Figure 3.

DFMO inhibits cell migration and invasion, increases p27^Kip1 expression, and increases the phosphorylation of Akt (Ser473) and GSK3-β (Ser9). (A) MYCN2 cells were treated with or without doxycycline and 5 mM DFMO ± 10 μM putrescine, 10 μM spermidine or 10 μM spermine for 72 h and the wound healing assay was performed. DFMO inhibited cell migration. Supplementing the external media with putrescine, spermidine or spermine reversed the effects of DFMO. Results are represented as mean ± SD, n=3. (B and C) MYCN2 cells were treated with doxycycline ± 5 mM DFMO. The external medium was supplemented with ± 10 μM putrescine, 10 μM spermidine or 10 μM spermine or left untreated for 72 h of DFMO treatment or the last 24 h of DFMO treatment. Transwell migration (B) and invasion (C) assays were performed. DFMO inhibited NB migration and invasion. Supplementing external media with polyamines for 72 h of DFMO treatment reversed the effects of DFMO. Supplementing external media with polyamines for the last 24 h of DFMO treatment only partially reversed the effects of DFMO. (A–C) Results are represented as mean ± SD, n=3 in duplicates. ^*Statistically significant difference between values obtained from DFMO-treated vs. untreated cells. ^†Statistically significant difference between values obtained from DFMO-treated cells and cells treated with both DFMO and putrescine, spermidine or spermine (P<0.05). (D) MYCN2 cells were treated with or without doxycycline and 5 mM DFMO ± 10 μM putrescine, 10 μM spermidine or 10 μM spermine for 72 h. DFMO treatment induced p27^Kip1 accumulation, downregulation of MYCN expression, and increased the phosphorylation of Akt (Ser473) and GSK3-β (Ser9). Tubulin was used as a loading control. Analysis was performed in three independent experiments (n=3). Doxy, doxycyline; put, putrescine; spd, spermidine; spm, spermine.