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The anti‐hypertensive drug prazosin inhibits glioblastoma growth via the PKC δ‐dependent inhibition of the AKT pathway
2016; Springer Nature; Volume: 8; Issue: 5 Linguagem: Inglês
10.15252/emmm.201505421
ISSN1757-4684
AutoresSuzana Assad Kahn, Sílvia Lima Costa, Sharareh Gholamin, Ryan T. Nitta, Luiz Gustavo Dubois, Marie Fève, Maria Zeniou, Paulo Lucas Cerqueira Coelho, Elias A. El-Habr, Josette Cadusseau, Pascale Varlet, Siddhartha S. Mitra, Bertrand Devaux, Marie‐Claude Kilhoffer, Samuel Cheshier, Vivaldo Moura‐Neto, Jacques Haiech, Marie‐Pierre Junier, Hervé Chneiweiss,
Tópico(s)Cancer Cells and Metastasis
ResumoResearch Article4 April 2016Open Access Source DataTransparent process The anti-hypertensive drug prazosin inhibits glioblastoma growth via the PKCδ-dependent inhibition of the AKT pathway Suzana Assad Kahn Suzana Assad Kahn INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Silvia Lima Costa Silvia Lima Costa INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Neurochemistry and Cell Biology Laboratory Universidade Federal da Bahia, Salvador-Bahia, Brazil Search for more papers by this author Sharareh Gholamin Sharareh Gholamin Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Ryan T Nitta Ryan T Nitta Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Luiz Gustavo Dubois Luiz Gustavo Dubois INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, Brazil Search for more papers by this author Marie Fève Marie Fève Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Maria Zeniou Maria Zeniou Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Paulo Lucas Cerqueira Coelho Paulo Lucas Cerqueira Coelho INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Neurochemistry and Cell Biology Laboratory Universidade Federal da Bahia, Salvador-Bahia, Brazil Search for more papers by this author Elias El-Habr Elias El-Habr INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Search for more papers by this author Josette Cadusseau Josette Cadusseau UMR INSERM 955-Team 10, Faculté des Sciences et Technologies UPEC, Créteil, France Search for more papers by this author Pascale Varlet Pascale Varlet Department of Neuropathology, Sainte-Anne Hospital, Paris, France Paris Descartes University, Paris, France Search for more papers by this author Siddhartha S Mitra Siddhartha S Mitra Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Bertrand Devaux Bertrand Devaux INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Paris Descartes University, Paris, France Department of Neurosurgery, Sainte-Anne Hospital, Paris, France Search for more papers by this author Marie-Claude Kilhoffer Marie-Claude Kilhoffer Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Samuel H Cheshier Samuel H Cheshier Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Vivaldo Moura-Neto Vivaldo Moura-Neto Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, Brazil Search for more papers by this author Jacques Haiech Jacques Haiech Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Marie-Pierre Junier Marie-Pierre Junier INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Search for more papers by this author Hervé Chneiweiss Corresponding Author Hervé Chneiweiss INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Search for more papers by this author Suzana Assad Kahn Suzana Assad Kahn INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Silvia Lima Costa Silvia Lima Costa INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Neurochemistry and Cell Biology Laboratory Universidade Federal da Bahia, Salvador-Bahia, Brazil Search for more papers by this author Sharareh Gholamin Sharareh Gholamin Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Ryan T Nitta Ryan T Nitta Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Luiz Gustavo Dubois Luiz Gustavo Dubois INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, Brazil Search for more papers by this author Marie Fève Marie Fève Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Maria Zeniou Maria Zeniou Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Paulo Lucas Cerqueira Coelho Paulo Lucas Cerqueira Coelho INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Neurochemistry and Cell Biology Laboratory Universidade Federal da Bahia, Salvador-Bahia, Brazil Search for more papers by this author Elias El-Habr Elias El-Habr INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Search for more papers by this author Josette Cadusseau Josette Cadusseau UMR INSERM 955-Team 10, Faculté des Sciences et Technologies UPEC, Créteil, France Search for more papers by this author Pascale Varlet Pascale Varlet Department of Neuropathology, Sainte-Anne Hospital, Paris, France Paris Descartes University, Paris, France Search for more papers by this author Siddhartha S Mitra