for the induction of ceramide and apoptosis in response to fenretinide. Downstream of ceramide, apoptosis in response to fenretinide is mediated by increased glucosylceramide synthase activity resulting in increased levels of gangliosides GD3 and GD2 via GD3 synthase. GD3 is a key signalling intermediate leading to apoptosis via the activation of 12-Lipoxygenase, and the parallel induction of GD2 suggests that fenretinide might enhance the response of neuroblastoma to therapy with anti-GD2 antibodies.
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Affiliation(s)
- Penny E Lovat
- Northern Institute for Cancer Research, University of Newcastle Upon Tyne, 4th Floor, Cookson Building, Newcastle Upon Tyne, NE2 4HH, UK
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Zhao YB, Yang HY, Zhang XW, Chen GY. Mutation in D-loop region of mitochondrial DNA in gastric cancer and its significance. World J Gastroenterol 2005; 11:3304-6. [PMID: 15929189 PMCID: PMC4316070 DOI: 10.3748/wjg.v11.i21.3304] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [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 mutation in D-loop region of mitoc-hondrial DNA in gastric cancer and its influence on the changes of reactive oxygen species (ROS) and cell cycle.
METHODS: The D-loop region was amplified by PCR and sequenced. Reactive oxygen species and cell cycle were detected by flow cytometry in 20 specimens from gastric cancer and adjacent normal tissues. According to the sequence results, gastric cancer tissue was divided into mutation group and control group. Reactive oxygen species, apoptosis and proliferation in the two groups were compared.
RESULTS: Among the 20 gastric cancer specimens, 18 mutations were identified in 7 patients, the mutation rate being 35%. There were four microsatellite instabilities in the mutations. No mutation was found in the adjacent tissues. Reactive oxygen species, apoptosis, and proliferation in the mutation group were all significantly higher than those in control group.
CONCLUSION: Mutation in D-loop region plays a role in the genesis and development of gastric cancer.
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Affiliation(s)
- Yi-Bing Zhao
- Department of Gastrointestinal Surgery, Jiangsu Provincial Hospital, Nanjing 210029, Jiangsu Province, China.
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Corazzari M, Lovat PE, Oliverio S, Di Sano F, Donnorso RP, Redfern CPF, Piacentini M. Fenretinide: A p53-independent way to kill cancer cells. Biochem Biophys Res Commun 2005; 331:810-5. [PMID: 15865936 DOI: 10.1016/j.bbrc.2005.03.184] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2005] [Indexed: 11/19/2022]
Abstract
The synthetic retinoid fenretinide [N-(4 hydroxyphenyl)retinamide] induces apoptosis of cancer cells and acts synergistically with chemotherapeutic drugs, thus providing opportunities for novel approaches to cancer therapy. The upstream signaling events induced by fenretinide include an increase in intracellular levels of ceramide, which is subsequently metabolized to GD3. This ganglioside triggers the activation of 12-Lox (12-lipoxygenase) leading to oxidative stress and apoptosis via the induction of the transcription factor Gadd153 and the Bcl-2-family member protein Bak. Increased evidence suggests that the apoptotic pathway activated by fenretinide is p53-independent and this may represent a novel way to treat tumors resistant to DNA-damaging chemotherapeutic agents. Therefore, fenretinide offers increased clinical benefit as a novel agent for cancer therapy, able to complement the action of existing chemotherapeutic treatment regimes. Furthermore, synergy between fenretinide and chemotherapeutic drugs may facilitate the use of chemotherapeutic drugs at lower concentrations, with possible reduction in treatment-associated morbidity.
