title: Tetraarsenic oxide-induced inhibition of malignant glioma cell invasion in vitro via a decrease in matrix metalloproteinase secretion and protein kinase B phosphorylation.
abstract: OBJECTIVE
  Local invasiveness of malignant glioma is a major reason for the failure of current treatments including surgery and radiation therapy. Tetraarsenic oxide (As4O6 [TAO]) is a trivalent arsenic compound that has potential anticancer and antiangiogenic effects in selected cancer cell lines at a lower concentration than arsenic trioxide (As2O3 [ATO]), which has been more widely tested in vitro and in vivo. The authors tried to determine the cytotoxic concentration of TAO in malignant glioma cell lines and whether TAO would show anti-invasive effects under conditions independent of cell death or apoptosis.
  
  
  METHODS
  The human phosphatase and tensin homolog (PTEN)-deficient malignant glioma cell lines U87MG, U251MG, and U373MG together with PTEN-functional LN428 were cultured with a range of micromolar concentrations of TAO. The invasiveness of the glioma cell lines was analyzed. The effect of TAO on matrix metalloproteinase (MMP) secretion and membrane type 1 (MT1)-MMP expression was measured using gelatin zymography and Western blot, respectively. Akt, or protein kinase B, activity, which is a downstream effector of PTEN, was assessed with a kinase assay using glycogen synthesis kinase-3β (GSK-3β) as a substrate and Western blotting of phosphorylated Akt.
  
  
  RESULTS
  Tetraarsenic oxide inhibited 50% of glioma cell proliferation at 6.3-12.2 μM. Subsequent experiments were performed under the same TAO concentrations and exposure times, avoiding the direct tumoricidal effect of TAO, which was confirmed with apoptosis markers. An invasion assay revealed a dose-dependent decrease in invasiveness under the influence of TAO. Both the gelatinolytic activity of MMP-2 and MT1-MMP expression decreased in a dose-dependent manner in all cell lines, which was in accordance with the invasion assay results. The TAO decreased kinase activity of Akt on GSK-3β assay and inhibited Akt phosphorylation in a dose-dependent manner in all cell lines regardless of their PTEN status.
  
  
  CONCLUSIONS
  These results showed that TAO effectively inhibits proliferation of glioblastoma cell lines and also exerts an anti-invasive effect via decreased MMP-2 secretion, decreased MT1-MMP expression, and the inhibition of Akt phosphorylation under conditions devoid of cytotoxicity. Further investigations using an in vivo model are needed to evaluate the potential role of TAO as an anti-invasive agent.
authors.affiliation: Neuro-Oncology Clinic, National Cancer Center, Goyang;
authors.forename: Ho-Shin
authors.initials: HS
authors.lastname: Gwak
authors.forename: Myung-Jin
authors.initials: MJ
authors.lastname: Park
authors.forename: In-Chul
authors.initials: IC
authors.lastname: Park
authors.forename: Sang Hyeok
authors.initials: SH
authors.lastname: Woo
authors.forename: Hyeon-Ok
authors.initials: HO
authors.lastname: Jin
authors.forename: Chang Hun
authors.initials: CH
authors.lastname: Rhee
authors.forename: Hee-Won
authors.initials: HW
authors.lastname: Jung
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000201:enzymology / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005909:Glioblastoma / Q000201:enzymology / Q000473:pathology*; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009361:Neoplasm Invasiveness; D010087:Oxides / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D001152:Arsenicals; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D010087:Oxides; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14; D000077237:Arsenic Trioxide
keywords: AP-1 = activator protein-1; ATO = arsenic trioxide; BBB = blood-brain barrier; ECM = extra-cellular matrix; EGTA = ethylene glycol tetraacetic acid; FACS = flow-activated cell sorter; GBM = glioblastoma; GSK-3β = glycogen synthase kinase-3β; IC50 = 50% inhibitory concentration; MMP = matrix metalloproteinase; MT1 = membrane type 1; PBS = phosphate-buffered saline; PI3K = phosphoinositide 3-kinase; PTEN = phosphatase and tensin homolog; ROS = reactive oxygen species; SDS = sodium dodecyl sulfate; SDS-PAGE = SDS–polyacrylamide gel electrophoresis; TAO = tetraarsenic oxide; glioblastoma; invasion; matrix metalloproteinase; oncology; phospho-Akt = phosphorylated active Akt; protein kinase B; tetraarsenic oxide