Siddhartha S Mitra Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Bertrand Devaux Bertrand Devaux INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Paris Descartes University, Paris, France Department of Neurosurgery, Sainte-Anne Hospital, Paris, France Search for more papers by this author Marie-Claude Kilhoffer Marie-Claude Kilhoffer Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Samuel H Cheshier Samuel H Cheshier Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA Search for more papers by this author Vivaldo Moura-Neto Vivaldo Moura-Neto Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, Brazil Search for more papers by this author Jacques Haiech Jacques Haiech Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France Search for more papers by this author Marie-Pierre Junier Marie-Pierre Junier INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Search for more papers by this author Hervé Chneiweiss Corresponding Author Hervé Chneiweiss INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France Search for more papers by this author Author Information Suzana Assad Kahn1,2,3,4, Silvia Lima Costa1,2,3,5, Sharareh Gholamin4, Ryan T Nitta4, Luiz Gustavo Dubois1,2,3,6, Marie Fève7, Maria Zeniou7, Paulo Lucas Cerqueira Coelho1,2,3,5, Elias El-Habr1,2,3, Josette Cadusseau8, Pascale Varlet9,10, Siddhartha S Mitra4, Bertrand Devaux1,2,10,11, Marie-Claude Kilhoffer7, Samuel H Cheshier4, Vivaldo Moura-Neto6, Jacques Haiech7, Marie-Pierre Junier1,2,3,‡ and Hervé Chneiweiss 1,2,3,‡ 1INSERM, UMR-S 1130, Neuroscience Paris Seine-IBPS, Paris, France 2CNRS, UMR 8246, Neuroscience Paris Seine-IBPS, Paris, France 3Sorbonne Universités, UPMC Université Paris 06, UMR-S 8246, Neuroscience Paris Seine-IBPS, Paris, France 4Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine and Division of Pediatric Neurosurgery, Lucile Packard Children's Hospital, Stanford University, Stanford, CA, USA 5Neurochemistry and Cell Biology Laboratory Universidade Federal da Bahia, Salvador-Bahia, Brazil 6Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, Brazil 7Laboratoire d'Innovation Thérapeutique, Laboratoire d'Excellence Medalis, Faculté de Pharmacie, Université de Strasbourg/CNRS UMR7200, Illkirch, France 8UMR INSERM 955-Team 10, Faculté des Sciences et Technologies UPEC, Créteil, France 9Department of Neuropathology, Sainte-Anne Hospital, Paris, France 10Paris Descartes University, Paris, France 11Department of Neurosurgery, Sainte-Anne Hospital, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 44 27 52 94; E-mail: [email protected] EMBO Mol Med (2016)8:511-526https://doi.org/10.15252/emmm.201505421 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract A variety of drugs targeting monoamine receptors are routinely used in human pharmacology. We assessed the effect of these drugs on the viability of tumor-initiating cells isolated from patients with glioblastoma. Among the drugs targeting monoamine receptors, we identified prazosin, an α1- and α2B-adrenergic receptor antagonist, as the most potent inducer of patient-derived glioblastoma-initiating cell death. Prazosin triggered apoptosis of glioblastoma-initiating cells and of their differentiated progeny, inhibited glioblastoma growth in orthotopic xenografts of patient-derived glioblastoma-initiating cells, and increased survival of glioblastoma-bearing mice. We found that prazosin acted in glioblastoma-initiating cells independently from adrenergic receptors. Its off-target activity occurred via a PKCδ-dependent inhibition of the AKT pathway, which resulted in caspase-3 activation. Blockade of PKCδ activation prevented all molecular changes observed in prazosin-treated glioblastoma-initiating cells, as well as prazosin-induced apoptosis. Based on these data, we conclude that prazosin, an FDA-approved drug for the control of hypertension, inhibits glioblastoma growth through a PKCδ-dependent mechanism. These findings open up promising prospects for the use of prazosin as an adjuvant therapy for glioblastoma patients. Synopsis Prazosin, an FDA-approved drug for hypertension with a record of over 40 years of safe and effective clinical use, curbs intracranial glioblastoma growth in mice in a preclinical setting and is thus as a potential anti-glioblastoma adjuvant drug. Prazosin induces glioblastoma cell apoptosis. PKCδ-dependent inhibition of AKT pathway is identified as a possible mechanism for prazosin-induced glioblastoma apoptosis, independently of adrenergic receptors. Glioblastoma growth in orthotopic xenograft mouse models was inhibited by prazosin and, as a consequence, mice survival increased. Introduction Glioblastoma is the most common and aggressive form of primary malignant brain tumors. Highly vascularized, infiltrating, and resistant to current therapies, glioblastomas affect