title: The microtubule stabilizer patupilone counteracts ionizing radiation-induced matrix metalloproteinase activity and tumor cell invasion.
abstract: BACKGROUND
  Ionizing radiation (IR) in combination with microtubule stabilizing agents (MSA) is a promising combined treatment modality. Supra-additive treatment responses might result from direct tumor cell killing and cooperative indirect, tumor cell-mediated effects on the tumor microenvironment. Here we investigated deregulation of matrix metalloproteinase (MMP) activity, as an important component of the tumor microenvironment, by the combined treatment modality of IR with the clinically relevant MSA patupilone.
  
  
  METHODS
  Expression, secretion and activity of MMPs and related tissue inhibitors of metalloproteinases (TIMPs) were determined in cell extracts and conditioned media derived from human fibrosarcoma HT1080 and human glioblastoma U251 tumor cells in response to treatment with IR and the MSA patupilone. Treatment-dependent changes of the invasive capacities of these tumor cell lines were analysed using a Transwell invasion assay. Control experiments were performed using TIMP-directed siRNA and TIMP-directed inhibitory antibodies.
  
  
  RESULTS
  Enzymatic activity of secreted MMPs was determined after treatment with patupilone and irradiation in the human fibrosarcoma HT1080 and the human glioblastoma U251 tumor cell line. IR enhanced the activity of secreted MMPs up to 2-fold and cellular pretreatment with low dose patupilone (0.05-0.2 nM) counteracted specifically the IR-induced MMP activity. The cell invasive capacity of HT1080 and U251 cells was increased after irradiation with 2 Gy by 30% and 50%, respectively, and patupilone treatment completely abrogated IR-induced cell invasion. Patupilone did not alter the level of MMP expression, but interestingly, the protein level of secreted TIMP-1 and TIMP-2 was lower after combined treatment than after irradiation treatment alone. Furthermore, siRNA depletion of TIMP-1 or TIMP-2 prevented IR-mediated induction of MMP activity and cell invasion.
  
  
  CONCLUSIONS
  These results indicate that patupilone counteracts an IR-induced MMP activation process by the reduction of secreted TIMP-1 and TIMP-2 proteins, which are required for activation of MMPs. Since IR-induced MMP activity could contribute to tumor progression, treatment combination of IR with patupilone might be of great clinical benefit for tumor therapy.
authors: Laboratory for Molecular Radiobiology, University Hospital Zurich, Rämistrasse 100, Zurich, 8091, Switzerland. martin.pruschy@usz.ch.
authors: Polina
authors: P
authors: Furmanova-Hollenstein
authors: Angela
authors: A
authors: Broggini-Tenzer
authors: Matthias
authors: M
authors: Eggel
authors: Anne-Laure
authors: AL
authors: Millard
authors: Martin
authors: M
authors: Pruschy
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D034261:Epothilones / Q000494:pharmacology*; D006801:Humans; D020782:Matrix Metalloproteinases / Q000378:metabolism* / Q000528:radiation effects; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D009361:Neoplasm Invasiveness; D009369:Neoplasms / Q000378:metabolism; D034741:RNA, Small Interfering; D011839:Radiation, Ionizing; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism* / Q000528:radiation effects; D014162:Transfection; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D034261:Epothilones; D034741:RNA, Small Interfering; D019714:Tissue Inhibitor of Metalloproteinases; D050257:Tubulin Modulators; D020782:Matrix Metalloproteinases; C093788:epothilone B
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