Accepted Article
Received Date 12-May-2015 Revised Date 05-Nov-2015 Accepted Date 04-Dec-2015 Article Type Regular Article
Dimethylfumarate effectively inhibits lymphangiogenesis via p21 induction and G1 cell cycle arrest
Eva Maria Valesky1, Igor Hrgovic1, Monika Doll1, Xiao-Fan Wang2, Andreas Pinter1, Johannes Kleemann1, Roland Kaufmann1, Stefan Kippenberger1, Markus Meissner1
1
Department of Dermatology, Venereology and Allergology, Goethe-University, Frankfurt am
Main, Germany; 2Department of Pharmacology&Cancer Biology, Duke University School of Medicine, C218 LSRC, Box 3813, Durham, NC 27710 USA.
Correspondence to: Markus Meissner, MD, Department of Dermatology, Venereology and Allergology, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E mail:
[email protected]
Running title: Dimethylfumarate suppress lymphangiogenesis
Key words: Dimethylfumarate, p21, lymphendothelial, lymphangiogenesis
Abbreviations used: DLEC, dermal lymphendothelial cell; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; DMF, Dimethylfumarate; BrdU, Bromodesoxyuridine; LDH, lactatedehydrogenase; DMSO, Dimethylsulfoxide
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/exd.12907 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Different pathologies, like lymphedema, cancer or psoriasis, are associated with abnormal lymphatic vessel formation. Therefore, influencing lymphangiogenesis is an interesting target. Recent evidence suggests that dimethylfumarate (DMF), an anti-psoriatic
agent,
might
have
anti-tumorigenic
and
anti-lymphangiogenic
properties.
To prove this assumption, we performed proliferation and functional assays with primary human dermal lymphendothelial cells (DLEC). We could demonstrated that DMF suppresses DLEC proliferation and formation of capillary-like structures. Underlying apoptotic mechanisms could be ruled out. Cell cycle analysis demonstrated a pronounced G1-arrest. Further evaluations revealed increases in p21 expression. In addition, DMF suppressed Cyclin D1 and Cyclin A expression in a concentration dependent manner. p21 knockdown experiments demonstrated a p21dependent mechanism of regulation. Further analysis showed an increased p21 mRNA expression after DMF treatment. This transcriptional regulation was enforced by post-transcriptional and post-translational mechanisms. In addition, we could demonstrate, that the combination of a proteasomal inhibitor and DMF superinduced the p21 expression.
Hence, DMF is a new anti-lymphangiogenic compound and might be used in various illnesses associated with increased lymphangiogenesis.
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Accepted Article
Recently,
our
group
demonstrated
that
DMF
inhibits
angiogenesis
via
downregulation of VEGFR-2 expression (13). These results were the first hint that DMF also has distinct anti-angiogenic actions (14,15). Several angiogenesis inhibitors with different molecular targets were approved in recent years for the treatment of cancer. The VEGF antibody bevacizumab is a standard treatment for advanced stages of colon, breast, small-cell lung and ovarian cancers and renal cell carcinomas that offers significant survival advantages and is usually administered in combination with other conventional chemotherapeutic agents. As a result, antiangiogenic therapy has become an important pillar of cancer treatment options (16). Lymphangiogenesis plays a crucial role in the initial metastasis (17). In particular, the tumor and the microenvironment under its influence, such as tumor-associated macrophages, appear to be critically involved in the induction of lymphangiogenesis in both the area around the tumor and in the directly draining lymph nodes (18). In addition to lymphangiogenesis, the direct interaction of tumor cells with the lymphatic endothelial cells plays a crucial role in lymphatic metastasis (19). Therefore, the pharmacological suppression of lymphangiogenesis seems to be an effective way to circumvent lymphatic metastasis and thus might influence patient outcomes. Evidence from Valero et al. demonstrated in a SCID mouse model that DMF seems to inhibit lymphangiogenesis and therefore lymph node metastasis (9). The underlying mechanisms of this observation have yet not been elucidated.
Whether DMF, besides its known anti-angiogenic action, inhibits lymphangiogenesis remains unclear. In this study, we investigated the influence of DMF on lymphangiogenesis and its underlying mechanisms of action.
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Accepted Article
Fluorescence-activated cell sorting analysis Cells were incubated with 100µM DMF and Ethanol as a control for 24 h after blocking the S-phase of the cell-cycle by treatment with serum depleted-medium for 24 hours. The cells were fixed in ice-cold 70% ethanol. Cells were incubated in PBS containing 40 g/mL RNase A for 30 minutes at 37°C and resuspended in PBS containing 50 g/mL propidium iodide. Analysis of cell cycle was assessed by a BD FACScan Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Annexin V-fluorescein isothiocyanate/propidium iodide (AV-FITC/PI) doublestaining assay LECs were treated with DMF (50µM and 100µM), solvent or staurosporin (1 µM). AV-FITC (Becton Dickinson, Franklin Lakes, NJ, USA) at a final concentration of 1 µg/ml and 250 ng of PI were added to a mixture containing 100 µl each of cell resuspension and binding buffer (BD Biosciences, USA). The mixture was incubated in the dark for 15 min at room temperature. Cells were washed once and resuspended in 400 µl of binding buffer. Analysis of Annexin-V/propidium iodide was assessed by a BD FACScan Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Tube-formation Assay The Matrigel tube formtaion assay was performed according to the description by Hernandez and coworkers with minor modifications (20). DLECs were treated with vehicle (0.1% DMSO) or DMF (100 μM) for 24 hours; the cells were then photographed using an inverted phase-contrast photomicroscope.
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Accepted Article
GAPDH were performed with the One Step RT-PCR Kit (Qiagen, Hilden, Germany). PCR products were resolved by 1-2% agarose gel electrophoresis, and ethidium bromide stained bands were visualized using an ultraviolet transilluminator. The primer sets for GAPDH were previously published (21).
Real time PCR analysis Total cellular RNA was isolated by the RNeasy Mini Procedure (Qiagen, Hilden, Germany) after DNase digestion. 750 ng of RNA was used for first strand cDNA synthesis using QuantiTect RT-Kit (Qiagen, Hilden, Germany). Real time PCR reactions were performed with SYBRgreen dye technique on a Light Cycler system (Roche Diagnostics, Mannheim, Germany).
Transient transfection and analysis of reporter gene expression DLECs (0,8×105 cells/well in 12-well plates) were transfected with 1 µg of the appropriate firefly luciferase construct and 0.5 µg phRG-TK vector (Promega, Madison, WI, USA) using the SuperFect transfection reagent (Qiagen). Human p21 reporter gene construct was generously provided by Prof. Xiao-Fan Wang (Duke University School of Medicine, Durham, NC, USA). The LightSwitch™ p21-3´UTR plasmid was purchased from ActifMotive (La Hulpe, Belgium). Twenty-four hours after transfection, cells were treated with vehicle (0.3% DMSO; control) or with DMF (100µM) for 24 hours. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, US). Mock transfections with the empty luciferase vectors served as negative controls.
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Accepted Article
Statistical analysis The data are expressed as the mean ± SEM from at least three independent experiments.
Statistical analyses were performed using the Student’s t-test. *p
