title: TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells.
abstract: BACKGROUND
  Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer.
  
  
  METHODS
  The present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses.
  
  
  RESULTS
  Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected.
  
  
  CONCLUSIONS
  We report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.
authors.affiliation: Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Ann-Marie
authors.initials: AM
authors.lastname: Simeone
authors.forename: Vanity
authors.initials: V
authors.lastname: McMurtry
authors.forename: René
authors.initials: R
authors.lastname: Nieves-Alicea
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: Larry K
authors.initials: LK
authors.lastname: Keefer
authors.forename: Marcella M
authors.initials: MM
authors.lastname: Johnson
authors.forename: Ana M
authors.initials: AM
authors.lastname: Tari
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001391:Azo Compounds / Q000494:pharmacology*; D001485:Basement Membrane / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003094:Collagen / Q000737:chemistry; D004338:Drug Combinations; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007797:Laminin / Q000737:chemistry; D008956:Models, Chemical; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009569:Nitric Oxide / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D011355:Prodrugs / Q000494:pharmacology*; D011509:Proteoglycans / Q000737:chemistry; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D001391:Azo Compounds; D004338:Drug Combinations; D007797:Laminin; C474958:O(2)-(2,4-dinitrophenyl) 1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate; D010879:Piperazines; D011355:Prodrugs; D011509:Proteoglycans; C059485:matrigel; D019716:Tissue Inhibitor of Metalloproteinase-2; D009569:Nitric Oxide; D003094:Collagen