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Myatt SS, Redfern CPF, Burchill SA. p38MAPK-Dependent Sensitivity of Ewing's Sarcoma Family of Tumors to Fenretinide-Induced Cell Death. Clin Cancer Res 2005; 11:3136-48. [PMID: 15837770 DOI: 10.1158/1078-0432.ccr-04-2050] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE There is an urgent need for new therapeutic strategies in Ewing's sarcoma family of tumors (ESFT). In this study, we have evaluated the effect of fenretinide [N-(4-hydroxyphenyl)retinamide] in ESFT models. EXPERIMENTAL DESIGN The effect of fenretinide on viable cell number and apoptosis of ESFT cell lines and spheroids and growth of s.c. ESFT in nu/nu mice was investigated. The role of the stress-activated kinases p38(MAPK) and c-Jun NH(2)-terminal kinase in fenretinide-induced death was investigated by Western blot and inhibitor experiments. Accumulation of reactive oxygen species (ROS) and changes in mitochondrial transmembrane potential were investigated by flow cytometry. RESULTS Fenretinide induced cell death in all ESFT cell lines examined in a dose- and time-dependent manner. ESFT cells were more sensitive to fenretinide than the neuroblastoma cell lines examined. Furthermore, fenretinide induced cell death in ESFT spheroids and delayed s.c. ESFT growth in mice. p38(MAPK) was activated within 15 minutes of fenretinide treatment and was dependent on ROS accumulation. Inhibition of p38(MAPK) activity partially rescued fenretinide-mediated cell death in ESFT but not in SH-SY5Y neuroblastoma cells. c-Jun NH(2)-terminal kinase was activated after 4 hours and was dependent on ROS accumulation but not on activation of p38(MAPK). After 8 hours, fenretinide induced mitochondrial depolarization (Deltapsi(m)) and release of cytochrome c into the cytoplasm in a ROS- and p38(MAPK)-dependent manner. CONCLUSIONS These data show that the high sensitivity of ESFT cells to fenretinide is dependent in part on the rapid and sustained activation of p38(MAPK). The efficacy of fenretinide in preclinical models demands the evaluation of fenretinide as a potential therapeutic agent in ESFT.
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Affiliation(s)
- Stephen S Myatt
- Candlelighter's Children's Cancer Research Laboratory, Cancer Research UK Clinical Centre, Leeds, United Kingdom
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Lovat PE, Di Sano F, Corazzari M, Fazi B, Donnorso RP, Pearson ADJ, Hall AG, Redfern CPF, Piacentini M. Gangliosides link the acidic sphingomyelinase-mediated induction of ceramide to 12-lipoxygenase-dependent apoptosis of neuroblastoma in response to fenretinide. J Natl Cancer Inst 2004; 96:1288-99. [PMID: 15339967 DOI: 10.1093/jnci/djh254] [Citation(s) in RCA: 72] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
BACKGROUND The lipid second messenger ceramide, which is generated by acidic and neutral sphingomyelinases or ceramide synthases, is a common intermediate of many apoptotic pathways. Metabolism of ceramide involves several enzymes, including glucosylceramide synthase and GD3 synthase, and results in the formation of gangliosides (GM3, GD3, and GT3), which in turn promote the generation of reactive oxygen species (ROS) and apoptosis. Fenretinide, a retinoic acid derivative, is thought to induce apoptosis via increases in ceramide levels, but the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. METHODS SH-SY5Y and HTLA230 neuroblastoma cells were treated with fenretinide in the presence or absence of inhibitors of enzymes important in ceramide metabolism (fumonisin B1, inhibitor of ceramide synthase; desipramine, inhibitor of acidic and neutral sphingomyelinases; and PDMP, inhibitor of glucosylceramide). Small interfering RNAs were used to specifically block acidic sphingomyelinase or GD3 synthase activities. Apoptosis, ROS, and GD3 expression were measured by flow cytometry. RESULTS In neuroblastoma cells, ROS generation and apoptosis were associated with fenretinide-induced increased levels of ceramide, glucosylceramide synthase activity, GD3 synthase activity, and GD3. Fenretinide also induced increased levels of GD2, a ganglioside derived from GD3. Inhibition of acidic sphingomyelinase but not of neutral sphingomyelinase or ceramide synthase, blocked fenretinide-induced increases in ceramide, ROS, and apoptosis. Exogenous GD3 induced ROS and apoptosis in SH-SY5Y cells but not in SH-SY5Y cells treated with baicalein, a specific 12-lipoxygenase inhibitor. Exogenous GD2 did not induce apoptosis. CONCLUSIONS A novel pathway of fenretinide-induced apoptosis is mediated by acidic sphingomyelinase, glucosylceramide synthase, and GD3 synthase, which may represent targets for future drug development. GD3 may be a key signaling intermediate leading to apoptosis via the activation of 12-lipoxygenase.
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Affiliation(s)
- Penny E Lovat
- Northern Institute for Cancer Research, University of Newcastle upon Tyne, Newcastle upon Tyne, UK
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Abstract
Execution of the apoptotic program involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can also be found when caspases are inhibited. Moreover, under pathological conditions, apoptosis and nonapoptotic death paradigms are often interwined, which suggest that, in vivo, cells may use diverging execution pathways. Molecular switches between apoptosis and necrosis include adenosine triphosphate-dependent steps in the activation of caspases or steps sensitive to reactive oxygen/nitrogen species. In turn, caspase activation can cause necrosis by promoting ion overload.
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