Tetraarsenic oxide–induced inhibition of malignant glioma cell invasion in vitro via a decrease in matrix metalloproteinase secretion and protein kinase B phosphorylation

Laboratory investigation

Ho-Shin Gwak M.D., Ph.D.1, Myung-Jin Park Ph.D.2, In-Chul Park Ph.D.3, Sang Hyeok Woo Ph.D.3, Hyeon-Ok Jin M.S.3, Chang Hun Rhee M.D., Ph.D.4, and Hee-Won Jung M.D., Ph.D.5
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  • 1 Neuro-Oncology Clinic, National Cancer Center, Goyang;
  • | 2 Research Center for Radio-senescence;
  • | 3 Division of Radiation Cancer Research;
  • | 4 Department of Neurosurgery, Korea Institute of Radiological and Medical Sciences; and
  • | 5 Department of Neurosurgery, Seoul National University, College of Medicine, Seoul, Republic of Korea
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Object

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.

Abbreviations used in this paper:

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; phospho-Akt = phosphorylated active Akt; 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.

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