patients at different ages with a median survival shorter than 18 months (Schechter, 1999). Isolation of tumor cells with stem-like properties from glioblastoma has resulted in a novel understanding of tumor behavior. These glioblastoma-initiating cells (GICs) exhibit long-term self-renewal and initiate tumors, contributing to the generation of all subtypes of cells that compose the tumor (Chen et al, 2012b; Cheng et al, 2013). A growing body of evidence implies GICs as crucial determinants of tumor behavior, including proliferation, invasion, and—most importantly—as major culprits of glioblastoma resistance to the standard of care treatments (Bao et al, 2006; Stupp & Hegi, 2007; Murat et al, 2008; Diehn et al, 2009; Chen et al, 2012a). Thus, targeting GICs represents one of the main therapeutic challenges to significantly improve glioblastoma treatments. Investigation of GIC properties has led to early recognition of their sensitivity to molecular changes in the micro-environment, exemplified by the loss of their stem and tumor-initiating properties following serum treatment (Singh et al, 2003; Lee et al, 2006; Gunther et al, 2008; Liu et al, 2009; Silvestre et al, 2011). Within the brain, tumor cells are exposed to extracellular signals from the nervous parenchyma. GICs and endothelial cells have been shown to exert reciprocal control of their properties through release of extracellular factors (Galan-Moya et al, 2011; Thirant et al, 2012). Of special interest are molecular signals arising from diffuse neurotransmitter systems, which can affect GIC behavior. These monoaminergic modulator systems control broad central nervous system functions such as arousal, sleep, food intake, and mood, which are disrupted in several neuropathological situations. Although α1-adrenergic receptor (α1-AR) agonists stimulate rodent neural stem cell (NSC) proliferation and protect them from stress-induced death (Ohashi et al, 2007; Gupta et al, 2009), their effects on GICs are unknown. Many molecules targeting the G protein-coupled receptors (GPCR) of these monoaminergic systems have been developed and used safely and effectively for over 40 years in human pharmacology. Determining whether GIC properties could be manipulated by such pharmacological compounds should help proposing adjuvants to current chemotherapies, or conversely identifying treatments that may promote tumor progression. Here, we found that prazosin, a non-selective α1-AR and a selective α2B-AR antagonist, induced apoptosis in patient-derived GICs in vitro, and inhibited expansion of tumors initiated by GICs in vivo. The effect of prazosin occurred via a PKCδ-dependent inhibition of AKT pathway. This effect was independent from adrenergic receptors, revealing a novel off-target activity of prazosin and a novel therapeutic application for this FDA-approved drug. Results Prazosin induces GIC death and inhibits glioblastoma growth In this study, we used two collections of patient-derived GICs endowed with stem-like properties isolated in two distinct laboratories (Patru et al, 2010; Silvestre et al, 2011; Fareh et al, 2012; Thirant et al, 2012). A major feature of these cells is their resistance to the currently used chemotherapy temozolomide (Patru et al, 2010). The effects of α-AR antagonists on GIC viability were determined following a 3-day treatment on a patient-derived GIC culture, TG1 (Patru et al, 2010; Fareh et al, 2012; Thirant et al, 2012). The following antagonists, all known to act as α-AR antagonists in the nanomolar range (http://www.bindingdb.org/bind/ByLigandName.jsp), were used: prazosin (α1-AR and α2B-AR antagonist), BMY 7378 (α1D-AR antagonist), terazosin (α2B-AR antagonist), ARC 239 (α2B-AR antagonists), and doxazosin (α1-AR antagonists). Only prazosin inhibited GIC viability in a robust and concentration-dependent manner (Fig 1A). Prazosin-induced GIC death was also observed after 24 h of treatment (Fig 1B). Prazosin-induced cell death was observed in all patient-derived GIC cultures tested (TG1, TG16, GBM5, and GBM44, Fig 1C). Of note, either GICs bearing the wild-type (e.g. TG1) or a mutant form of TP53 lacking DNA binding activity (e.g. TG16) (Silvestre et al, 2011) were sensitive to prazosin treatment (Fig 1C), indicating that prazosin effect was independent of the transcription factor P53, a well-known regulator of cell survival. In addition, we explored whether GICs having escaped a first 72-h prazosin treatment were responsive to a second prazosin treatment. The results showed that GICs remained sensitive to 30 μM prazosin (Fig EV1D). The viability of human fetal brain-derived neural stem cells/neural progenitor cells (NSC24, NSC25, NSC5031, and NSC8853), on the other hand, was only marginally decreased at prazosin concentrations of 10 μM or higher (Fig 1C). Extreme limiting dilution assay (ELDA) was used to further evaluate the targeting of GICs by prazosin. Frequency of sphere-forming cells, a surrogate property of GICs (Flavahan et al, 2013), was drastically reduced by prazosin, dropping from 1/3.88 to 1/248 for TG1 (P = 1.13 10−10) and from 1/6.32 to 1/31 for GBM44 (P = 0.0331) (Figs 1D and EV2). In addition, we sorted the GIC according to their expression of EGFR, a marker of malignancy, and of CD133 and CD15, frequently used as GIC markers (Son et al, 2009). Prazosin also inhibited the survival of every population subtype, including EGFR+/CD133+/CD15+ cells (Fig 1E). To further evaluate whether the effectiveness of prazosin is influenced by the stem and/or differentiated state of the cells, NSCs and GICs were differentiated along the astroglial, oligodendroglial, and neuronal lineages (Fig 1F). Prazosin inhibited also the survival of differentiated glioblastoma cells while minimally affecting differentiated NSCs (Fig 1G). Figure 1. Prazosin inhibits GIC survival Viability analysis of GICs treated with the α-AR antagonists prazosin, ARC 239, doxazosin, BMY 7378, and terazosin. GICs were treated with the antagonists or corresponding vehicles for 72 h, and viability was assayed using WST-1. *P < 0.05, n = 4, two-sided Mann–Whitney U-test. Quantification of GIC survival using trypan blue exclusion after 24 and 72 h of treatment with prazosin. *P = 0.0286, n = 4, two-sided Mann–Whitney U-test. Viability analysis of patient-derived GICs (TG1, TG16, GBM5, GBM44) and NSCs (NSC24, NSC25, NSC5031, NSC8853) treated with prazosin for 72 h. *P = 0.0286, n = 4, two-sided Mann–Whitney U-test. Analysis of the sphere-forming capabilities of GICs using the extreme limiting dilution assay. Cells were seeded in presence of vehicle or 30 µM prazosin (PRZ). Sphere formation was scored 10 days post-seeding. Frequency of sphere-forming cells: Control = 1/3.88 (lower 8.61, upper 1.95); prazosin 1/248 (lower 1,003, upper 62), n = 12, P = 1.13 × 10−10. Overall test for difference in stem cell number between groups. Viability analysis of GICs treated with prazosin after sorting cells according to their expression of EGFR, and the neural stem cell markers CD15 and CD133. Prazosin inhibits cell viability regardless of CD133 or CD15 expression. *P = 0.0286, n = 4, two-sided Mann–Whitney U-test. Immunocytochemical staining of NSCs and GICs cultured in media favoring neuronal (β3-tubulin), astroglial (GFAP), or oligodendroglial (O4) differentiation (Diff media). β3-tub: β3-tubulin. Scale bar: 20 μm. Viability analysis of differentiated NSCs and GICs treated with prazosin for 72 h. Diff: differentiation. *P = 0.0286, n = 4, two-sided Mann–Whitney U-test. Data information: Results in (A-C, E, G) are presented as mean ± SD in biological quadruplicates from three independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Prazosin induces GIC apoptosis Flow cytometry analysis for CD15, Annexin V and DAPI in prazosin-treated GBM44 cells. Prazosin induces apoptosis in both CD15+ and CD15− glioblastoma cells. TUNEL staining shows increased numbers of tumor cells undergoing apoptosis following in vivo prazosin treatment of GBM44-bearing mice. Right panel: quantification of TUNEL-positive glioblastoma cells in vehicle- versus prazosin-treated mice. Protocol design is schematized in Fig 2A. Mice were sacrificed 48 h after the last prazosin injection. Scale bar: 50 μm. Results are presented as mean ± SD in biological quadruplicates from three independent experiments. *P < 0.05, two-sided Mann–Whitney U-test. In vivo prazosin treatment does not alter angiogenesis. Representative H&E images of tumors initiated with GBM44 grafting. Mice were treated according to the protocol depicted in Fig 2A and sacrificed 2 days after the last prazosin injection. Arrowheads point to blood vessels. Scale bar: 50 μm. Viability analysis of GICs that escaped prazosin treatment. GICs having escaped a first prazosin treatment are responsive to a second prazosin treatment at 30 µM. GICs were treated with prazosin for 72 h. The medium was then replaced with fresh medium, and the cells were allowed to recover for 2 weeks prior to be exposed to prazosin for 72 h. