ascorbate-2-phosphate and 5-5-dimethyl-1-pyrroline-1-oxide

Our previous study showed that tumor invasion of human fibrosarcoma cells HT-1080 is hardly inhibited by ascorbic acid itself (Asc), but inhibited by 2-O-phosphorylated Asc-6-O-palmitylester (Asc2P6Plm) more markedly than 2-O-phosphorylated Asc or Asc-6-O-palmitylester, and that the inhibitory effect may be attributed to an increase in intracellular Asc derived from Asc2P6Plm. In the present study, the mechanism underlying the inhibitory effect of Asc2P6Plm on tumor invasion was analyzed. Migratory ability of the tumor cells was shown to be inhibited in a dose-dependent manner by either treatment with Asc2P6Plm at 50-300 micro M for 1 h or at 10-50 micro M for 18 h as assessed by cell sheet scratching assay. Hydroxyl radicals in homogenates of Asc2P6Plm-treated HT-1080 cells were markedly diminished relative to those of non-treated cells as evaluated by electron spin resonance method using the spin trapping agent DMPO. This may be closely related to attenuation of intracellular gross reactive oxygen species by Asc2P6Plm as was shown with the redox indicator CDCFH-DA. Actin was localized in the vicinity of the cell membrane abundantly in non-treated cells, but was diminished in a time-dependent manner in Asc2P6Plm-treated cells together with disappearance of pseudopods as shown with the actin-directed agent NBD-phallacidin and by immunocytochemical stain. The cell adhesion-controling molecule RhoA was increased time-dependently in the cytoplasm of Asc2P6Plm-treated cells as shown by Western blots. Thus the inhibition of tumor invasion by Asc2P6Plm was shown to be attributed to decrease in both the cell migratory ability and the actin localization near the cell membrane, which may result from an increase in cytoplasmic RhoA and reduction of intracellular ROS that is achieved by enrichment of intracellular Asc derived from Asc2P6Plm.

Topics: Actins; Ascorbic Acid; Blotting, Western; Cell Line, Tumor; Cell Movement; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Humans; Hydroxyl Radical; Immunohistochemistry; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasms; Reactive Oxygen Species; rhoA GTP-Binding Protein; Spin Labels; Time Factors