title: The histone deacetylase inhibitor trichostatin a decreases lymphangiogenesis by inducing apoptosis and cell cycle arrest via p21-dependent pathways.
abstract: The formation of new lymphatic vessels provides an additional route for tumour cells to metastasize. Therefore, inhibiting lymphangiogenesis represents an interesting target in cancer therapy. First evidence suggests that histone deacetylase inhibitors (HDACi) may mediate part of their antitumor effects by interfering with lymphangiogenesis. However, the underlying mechanisms of HDACi induced anti-lymphangiogenic properties are not fully investigated so far and in part remain unknown.
  
  
  
  Human lymphatic endothelial cells (LEC) were cultured in vitro and treated with or without HDACi. Effects of HDACi on proliferation and cell cycle progress were analysed by BrdU assay and flow cytometry. Apoptosis was measured by quantifying mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. In vitro lymphangiogenesis was investigated using the Matrigel short term lymphangiogenesis assay. The effects of TSA on cell cycle regulatory proteins and apoptosis-related proteins were examined by western blotting, immunofluorescence staining and semi-quantitative RT-PCR. Protein- and mRNA half-life of p21 were analysed by western blotting and quantitative RT-PCR. The activity of the p21 promoter was determined using a dual luciferase assay and DNA-binding activity of Sp1/3 was investigated using EMSA. Furthermore, siRNA assays were performed to analyse the role of p21 and p53 on TSA-mediated anti-lymphangiogenic effects.
  
  
  
  We found that HDACi inhibited cell proliferation and that the pan-HDACi TSA induced G0/G1 arrest in LEC. Cell cycle arrest was accompanied by up-regulation of p21, p27 and p53. Additionally, we observed that p21 protein accumulated in cellular nuclei after treatment with TSA. Moreover, we found that p21 mRNA was significantly up-regulated by TSA, while the protein and mRNA half-life remained largely unaffected. The promoter activity of p21 was enhanced by TSA indicating a transcriptional mechanism. Subsequent EMSA analyses showed increased constitutive Sp1/3-dependent DNA binding in response to HDACi. We demonstrated that p53 was not required for TSA induced p21 expression and growth inhibition of LECs. Interestingly, siRNA-mediated p21 depletion almost completely reversed the anti-proliferative effects of TSA in LEC. In addition, TSA induced apoptosis by cytochrome c release contributed to activating caspases-9, -7 and -3 and downregulating the anti-apoptotic proteins cIAP-1 and -2.
  
  
  
  In conclusion, we demonstrate that TSA - a pan-HDACi - has distinct anti-lymphangiogenic effects in primary human lymphatic endothelial cells by activating intrinsic apoptotic pathway and cell cycle arrest via p21-dependent pathways.
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany. igor.hrgovic@kgu.de.|Klinik für Dermatologie, Venerologie und Allergologie, Klinikum der J. W. Goethe-Universität, Theodor-Stern-Kai 7, Frankfurt am Main, D-60590, Germany. igor.hrgovic@kgu.de.
authors.forename: Igor
authors.initials: I
authors.lastname: Hrgovic
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Doll
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Kleemann
authors.affiliation: Department of Pharmacology & Cancer Biology, Duke University School of Medicine, C218 LSRC, Box 3813, Durham, NC, 27710, USA.
authors.forename: Xiao-Fan
authors.initials: XF
authors.lastname: Wang
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany.
authors.forename: Nadja
authors.initials: N
authors.lastname: Zoeller
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pinter
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kippenberger
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Kaufmann
authors.affiliation: Department of Dermatology, Venereology and Allergology, Goethe University, Theodor-Stern Kai 7, Frankfurt/Main, 60590, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Meissner
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D020405:Dermis / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004729:Endothelium, Lymphatic / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D042583:Lymphangiogenesis / Q000187:drug effects*; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D051017:Apoptosis Regulatory Proteins; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C012589:trichostatin A
keywords: G0/G1 cell cycle arrest; Histon deacetylase inhibitors (HDACi); Lymphangiogenesis; intrinsic apoptotic pathway; p21