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Extreme limiting dilution assay of GBM44Prazosin inhibits the sphere-forming capability of GICs. Extreme limiting dilution assay. GBM44 cells were seeded in presence of vehicle or 10 µM prazosin (PRZ). Sphere formation was scored 21 days post-seeding. Control = 1/6.32 (lower 15.9, upper 2.72); prazosin 1/248 (lower 85.3, upper 11.4), n = 6, P = 0.0331, overall test for difference in stem cell number between groups. Download figure Download PowerPoint We then assessed the in vivo effect of prazosin on orthotopic glioblastoma xenografts from GICs derived from human glioblastoma samples (GBM5 and GBM44). EGFR+/CD133+ cells, which constitute a population of GICs with a high degree of self-renewal and tumor-initiating ability (Mazzoleni et al, 2010; Emlet et al, 2014), were sorted from primary patient-derived glioblastoma samples and transduced with a GFP-luciferase construct prior to injection into mice brains for further bioluminescence imaging. Forty-five-day-long treatments were initiated once the presence of tumor masses was confirmed by in vivo bioluminescent imaging (Fig 2A). Prazosin inhibited glioblastoma growth compared to control in both xenograft models (Fig 2B–D), and Kaplan–Meier analysis showed a significant improvement in survival of the groups treated with prazosin as compared to the control groups (Fig 2B and C). Histological analysis performed at the end of the treatment period confirmed that prazosin-treated mice presented smaller tumors than vehicle-treated mice (Fig 2D). Of note, tumors from vehicle- and prazosin-treated mice presented similar blood vessels density, suggesting that prazosin did not affect angiogenesis (Fig EV1C). Flow cytometry analysis of GFP-positive tumor cells showed a significant decrease in human CD133-positive cells in prazosin-treated mice, suggesting removal of GICs along with the non-GICs (Fig 2E). To further demonstrate that prazosin affects GICs, we evaluated its effects on a major property of cancer stem cells, tumor initiation. GFP-positive tumor cells from primary tumors were isolated (see Materials and Methods section) and re-injected into new groups of mice (Fig 2F). All mice that were grafted with glioblastoma cells isolated from vehicle-treated mice developed tumors (8/8 cases, Fig 2F). However, only 4/8 mice injected with glioblastoma cells isolated from prazosin-treated mice developed tumors (Fig 2F). Moreover, mice injected with glioblastoma cells isolated from prazosin-treated mice showed a statistically significant survival benefit (P = 0.0047) (Fig 2F). We also tested lower doses of prazosin (0.15 mg/kg instead of 1.5 mg/kg) compatible with the human daily regimen for treatment of hypertension (see Discussion section). The lower dose of prazosin also induced a significant reduction in tumor growth and increased survival of glioblastoma-bearing mice (Fig 2G). To verify whether prazosin effects could also be observed in an immunocompetent syngeneic mouse model, we implanted the mouse glioblastoma-like cell line GL261, transduced with GFP-luciferase, in C57Bl/6 mice brains. Prazosin induced GL261 cell death in vitro (Fig 3A) and significantly inhibited tumor growth in vivo (Fig 3B–D), an effect associated with a survival benefit (Fig 3C). Finally, using this glioblastoma model coupled with intraperitoneal injections of the green-fluorescent derivative of prazosin, BODIPY FL prazosin, we observed a marked accumulation of prazosin in the tumor within 2 h post-treatment (Fig 3E). Taken altogether, these data show that prazosin inhibits tumor growth initiated by GICs in vivo and increases the survival of glioblastoma-bearing mice including at low doses akin to those used in human treatments. Figure 2. Prazosin inhibits glioblastoma growth in vivo A. Schematic representation of prazosin treatment in vivo. EGFR+/CD133+ GICs directly isolated from primary human glioblastoma samples (GBM44 and GBM5) were transduced with a GFP-luciferase construct and orthotopically implanted into the striatum of NSG mice. Treatment was initiated once the tumors were detected and bioluminescent analyses of tumor growth were performed after 45 days of treatment. B, C. In vivo effect of prazosin treatment (1.5 mg/kg) on glioblastoma growth. Tumors were initiated with GBM44 GICs (B) or GMB5 GICs (C). Left panels: Bioluminescent in vivo images of tumors in mice treated with prazosin (PRZ) or vehicle for 45 days. Middle panels: Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P = 0.0002 and *P = 0.003 for GBM44 and GBM5, respectively, n = 8, two-sided Mann–Whitney U-test. Right panels: Kaplan–Meyer survival curves of mice treated with prazosin (PRZ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle, log-rank Mantel–Cox test. The treatment period is shaded in gray. D. Example of hematoxylin/eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin (PRZ) or vehicle. i: tumor infiltration. n: tumor necrosis. Scale bar: 2 mm. GBM44 GICs. E. Left panel: Representative flow cytometry plots depicting the percentage of CD133+ glioblastoma cells (GFP+) isolated from mice treated with prazosin (PRZ) or vehicle. Tumors were initiated with GBM44 GICs. Right panel: Quantification of flow cytometry analyses of CD133 expression by GFP+-tumor cells isolated from xenografts of three prazosin-treated (PRZ) mice and three vehicle-treated mice. *P = 0.0003, n = 6, two-sided Mann–Wh
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